Differential Expression of Metallothionein Isoforms in Terrestrial Snail Embryos Reflects Early Life Stage Adaptation to Metal Stress

PubMed Central

Baurand, Pierre-Emmanuel; Pedrini-Martha, Veronika; de Vaufleury, Annette; Niederwanger, Michael; Capelli, Nicolas; Scheifler, Renaud; Dallinger, Reinhard

2015-01-01

The aim of this study was to analyze the expression of three metallothionein (MT) isoform genes (CdMT, CuMT and Cd/CuMT), already known from adults, in the Early Life Stage (ELS) of Cantareus aspersus. This was accomplished by detection of the MT isoform-specific transcription adopting Polymerase Chain Reaction (PCR) amplification and quantitative Real Time (qRT)-PCR of the three MT genes. Freshly laid eggs were kept for 24 hours under control conditions or exposed to three cadmium (Cd) solutions of increasing concentration (5, 10, and 15 mg Cd/L). The transcription of the three MT isoform genes was detected via PCR in 1, 6 and 12-day-old control or Cd-exposed embryos. Moreover, the transcription of this isoform genes during development was followed by qRT-PCR in 6 and 12-day-old embryos. Our results showed that the CdMT and Cd/CuMT genes, but not the CuMT gene, are expressed in embryos at the first day of development. The transcription of the 3 MT genes in control embryos increased with development time, suggesting that the capacities of metal regulation and detoxification may have gradually increased throughout embryogenesis. However in control embryos, the most highly expressed MT gene was that of the Cd/CuMT isoform, whose transcription levels greatly exceeded those of the other two MT genes. This contrasts with the minor significance of this gene in adult snails and suggests that in embryos, this isoform may play a comparatively more important role in metal physiology compared to adult individuals. This function in adult snails appears not to be related to Cd detoxification. Instead, snail embryos responded to Cd exposure by over-expression of the CdMT gene in a concentration-dependent manner, whereas the expression of the Cd/CuMT gene remained unaffected. Moreover, our study demonstrates the ability of snail embryos to respond very early to Cd exposure by up-regulation of the CdMT gene. PMID:25706953

MET Signaling Mediates Intestinal Crypt-Villus Development, Regeneration, and Adenoma Formation and Is Promoted by Stem Cell CD44 Isoforms.

PubMed

Joosten, Sander P J; Zeilstra, Jurrit; van Andel, Harmen; Mijnals, R Clinton; Zaunbrecher, Joost; Duivenvoorden, Annet A M; van de Wetering, Marc; Clevers, Hans; Spaargaren, Marcel; Pals, Steven T

2017-10-01

Resistance of metastatic human colorectal cancer cells to drugs that block epidermal growth factor (EGF) receptor signaling could be caused by aberrant activity of other receptor tyrosine kinases, activating overlapping signaling pathways. One of these receptor tyrosine kinases could be MET, the receptor for hepatocyte growth factor (HGF). We investigated how MET signaling, and its interaction with CD44 (a putative MET coreceptor regulated by Wnt signaling and highly expressed by intestinal stem cells [ISCs] and adenomas) affects intestinal homeostasis, regeneration, and adenoma formation in mini-gut organoids and mice. We established organoid cultures from ISCs stimulated with HGF or EGF and assessed intestinal differentiation by immunohistochemistry. Mice with total epithelial disruption of MET (Ah Cre /Met fl/fl /LacZ) or ISC-specific disruption of MET (Lgr5 Creert2 /Met fl/fl /LacZ) and control mice (Ah Cre /Met +/+ /LacZ, Lgr5 Creert2 /Met +/+ /LacZ) were exposed to 10 Gy total body irradiation; intestinal tissues were collected, and homeostasis and regeneration were assessed by immunohistochemistry. We investigated adenoma organoid expansion stimulated by HGF or EGF using adenomas derived from Lgr5 Creert2 /Met fl/fl /Apc fl/fl and Lgr5 Creert2 /Met +/+ /Apc fl/fl mice. The same mice were evaluated for adenoma prevalence and size. We also quantified adenomas in Ah Cre /Met fl/fl /Apc fl/+ mice compared with Ah Cre /Met +/+ /Apc fl/+ control mice. We studied expansion of organoids generated from crypts and adenomas, stimulated by HGF or EGF, that were derived from mice expressing different CD44 splice variants (Cd44 +/+ , Cd44 -/- , Cd44 s/s , or Cd44 v4-10/v4-10 mice). Crypts incubated with EGF or HGF expanded into self-organizing mini-guts with similar levels of efficacy and contained all differentiated cell lineages. MET-deficient mice did not have defects in intestinal homeostasis. Total body irradiation reduced numbers of proliferating crypts in Ah Cre /Met fl/fl /LacZ mice. Lgr5 Creert2 /Met fl/fl /LacZ mice had impaired regeneration of MET-deficient ISCs. Adenoma organoids stimulated with EGF or HGF expanded to almost twice the size of nonstimulated organoids. MET-deficient adenoma organoids did not respond to HGF stimulation, but did respond to EGF. ISC-specific disruption of Met (Lgr5 Creert2 /Met fl/fl /Apc fl/fl mice) caused a twofold increase in apoptosis in microadenomas, resulting in an approximately 50% reduction of microadenoma numbers and significantly reduced average adenoma size. Total epithelial disruption of Met (Ah Cre /Met fl/fl /Apc fl/+ mice) resulted in an approximate 50% reduction in (micro)adenoma numbers. Intestinal crypts from Cd44 -/- mice did not expand to the same extent as crypts from Cd44 +/+ mice on stimulation with HGF, but had the same response to EGF. The negative effect on HGF-mediated growth was overcome by expression of CD44v4-10, but not by CD44s. Similarly, HGF-mediated expansion of adenoma organoids required CD44v4-10. In studies of intestinal organoid cultures and mice with inducible deletion of MET, we found HGF receptor signaling to regulate intestinal homeostasis and regeneration, as well as adenoma formation. These activities of MET are promoted by the stem cell CD44 isoform CD44v4-10. Our findings provide rationale for targeting signaling via MET and CD44 during anti-EGF receptor therapy of patients with colorectal cancer or in patients resistant to EGF receptor inhibitors. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Enhanced expression of PKM2 associates with the biological properties of cancer stem cells from A549 human lung cancer cells.

PubMed

Guo, Chang-Ying; Yan, Chen; Luo, Lan; Goto, Shinji; Urata, Yoshishige; Xu, Jian-Jun; Wen, Xiao-Ming; Kuang, Yu-Kang; Tou, Fang-Fang; Li, Tao-Sheng

2017-04-01

Cancer cells express the M2 isoform of glycolytic enzyme pyruvate kinase (PKM2) for favoring the survival under a hypoxic condition. Considering the relative low oxygen microenvironment in stem cell niche, we hypothesized that an enhanced PKM2 expression associates with the biological properties of cancer stem cells. We used A549 human lung cancer cell line and surgical resected lung cancer tissue samples from patients for experiments. We confirmed the co-localization of PKM2 and CD44, a popular marker for cancer stem cells in lung cancer tissue samples from patients. The expression of PKM2 was clearly observed in approximately 80% of the A549 human lung cancer cells. Remarkably, enhanced expression of PKM2 was specially observed in these cells that also positively expressed CD44. Downregulation of PKM2 in CD44+ cancer stem cells by siRNA significantly impaired the potency for spheroid formation, decreased the cell survival under fetal bovine serum deprivation and hypoxic conditions, but increased their sensitivity to anti-cancer drug of cisplatin and γ-ray. The enhanced expression of PKM2 seems to associate with the biological properties of cancer stem cells from A549 human lung cancer cells. Selective targeting of PKM2 may provide a new strategy for cancer therapy, especially for patients with therapeutic resistance.

IL-10 production by B cells expressing CD5 with the alternative exon 1B.

PubMed

Garaud, Soizic; Le Dantec, Christelle; de Mendoza, Agnès Revol; Mageed, Rizgar A; Youinou, Pierre; Renaudineau, Yves

2009-09-01

B lymphocytes are divided into two subpopulations, B1 and B2 cells based on expression of the T cell-associated protein CD5. Natural B1 cells are further divided into B1a cells that express CD5 on their membrane and B1b cells that do not but share most other biological characteristics of B1a cells. Recent studies from our laboratory have revealed, in humans, the existence of two alternative isoforms of the CD5 protein. A cell surface CD5 isoform which uses exon 1A (E1A) of the gene in B1a cells, and an intracellular isoform which uses exon 1B (E1B) mainly in human B1b cells. Indeed, the protein isoform encoded by transcripts containing E1B lack the leader peptide and is, thus, retained in the cytoplasm of B cells. The restriction of interleukin (IL)-10 to B1 lymphocytes in the mouse raises the possibility that the human CD5-E1B-expressing B cells produce IL-10. This prediction was confirmed in the CD5 negative Jok-1 B cells transfected with cDNA for either isoforms resulted in high level IL-10 production. Our data indicate that E1B-CD5-expressing B cells have the capacity to interfere with the immune response through their ability to produce high levels of IL-10.

Na+/H+ exchanger isoform 1-induced osteopontin expression facilitates cardiac hypertrophy through p90 ribosomal S6 kinase.

PubMed

Abdulrahman, Nabeel; Jaspard-Vinassa, Beatrice; Fliegel, Larry; Jabeen, Aayesha; Riaz, Sadaf; Gadeau, Alain-Pierre; Mraiche, Fatima

2018-05-01

Cardiovascular diseases are the leading cause of death worldwide. One in three cases of heart failure is due to dilated cardiomyopathy. The Na + /H + exchanger isoform 1 (NHE1), a multifunctional protein and the key pH regulator in the heart, has been demonstrated to be increased in this condition. We have previously demonstrated that elevated NHE1 activity induced cardiac hypertrophy in vivo. Furthermore, the overexpression of active NHE1 elicited modulation of gene expression in cardiomyocytes including an upregulation of myocardial osteopontin (OPN) expression. To determine the role of OPN in inducing NHE1-mediated cardiomyocyte hypertrophy, double transgenic mice expressing active NHE1 and OPN knockout were generated and assessed by echocardiography and the cardiac phenotype. Our studies showed that hearts expressing active NHE1 exhibited cardiac remodeling indicated by increased systolic and diastolic left ventricular internal diameter and increased ventricular volume. Moreover, these hearts demonstrated impaired function with decreased fractional shortening and ejection fraction. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) mRNA was upregulated, and there was an increase in heart cell cross-sectional area confirming the cardiac hypertrophic effect. Moreover, NHE1 transgenic mice also showed increased collagen deposition, upregulation of CD44 and phosphorylation of p90 ribosomal s6 kinase (RSK), effects that were regressed in OPN knockout mice. In conclusion, we developed an interesting comparative model of active NHE1 transgenic mouse lines which express a dilated hypertrophic phenotype expressing CD44 and phosphorylated RSK, effects which were regressed in absence of OPN.

Specific Detection of CD56 (NCAM) Isoforms for the Identification of Aggressive Malignant Neoplasms with Progressive Development

PubMed Central

Gattenlöhner, Stefan; Stühmer, Thorsten; Leich, Ellen; Reinhard, Matthias; Etschmann, Benjamin; Völker, Hans-Ulrich; Rosenwald, Andreas; Serfling, Edgar; Christian Bargou, Ralf; Ertl, Georg; Einsele, Hermann; Müller-Hermelink, Hans-Konrad

2009-01-01

Alternative splicing of transcripts from many cancer-associated genes is believed to play a major role in carcinogenesis as well as in tumor progression. Alternative splicing of one such gene, the neural cell adhesion molecule CD56 (NCAM), impacts the progression, inadequate therapeutic response, and reduced total survival of patients who suffer from numerous malignant neoplasms. Although previous investigations have determined that CD56 exists in three major isoforms (CD56120kD, CD56140kD, and CD56180kD) with individual structural and functional properties, neither the expression profiles nor the functional relevance of these isoforms in malignant tumors have been consistently investigated. Using new quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) strategies and novel CD56 isoform-specific antibodies, CD56140kD was shown to be exclusively expressed in a number of highly malignant CD56+ neoplasms and was associated with the progression of CD56+ precursor lesions of unclear malignant potential. Moreover, only CD56140kD induced antiapoptotic/proliferative pathways and specifically phosphorylated calcium-dependent kinases that are relevant for tumorigenesis. We conclude, therefore, that the specific detection of CD56 isoforms will help to elucidate their individual functions in the pathogenesis and progression of malignant neoplasms and may have a positive impact on the development of CD56-based immunotherapeutic strategies. PMID:19246644

Functional role of human NK cell receptor 2B4 (CD244) isoforms.

PubMed

Mathew, Stephen O; Rao, Krithi K; Kim, Jong R; Bambard, Nowland D; Mathew, Porunelloor A

2009-06-01

2B4 (CD244), a member of the signaling lymphocyte-activation molecule (SLAM/CD150), is expressed on all NK cells, a subpopulation of T cells, monocytes and basophils. Human NK cells express two isoforms of 2B4, h2B4-A and h2B4-B that differ in a small portion of the extracellular domain. In the present investigation, we have studied the functions of h2B4-A and h2B4-B. Our study demonstrated that these two isoforms differ in their binding affinity for CD48, which results in differential cytotoxic activity as well as intracellular calcium release by NK cells upon target cell recognition. Analysis of the predicted 3-D structure of the two isoforms showed conformational differences that could account for their differences in binding affinity to CD48. h2B4-A was able to mediate natural cytotoxicity against CD48-expressing K562 target cells and induce intracellular calcium release, whereas h2B4-B showed no effects. NK-92MI, U937, THP-1, KU812, primary monocytes, basophils and NK cells showed expression of both h2B4-A and h2B4-B whereas YT and IL-2-activated NK cells did not show any h2B4-B expression. Stimulation of NK cells through 2B4 resulted in decreased mRNA levels of both h2B4-A and h2B4-B indicating that down-regulation of 2B4 isoforms may be an important factor in controlling NK cell activation during immune responses.

Characterization of polyethylene glycol-grafted polyethylenimine and superparamagnetic iron oxide nanoparticles (PEG-g-PEI-SPION) as an MRI-visible vector for siRNA delivery in gastric cancer in vitro and in vivo.

PubMed

Chen, Yinting; Lian, Guoda; Liao, Chengde; Wang, Weiwei; Zeng, Linjuan; Qian, Chenchen; Huang, Kaihong; Shuai, Xintao

2013-07-01

Gene therapy is a promising therapeutic method but is severely hampered due to its lack of an ideal delivery system. Therefore, in this study, a nonviral and magnetic resonance imaging (MRI) visible vector, polyethylene glycol-grafted polyethylenimine and superparamagnetic iron oxide nanoparticles (PEG-g-PEI-SPION) was used as a nanocarrier for small interfering RNA (siRNA) delivery in gastric cancer. Biophysical characterization of PEG-g-PEI-SPION was systematically analyzed, including size, zeta potential, siRNA condensation capacity, cell viability, transfection efficiency, cellular uptake, and MRI-visible function in vivo. Besides, CD44 variant isoform 6 (CD44v6), a protein marker for metastatic behavior in gastric cancer, and was chose as the target gene to further analyze the siRNA delivery function of PEG-g-PEI-SPION. Under comprehensive analysis, the appropriate N/P ratio of PEG-g-PEI-SPION/siRNA was 10, and siRNA targeting at human CD44v6 (siCD44v6) transferred by PEG-g-PEI-SPION was effective at downregulating the CD44v6 expression of gastric carcinoma cell line SGC-7901 in vitro. Moreover, knockdown of CD44v6 impaired migrating and invasive abilities of SGC-7901 cells. Furthermore, PEG-g-PEI-SPION was a highly efficient contrast agent for MRI scan in vivo. PEG-g-PEI-SPION was a promising nonviral vector with molecular image tracing capacity for cancer gene therapy. And CD44v6 was a potential target gene for the prevention and detection of metastatic behavior in gastric cancer.

WT1 isoform expression pattern in acute myeloid leukemia.

PubMed

Luna, Irene; Such, Esperanza; Cervera, Jose; Barragán, Eva; Ibañez, Mariam; Gómez-Seguí, Inés; López-Pavía, María; Llop, Marta; Fuster, Oscar; Dolz, Sandra; Oltra, Silvestre; Alonso, Carmen; Vera, Belén; Lorenzo, Ignacio; Martínez-Cuadrón, David; Montesinos, Pau; Senent, M Leonor; Moscardó, Federico; Bolufer, Pascual; Sanz, Miguel A

2013-12-01

WT1 plays a dual role in leukemia development, probably due to an imbalance in the expression of the 4 main WT1 isoforms. We quantify their expression and evaluate them in a series of AML patients. Our data showed a predominant expression of isoform D in AML, although in a lower quantity than in normal CD34+ cells. We found a positive correlation between the total WT1 expression and A, B and C isoforms. The overexpression of WT1 in AML might be due to a relative increase in A, B and C isoforms, together with a relative decrease in isoform D expression. Copyright © 2013 Elsevier Ltd. All rights reserved.

Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities

PubMed Central

Yang, Li-yun; Liu, Xiao-fang; Yang, Yang; Yang, Lin-lin; Liu, Kai-wen; Tang, Yu-bo; Zhang, Min; Tan, Min-jia; Cheng, Shan-mei; Xu, Ye-chun; Yang, Huai-yu; Liu, Zhi-jie; Song, Gao-jie; Huang, Wei

2017-01-01

CD97 belongs to the adhesion GPCR family characterized by a long ECD linked to the 7TM via a GPCR proteolytic site (GPS) and plays important roles in modulating cell migration and invasion. CD97 (EGF1-5) is a splicing variant of CD97 that recognizes a specific ligand chondroitin sulfate on cell membranes and the extracellular matrix. The aim of this study was to elucidate the extracellular molecular basis of the CD97 EGF1-5 isoform in protein expression, auto-proteolysis and cell adhesion, including epidermal growth factor (EGF)-like domain, GPCR autoproteolysis-inducing (GAIN) domain, as well as GPS mutagenesis and N-glycosylation. Both wild-type (WT) CD97-ECD and its truncated, GPS mutated, PNGase F-deglycosylated, and N-glycosylation site mutated forms were expressed and purified. The auto-proteolysis of the proteins was analyzed with Western blotting and SDS-PAGE. Small angle X-ray scattering (SAXS) and molecular modeling were used to determine a structural profile of the properly expressed receptor. Potential N-glycosylation sites were identified using MS and were modulated with PNGase F digestion and glyco-site mutations. A flow cytometry-based HeLa cell attachment assay was used for all aforementioned CD97 variants to elucidate the molecular basis of CD97-HeLa interactions. A unique concentration-dependent GPS auto-proteolysis was observed in CD97 EGF1-5 isoform with the highest concentration (4 mg/mL) per sample was self-cleaved much faster than the lower concentration (0.1 mg/mL), supporting an intermolecular mechanism of auto-proteolysis that is distinct to the reported intramolecular mechanism for other CD97 isoforms. N-glycosylation affected the auto-proteolysis of CD97 EGF1-5 isoform in a similar way as the other previously reported CD97 isoforms. SAXS data for WT and deglycosylated CD97ECD revealed a spatula-like shape with GAIN and EGF domains constituting the body and handle, respectively. Structural modeling indicated a potential interaction between the GAIN and EGF5 domains accounting for the absence of expression of the GAIN domain itself, although EGF5-GAIN was expressed similarly in the wild-type protein. For HeLa cell adhesion, the GAIN-truncated forms showed dramatically reduced binding affinity. The PNGase F-deglycosylated and GPS mutated forms also exhibited reduced HeLa attachment compared with WT CD97. However, neither N-glycosylation mutagenesis nor auto-proteolysis inhibition caused by N-glycosylation mutagenesis affected CD97-HeLa cell interactions. A comparison of the HeLa binding affinities of PNGase F-digested, GPS-mutated and N-glycosylation-mutated CD97 samples revealed diverse findings, suggesting that the functions of CD97 ECD were complex, and various technologies for function validation should be utilized to avoid single-approach bias when investigating N-glycosylation and auto-proteolysis of CD97. A unique mechanism of concentration-dependent auto-proteolysis of the CD97 EGF1-5 isoform was characterized, suggesting an intermolecular mechanism that is distinct from that of other previously reported CD97 isoforms. The EGF5 and GAIN domains are likely associated with each other as CD97 expression and SAXS data revealed a potential interaction between the two domains. Finally, the GAIN and EGF domains are also important for CD97-HeLa adhesion, whereas N-glycosylation of the CD97 GAIN domain and GPS auto-proteolysis are not required for HeLa cell attachment. PMID:27641734

Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities.

PubMed

Yang, Li-Yun; Liu, Xiao-Fang; Yang, Yang; Yang, Lin-Lin; Liu, Kai-Wen; Tang, Yu-Bo; Zhang, Min; Tan, Min-Jia; Cheng, Shan-Mei; Xu, Ye-Chun; Yang, Huai-Yu; Liu, Zhi-Jie; Song, Gao-Jie; Huang, Wei

2017-01-01

CD97 belongs to the adhesion GPCR family characterized by a long ECD linked to the 7TM via a GPCR proteolytic site (GPS) and plays important roles in modulating cell migration and invasion. CD97 (EGF1-5) is a splicing variant of CD97 that recognizes a specific ligand chondroitin sulfate on cell membranes and the extracellular matrix. The aim of this study was to elucidate the extracellular molecular basis of the CD97 EGF1-5 isoform in protein expression, auto-proteolysis and cell adhesion, including epidermal growth factor (EGF)-like domain, GPCR autoproteolysis-inducing (GAIN) domain, as well as GPS mutagenesis and N-glycosylation. Both wild-type (WT) CD97-ECD and its truncated, GPS mutated, PNGase F-deglycosylated, and N-glycosylation site mutated forms were expressed and purified. The auto-proteolysis of the proteins was analyzed with Western blotting and SDS-PAGE. Small angle X-ray scattering (SAXS) and molecular modeling were used to determine a structural profile of the properly expressed receptor. Potential N-glycosylation sites were identified using MS and were modulated with PNGase F digestion and glyco-site mutations. A flow cytometry-based HeLa cell attachment assay was used for all aforementioned CD97 variants to elucidate the molecular basis of CD97-HeLa interactions. A unique concentration-dependent GPS auto-proteolysis was observed in CD97 EGF1-5 isoform with the highest concentration (4 mg/mL) per sample was self-cleaved much faster than the lower concentration (0.1 mg/mL), supporting an intermolecular mechanism of auto-proteolysis that is distinct to the reported intramolecular mechanism for other CD97 isoforms. N-glycosylation affected the auto-proteolysis of CD97 EGF1-5 isoform in a similar way as the other previously reported CD97 isoforms. SAXS data for WT and deglycosylated CD97ECD revealed a spatula-like shape with GAIN and EGF domains constituting the body and handle, respectively. Structural modeling indicated a potential interaction between the GAIN and EGF5 domains accounting for the absence of expression of the GAIN domain itself, although EGF5-GAIN was expressed similarly in the wild-type protein. For HeLa cell adhesion, the GAIN-truncated forms showed dramatically reduced binding affinity. The PNGase F-deglycosylated and GPS mutated forms also exhibited reduced HeLa attachment compared with WT CD97. However, neither N-glycosylation mutagenesis nor auto-proteolysis inhibition caused by N-glycosylation mutagenesis affected CD97-HeLa cell interactions. A comparison of the HeLa binding affinities of PNGase F-digested, GPS-mutated and N-glycosylation-mutated CD97 samples revealed diverse findings, suggesting that the functions of CD97 ECD were complex, and various technologies for function validation should be utilized to avoid single-approach bias when investigating N-glycosylation and auto-proteolysis of CD97. A unique mechanism of concentration-dependent auto-proteolysis of the CD97 EGF1-5 isoform was characterized, suggesting an intermolecular mechanism that is distinct from that of other previously reported CD97 isoforms. The EGF5 and GAIN domains are likely associated with each other as CD97 expression and SAXS data revealed a potential interaction between the two domains. Finally, the GAIN and EGF domains are also important for CD97-HeLa adhesion, whereas N-glycosylation of the CD97 GAIN domain and GPS auto-proteolysis are not required for HeLa cell attachment.

Altered STAT4 Isoform Expression in Patients with Inflammatory Bowel Disease.

PubMed

Jabeen, Rukhsana; Miller, Lucy; Yao, Weiguo; Gupta, Sandeep; Steiner, Steven; Kaplan, Mark H

2015-10-01

Crohn's disease (CD) and ulcerative colitis (UC) are the major forms of inflammatory bowel disease, and pathogenesis involves a complex interplay among genetic, environmental, and immunological factors. We evaluated isoform expression of the IL-12-activated transcription factor STAT4 in children with CD and UC. We collected biopsy samples from both patients newly diagnosed with CD and with UC. We further collected blood samples from patients newly diagnosed with CD and with UC as well as from patients who had a flare-up after being in clinical remission, and we examined the ratios of STAT4β/STAT4α mRNA. In addition to STAT4 isoforms, we measured the expression of the cytokines TNFα, IFNγ, granulocyte macrophage-colony stimulating factor, and IL-17 using polymerase chain reaction of biopsy samples and multiplex analysis of patient serum samples. Ratios of STAT4β/STAT4α were increased in specific gastrointestinal tract segments in both patients with CD and those with UC that correlate with the location and severity of inflammation. In contrast, we did not observe changes in STAT4β/STAT4α ratios in biopsy specimens from patients with eosinophilic esophagitis. We also observed increased STAT4β/STAT4α ratios in the peripheral blood mononuclear cells of patients with UC and those with CD, compared with healthy controls. Ratios were normalized after patients were treated with steroids. Collectively, these data indicate that STAT4 isoforms could be an important noninvasive biomarker in the diagnosis and treatment of inflammatory bowel disease and that expression of these isoforms might provide further insight into the pathogenesis of IBD.

Novel mesenchymal and haematopoietic cell isoforms of the SHP-2 docking receptor, PZR: identification, molecular cloning and effects on cell migration.

PubMed Central

Zannettino, Andrew C W; Roubelakis, Maria; Welldon, Katie J; Jackson, Denise E; Simmons, Paul J; Bendall, Linda J; Henniker, Anthony; Harrison, Kate L; Niutta, Silvana; Bradstock, Kenneth F; Watt, Suzanne M

2003-01-01

SHP-2 (Src homology phosphatase type-2) is essential for haematopoietic skeletal and vascular development. Thus the identification of its binding partners is critically important. In the present study, we describe a unique monoclonal antibody, WM78, which interacts with PZR, a SHP-2 binding partner. Furthermore, we identify two novel isoforms of PZR, PZRa and PZRb, derived by differential splicing from a single gene transcription unit on human chromosome 1q24. All are type 1 transmembrane glycoproteins with identical extracellular and transmembrane domains, but differ in their cytoplasmic tails. The PZR intracellular domain contains two SHP-2 binding immunoreceptor tyrosine-based inhibitory motifs (VIY(246)AQL and VVY(263)ADI) which are not present in PZRa and PZRb. Using the WM78 monoclonal antibody, which recognizes the common extracellular domain of the PZR isoforms, we demonstrate that the PZR molecules are expressed on mesenchymal and haematopoietic cells, being present on the majority of CD34(+)CD38(+) and early clonogenic progenitors, and at lower levels on CD34(+)CD38(-) cells and the hierarchically more primitive pre-colony forming units. Interestingly, we show by reverse transcriptase-PCR that the PZR isoforms are differentially expressed in haematopoietic, endothelial and mesenchymal cells. Both PZR and PZRb are present in CD133(+) precursors and endothelial cells, PZRb predominates in mesenchymal and committed myelomonocytic progenitor cells, and all three isoforms occur in erythroid precursor cell lines. Importantly, using SHP-2 mutant (Delta 46-110) and SHP-2 rescue of embryonic fibroblasts stably expressing the PZR isoforms, we demonstrate for the first time that PZR, but not PZRa or PZRb, facilitates fibronectin- dependent migration of cells expressing a competent SHP-2 molecule. These observations will be instrumental in determining the mechanisms whereby PZR isoforms regulate cell motility. PMID:12410637

Tartrate-resistant acid phosphatase (TRAP/ACP5) promotes metastasis-related properties via TGFβ2/TβR and CD44 in MDA-MB-231 breast cancer cells.

PubMed

Reithmeier, Anja; Panizza, Elena; Krumpel, Michael; Orre, Lukas M; Branca, Rui M M; Lehtiö, Janne; Ek-Rylander, Barbro; Andersson, Göran

2017-09-15

Tartrate-resistant acid phosphatase (TRAP/ACP5), a metalloenzyme that is characteristic for its expression in activated osteoclasts and in macrophages, has recently gained considerable focus as a driver of metastasis and was associated with clinically relevant parameters of cancer progression and cancer aggressiveness. MDA-MB-231 breast cancer cells with different TRAP expression levels (overexpression and knockdown) were generated and characterized for protein expression and activity levels. Functional cell experiments, such as proliferation, migration and invasion assays were performed as well as global phosphoproteomic and proteomic analysis was conducted to connect molecular perturbations to the phenotypic changes. We identified an association between metastasis-related properties of TRAP-overexpressing MDA-MB-231 breast cancer cells and a TRAP-dependent regulation of Transforming growth factor (TGFβ) pathway proteins and Cluster of differentiation 44 (CD44). Overexpression of TRAP increased anchorage-independent and anchorage-dependent cell growth and proliferation, induced a more elongated cellular morphology and promoted cell migration and invasion. Migration was increased in the presence of the extracellular matrix (ECM) proteins osteopontin and fibronectin and the basement membrane proteins collagen IV and laminin I. TRAP-induced properties were reverted upon shRNA-mediated knockdown of TRAP or treatment with the small molecule TRAP inhibitor 5-PNA. Global phosphoproteomics and proteomics analyses identified possible substrates of TRAP phosphatase activity or signaling intermediates and outlined a TRAP-dependent regulation of proteins involved in cell adhesion and ECM organization. Upregulation of TGFβ isoform 2 (TGFβ2), TGFβ receptor type 1 (TβR1) and Mothers against decapentaplegic homolog 2 (SMAD2), as well as increased intracellular phosphorylation of CD44 were identified upon TRAP perturbation. Functional antibody-mediated blocking and chemical inhibition demonstrated that TRAP-dependent migration and proliferation is regulated via TGFβ2/TβR, whereas proliferation beyond basal levels is regulated through CD44. Altogether, TRAP promotes metastasis-related cell properties in MDA-MB-231 breast cancer cells via TGFβ2/TβR and CD44, thereby identifying a potential signaling mechanism associated to TRAP action in breast cancer cells.

CD44 expression is related to poor prognosis of hypopharyngeal squamous cell carcinoma.

PubMed

Uwa, Nobuhiro; Kataoka, Tatsuki R; Torii, Ikuko; Sato, Ayuko; Nishigami, Takashi; Song, Misa; Daimon, Takashi; Saeki, Nobuo; Sagawa, Kousuke; Mouri, Takeshi; Terada, Tomonori; Sakagami, Masafumi; Tsujimura, Tohru

2011-03-01

CD44 expression in hypopharyngeal squamous cell carcinomas (SCCs) is closely associated with poor prognosis for patients. CD44 may serve as a prognostic marker for hypopharyngeal SCCs. CD44, an adhesion molecule binding to extracellular matrix, is believed to participate in the progression of malignancies. To clarify the role of CD44 in the progression of hypopharyngeal SCCs, we examined CD44 expression in relation to clinical parameters in hypopharyngeal SCCs. Biopsy specimens of hypopharyngeal SCCs were collected from 40 untreated patients, and their CD44 expression was examined immunohistochemically. Hypopharyngeal SCCs were classified into two groups: CD44-low SCCs comprising < 50% CD44-positive tumor cells and CD44-high SCCs comprising ≥ 50% CD44-positive tumor cells. The relation between CD44 expression and various parameters (clinical T and N stages, distant metastasis, and pathological T and N stages) was analyzed by Fisher's exact test. The relation between CD44 expression and the 5-year disease-free survival (DFS) rate was also analyzed by log rank test. The CD44 expression in hypopharyngeal SCCs was related to pathological N stage, but not to clinical T and N stages and pathological T stage, of the patients. Distant metastasis during the follow-up occurred more frequently in patients with CD44-high SCCs than those with CD44-low SCCs. The 5-year DFS was significantly lower in the former than in the latter.

CD44s and CD44v6 Expression in Head and Neck Epithelia

PubMed Central

Mack, Brigitte; Gires, Olivier

2008-01-01

Background CD44 splice variants are long-known as being associated with cell transformation. Recently, the standard form of CD44 (CD44s) was shown to be part of the signature of cancer stem cells (CSCs) in colon, breast, and in head and neck squamous cell carcinomas (HNSCC). This is somewhat in contradiction to previous reports on the expression of CD44s in HNSCC. The aim of the present study was to clarify the actual pattern of CD44 expression in head and neck epithelia. Methods Expression of CD44s and CD44v6 was analysed by immunohistochemistry with specific antibodies in primary head and neck tissues. Scoring of all specimens followed a two-parameters system, which implemented percentages of positive cells and staining intensities from − to +++ (score = %×intensity; resulting max. score 300). In addition, cell surface expression of CD44s and CD44v6 was assessed in lymphocytes and HNSCC. Results In normal epithelia CD44s and CD44v6 were expressed in 60–95% and 50–80% of cells and yielded mean scores with a standard error of a mean (SEM) of 249.5±14.5 and 198±11.13, respectively. In oral leukoplakia and in moderately differentiated carcinomas CD44s and CD44v6 levels were slightly increased (278.9±7.16 and 242±11.7; 291.8±5.88 and 287.3±6.88). Carcinomas in situ displayed unchanged levels of both proteins whereas poorly differentiated carcinomas consistently expressed diminished CD44s and CD44v6 levels. Lymphocytes and HNSCC lines strongly expressed CD44s but not CD44v6. Conclusion CD44s and CD44v6 expression does not distinguish normal from benign or malignant epithelia of the head and neck. CD44s and CD44v6 were abundantly present in the great majority of cells in head and neck tissues, including carcinomas. Hence, the value of CD44s as a marker for the definition of a small subset of cells (i.e. less than 10%) representing head and neck cancer stem cells may need revision. PMID:18852874

Screening and Identification of a Phage Display Derived Peptide That Specifically Binds to the CD44 Protein Region Encoded by Variable Exons.

PubMed

Zhang, Dan; Jia, Huan; Li, Weiming; Hou, Yingchun; Lu, Shaoying; He, Shuixiang

2016-01-01

CD44, especially the isoforms with variable exons (CD44v), is a promising biomarker for the detection of cancer. To develop a CD44v-specific probe, we screened a 7-mer phage peptide library against the CD44v3-v10 protein using an improved subtractive method. The consensus sequences with the highest frequency (designated CV-1) emerged after four rounds of panning. The binding affinity and specificity of the CV-1 phage and the synthesized peptide for the region of CD44 encoded by the variable exons were confirmed using enzyme-linked immunosorbent assay and competitive inhibition assays. Furthermore, the binding of the CV-1 probe to gastric cancer cells and tissues was validated using immunofluorescence and immunohistochemistry assays. CV-1 sensitively and specifically bound to CD44v on cancer cells and tissues. Thus, CV-1 has the potential to serve as a promising probe for cancer molecular imaging and target therapy. © 2015 Society for Laboratory Automation and Screening.

Intracellular Domain Fragment of CD44 Alters CD44 Function in Chondrocytes*

PubMed Central

Mellor, Liliana; Knudson, Cheryl B.; Hida, Daisuke; Askew, Emily B.; Knudson, Warren

2013-01-01

The hyaluronan receptor CD44 undergoes sequential proteolytic cleavage at the cell surface. The initial cleavage of the CD44 extracellular domain is followed by a second intramembranous cleavage of the residual CD44 fragment, liberating the C-terminal cytoplasmic tail of CD44. In this study conditions that promote CD44 cleavage resulted in a diminished capacity to assemble and retain pericellular matrices even though sufficient non-degraded full-length CD44 remained. Using stable and transient overexpression of the cytoplasmic domain of CD44, we determined that the intracellular domain interfered with anchoring of the full-length CD44 to the cytoskeleton and disrupted the ability of the cells to bind hyaluronan and assemble a pericellular matrix. Co-immunoprecipitation assays were used to determine whether the mechanism of this interference was due to competition with actin adaptor proteins. CD44 of control chondrocytes was found to interact and co-immunoprecipitate with both the 65- and 130-kDa isoforms of ankyrin-3. Moreover, this interaction with ankyrin-3 proteins was diminished in cells overexpressing the CD44 intracellular domain. Mutating the putative ankyrin binding site of the transiently transfected CD44 intracellular domain diminished the inhibitory effects of this protein on matrix retention. Although CD44 in other cells types has been shown to interact with members of the ezrin/radixin/moesin (ERM) family of adaptor proteins, only modest interactions between CD44 and moesin could be demonstrated in chondrocytes. The data suggest that release of the CD44 intracellular domain into the cytoplasm of cells such as chondrocytes exerts a competitive or dominant-negative effect on the function of full-length CD44. PMID:23884413

Non-Small Cell Lung Cancer Cells Expressing CD44 Are Enriched for Stem Cell-Like Properties

PubMed Central

Leung, Elaine Lai-Han; Fiscus, Ronald R.; Tung, James W.; Tin, Vicky Pui-Chi; Cheng, Lik Cheung; Sihoe, Alan Dart-Loon; Fink, Louis M.; Ma, Yupo; Wong, Maria Pik

2010-01-01

Background The cancer stem cell theory hypothesizes that cancers are perpetuated by cancer stem cells (CSC) or tumor initiating cells (TIC) possessing self-renewal and other stem cell-like properties while differentiated non-stem/initiating cells have a finite life span. To investigate whether the hypothesis is applicable to lung cancer, identification of lung CSC and demonstration of these capacities is essential. Methodology/Principal Finding The expression profiles of five stem cell markers (CD34, CD44, CD133, BMI1 and OCT4) were screened by flow cytometry in 10 lung cancer cell lines. CD44 was further investigated by testing for in vitro and in vivo tumorigenecity. Formation of spheroid bodies and in vivo tumor initiation ability were demonstrated in CD44+ cells of 4 cell lines. Serial in vivo tumor transplantability in nude mice was demonstrated using H1299 cell line. The primary xenografts initiated from CD44+ cells consisted of mixed CD44+ and CD44− cells in similar ratio as the parental H1299 cell line, supporting in vivo differentiation. Semi-quantitative Real-Time PCR (RT-PCR) showed that both freshly sorted CD44+ and CD44+ cells derived from CD44+-initiated tumors expressed the pluripotency genes OCT4/POU5F1, NANOG, SOX2. These stemness markers were not expressed by CD44− cells. Furthermore, freshly sorted CD44+ cells were more resistant to cisplatin treatment with lower apoptosis levels than CD44− cells. Immunohistochemical analysis of 141 resected non-small cell lung cancers showed tumor cell expression of CD44 in 50.4% of tumors while no CD34, and CD133 expression was observed in tumor cells. CD44 expression was associated with squamous cell carcinoma but unexpectedly, a longer survival was observed in CD44-expressing adenocarcinomas. Conclusion/Significance Overall, our results demonstrated that stem cell-like properties are enriched in CD44-expressing subpopulations of some lung cancer cell lines. Further investigation is required to clarify the role of CD44 in tumor cell renewal and cancer propagation in the in vivo environment. PMID:21124918

Differential regulation of CD44 expression by lipopolysaccharide (LPS) and TNF-alpha in human monocytic cells: distinct involvement of c-Jun N-terminal kinase in LPS-induced CD44 expression.

PubMed

Gee, Katrina; Lim, Wilfred; Ma, Wei; Nandan, Devki; Diaz-Mitoma, Francisco; Kozlowski, Maya; Kumar, Ashok

2002-11-15

Alterations in the regulation of CD44 expression play a critical role in modulating cell adhesion, migration, and inflammation. LPS, a bacterial cell wall component, regulates CD44 expression and may modulate CD44-mediated biological effects in monocytic cells during inflammation and immune responses. In this study, we show that in normal human monocytes, LPS and LPS-induced cytokines IL-10 and TNF-alpha enhance CD44 expression. To delineate the mechanism underlying LPS-induced CD44 expression, we investigated the role of the mitogen-activated protein kinases (MAPKs), p38, p42/44 extracellular signal-regulated kinase, and c-Jun N-terminal kinase (JNK) by using their specific inhibitors. We demonstrate the involvement, at least in part, of p38 MAPK in TNF-alpha-induced CD44 expression in both monocytes and promonocytic THP-1 cells. However, neither p38 nor p42/44 MAPKs were involved in IL-10-induced CD44 expression in monocytes. To further dissect the TNF-alpha and LPS-induced signaling pathways regulating CD44 expression independent of IL-10-mediated effects, we used IL-10 refractory THP-1 cells as a model system. Herein, we show that CD44 expression induced by the LPS-mediated pathway predominantly involved JNK activation. This conclusion was based on results derived by transfection of THP-1 cells with a dominant-negative mutant of stress-activated protein/extracellular signal-regulated kinase kinase 1, and by exposure of cells to JNK inhibitors dexamethasone and SP600125. All these treatments prevented CD44 induction in LPS-stimulated, but not in TNF-alpha-stimulated, THP-1 cells. Furthermore, we show that CD44 induction may involve JNK-dependent early growth response gene activation in LPS-stimulated monocytic cells. Taken together, these results suggest a predominant role of JNK in LPS-induced CD44 expression in monocytic cells.

Forced expression of the Ikaros 6 isoform in human placental blood CD34(+) cells impairs their ability to differentiate toward the B-lymphoid lineage.

PubMed

Tonnelle, C; Bardin, F; Maroc, C; Imbert, A M; Campa, F; Dalloul, A; Schmitt, C; Chabannon, C

2001-11-01

Studies in mice suggest that the Ikaros (Ik) gene encodes several isoforms and is a critical regulator of hematolymphoid differentiation. Little is known on the role of Ikaros in human stem cell differentiation. Herein, the biological consequences of the forced expression of Ikaros 6 (Ik6) in human placental blood CD34(+) progenitors are evaluated. Ik6 is one of the isoforms produced from the Ikaros premessenger RNA by alternative splicing and is thought to behave as a dominant negative isoform of the gene product because it lacks the DNA binding domain present in transcriptionally active isoforms. The results demonstrate that human cord blood CD34(+) cells that express high levels of Ik6 as a result of retrovirally mediated gene transfer have a reduced capacity to produce lymphoid B cells in 2 independent assays: (1) in vitro reinitiation of human hematopoiesis during coculture with the MS-5 murine stromal cell line and (2) xenotransplantation in nonobese diabetic-severe combined immunodeficient mice. These results suggest that Ikaros plays an important role in stem cell commitment in humans and that the balance between the different isoforms is a key element of this regulatory system; they support the hypothesis that posttranscriptional events can participate in the control of human hematopoietic differentiation.

Prognostic value of CD44 expression in penile squamous cell carcinoma: a pilot study.

PubMed

Minardi, Daniele; Lucarini, Guendalina; Filosa, Alessandra; Zizzi, Antonio; Simonetti, Oriana; Offidani, Anna Maria; d'Anzeo, Gianluca; Di Primio, Roberto; Montironi, Rodolfo; Muzzonigro, Giovanni

2012-10-01

Several studies have reported on the prognostic value of molecular markers for metastasis risk and survival in penile squamous cell carcinoma (SCC) patients. The usefulness of CD44 expression as such a marker has been studied in different tumors, but not in penile SCC. Our aim was to determine whether CD44 expression may serve as a prognostic marker for lymph node metastasis and survival in penile SCC patients. CD44 immunoistochemical expression was investigated in tissue specimens from 39 patients with penile SCC. CD44 cell positivity, staining intensity and distribution were analyzed and correlated with tumor stage, grade, lymph node status and disease-specific survival. CD44 expression was detected in epithelial cells of both intratumoral and normal tissues with different intensities and staining distributions. In normal tissues CD44 protein was mainly detected in cell membranes, whereas in the tumor compartments it was found in both the cell membranes and the cytoplasm. The intensities and percentages of CD44 expressing cells did not correlate with tumor stage and/or grade. Seventy-three percent of the patients with lymph node metastasis showed high intensities of CD44 staining, as compared to 44% of the patients without lymph node metastasis (P = 0.03). Lymph node-positive patients showed both cytoplasmic and membranous CD44 expression. High CD44 expression was found to be significantly correlated with a decreased 5 year overall survival (P = 0.01). CD44 levels and patterns of expression can be considered as markers for penile SCC aggressiveness and, in addition, may serve as predictive markers for lymph node metastasis, also in patients with clinically negative lymph nodes. CD44 expression may provide prognostic information for penile SCC patients, next to classical clinical-pathological factors.

mRNA Quantification of NIPBL Isoforms A and B in Adult and Fetal Human Tissues, and a Potentially Pathological Variant Affecting Only Isoform A in Two Patients with Cornelia de Lange Syndrome

PubMed Central

Puisac, Beatriz; Teresa-Rodrigo, María-Esperanza; Hernández-Marcos, María; Baquero-Montoya, Carolina; Gil-Rodríguez, María-Concepción; Visnes, Torkild; Bot, Christopher; Gómez-Puertas, Paulino; Kaiser, Frank J.; Ramos, Feliciano J.; Ström, Lena; Pié, Juan

2017-01-01

Cornelia de Lange syndrome (CdLS) is a congenital developmental disorder characterized by craniofacial dysmorphia, growth retardation, limb malformations, and intellectual disability. Approximately 60% of patients with CdLS carry a recognizable pathological variant in the NIPBL gene, of which two isoforms, A and B, have been identified, and which only differ in the C-terminal segment. In this work, we describe the distribution pattern of the isoforms A and B mRNAs in tissues of adult and fetal origin, by qPCR (quantitative polymerase chain reaction). Our results show a higher gene expression of the isoform A, even though both seem to have the same tissue distribution. Interestingly, the expression in fetal tissues is higher than that of adults, especially in brain and skeletal muscle. Curiously, the study of fibroblasts of two siblings with a mild CdLS phenotype and a pathological variant specific of the isoform A of NIPBL (c.8387A > G; P.Tyr2796Cys), showed a similar reduction in both isoforms, and a normal sensitivity to DNA damage. Overall, these results suggest that the position of the pathological variant at the 3´ end of the NIPBL gene affecting only isoform A, is likely to be the cause of the atypical mild phenotype of the two brothers. PMID:28241484

Expression of CD44s and CD44v6 in transitional cell carcinomas of the urinary bladder: comparison with tumour grade, proliferative activity and p53 immunoreactivity of tumour cells.

PubMed

Kuncová, Jitka; Urban, Michael; Mandys, Václav

2007-11-01

Alterations of CD44 glycoproteins have been shown to play an important role in progression of various malignancies, including urothelial cancer. We investigated expression patterns of CD44s and CD44v6 in transitional cell carcinoma (TCC) of the urinary bladder in relation to tumour grade, proliferative activity, and immunoreactivity for p53. The selected markers were detected immunohistochemically in 122 samples of TCC. We found a close relationship between CD44s and CD44v6 expression and tumour grade. The extension of positive staining for CD44s and CD44v6 towards the luminal surface was a predominant feature of differentiated carcinomas (grades 1 and 2), suggesting deranged maturation of cancer cells related to their neoplastic transformation. Heterogeneous expression of CD44s and CD44v6 predominated in poorly differentiated tumours (G3-4). However, areas of squamous differentiation within the high-grade tumours displayed strong immunoreactivity for both CD44s and CD44v6. The proliferative activity and p53 overexpression increased with the dedifferentiation of the tumour. The results of this study are discussed in relation to the significance of CD44 expression in TCC and to the explanation for controversial results reported in previous studies on the relationship between CD44 expression and the biological behaviour of urothelial cells.

Pancreatic cancer cells express CD44 variant 9 and multidrug resistance protein 1 during mitosis.

PubMed

Kiuchi, Shizuka; Ikeshita, Shunji; Miyatake, Yukiko; Kasahara, Masanori

2015-02-01

Pancreatic cancer is one of the most lethal cancers with high metastatic potential and strong chemoresistance. Its intractable natures are attributed to high robustness in tumor cells for their survival. We demonstrate here that pancreatic cancer cells (PCCs) with an epithelial phenotype upregulate cell surface expression of CD44 variant 9 (CD44v9), an important cancer stem cell marker, during the mitotic phases of the cell cycle. Of five human CD44(+) PCC lines examined, three cell lines, PCI-24, PCI-43 and PCI-55, expressed E-cadherin and CD44 variants, suggesting that they have an epithelial phenotype. By contrast, PANC-1 and MIA PaCa-2 cells expressed vimentin and ZEB1, suggesting that they have a mesenchymal phenotype. PCCs with an epithelial phenotype upregulated cell surface expression of CD44v9 in prophase, metaphase, anaphase and telophase and downregulated CD44v9 expression in late-telophase, cytokinesis and interphase. Sorted CD44v9-negative PCI-55 cells resumed CD44v9 expression when they re-entered the mitotic stage. Interestingly, CD44v9(bright) mitotic cells expressed multidrug resistance protein 1 (MDR1) intracellularly. Upregulated expression of CD44v9 and MDR1 might contribute to the intractable nature of PCCs with high proliferative activity. Copyright © 2014 Elsevier Inc. All rights reserved.

CD44 regulates cell migration in human colon cancer cells via Lyn kinase and AKT phosphorylation.

PubMed

Subramaniam, Venkateswaran; Vincent, Isabella R; Gardner, Helena; Chan, Emily; Dhamko, Helena; Jothy, Serge

2007-10-01

Colon cancer is among the leading causes of cancer death in North America. CD44, an adhesion and antiapoptotic molecule is overexpressed in colon cancer. Cofilin is involved in the directional motility of cells. In the present study, we looked at how CD44 might modulate cell migration in human colon cancer via cofilin. We used a human colon cancer cell line, HT29, which expresses CD44, HT29 where CD44 expression was knocked down by siRNA, SW620, a human colon cancer cell line which does not express CD44, stably transfected exons of CD44 in SW620 cells and the colon from CD44 knockout and wild-type mouse. Western blot analysis of siRNA CD44 lysates showed increased level of AKT phosphorylation and decreased level of cofilin expression. Similar results were also observed with SW620 cells and CD44 knockout mouse colon lysates. Experiments using the AKT phosphorylation inhibitor LY294002 indicate that AKT phosphorylation downregulates cofilin. Immunoprecipitation studies showed CD44 complex formation with Lyn, providing an essential link between CD44 and AKT phosphorylation. LY294002 also stabilized Lyn from phosphorylated AKT, suggesting an interaction between Lyn and AKT phosphorylation. Immunocytochemistry showed that cofilin and Lyn expression were downregulated in siRNA CD44 cells and CD44 knockout mouse colon. siRNA CD44 cells had significantly less migration compared to HT29 vector. Given the well-defined roles of CD44, phosphorylated AKT in apoptosis and cancer, these results indicate that CD44-induced cell migration is dependent on its complex formation with Lyn and its consequent regulation of AKT phosphorylation and cofilin expression.

Low molecular weight (LMW) heparin inhibits injury-induced femoral artery remodeling in mouse via upregulating CD44 expression.

PubMed

Zhao, Gaofeng; Shaik, Rahamthulla S; Zhao, Hang; Beagle, John; Kuo, Shuennwen; Hales, Charles A

2011-05-01

The mechanism of postangioplasty restenosis remains poorly understood. Low molecular weight (LMW) heparin has been shown to inhibit the proliferation of vascular smooth muscle cells (VSMCs), which is the principal characteristic of restenosis. Studies have shown that LMW heparin could bind to CD44. We hypothesized that LMW heparin might modulate CD44 expression thereby decreasing vascular remodeling. Vascular remodeling was induced in CD44(+/+) and CD44(-/-) mice and treated with LMW heparin. The arteries were harvested for histologic assessment and determination of CD44 expression. Bone marrow transplantation was introduced to further explore the role and functional sites of CD44. Effects of LMW heparin on growth capacity, CD44 expression were further studied using the cultured mouse VSMCs. Transluminal injury induced remarkable remodeling in mouse femoral artery (sham wall thickness percentage [WT%]: 3.4 ± 1.2% vs injury WT%: 31.8 ± 4.7%; P < .001). LMW heparin reduced the remodeling significantly (WT%: 17.8 ± 3.5%, P < .005). CD44(-/-) mice demonstrated considerably thicker arterial wall remodeling (WT%: 46.2 ± 7.6%, P = .0035), and CD44-chimeric mice exhibited equal contributions of the local and circulating CD44 signal to the neointima formation. LMW heparin markedly upregulated CD44 expression in the injured femoral arteries. In vitro, LMW heparin decreased mouse VSMC growth capacity and upregulated its CD44 expression simultaneously in a dose-dependent and time-dependent manner, which could be partially blocked by CD44 inhibitor. LMW heparin inhibits injury-induced femoral artery remodeling, at least partially, by upregulating CD44 expression. Copyright © 2011. Published by Mosby, Inc.

Role of CD44 in lymphokine-activated killer cell-mediated killing of melanoma.

PubMed

Sun, Jingping; Law, Gabriela P; McKallip, Robert J

2012-03-01

In the current study, we examined the potential significance of CD44 expression on lymphokine-activated killer (LAK) cells in their interaction and killing of melanoma cells. Stimulation of splenocytes with IL-2 led to a significant increase in the expression of CD44 on T cells, NK cells, and NKT cells. Treatment of melanoma-bearing CD44 WT mice with IL-2 led to a significant reduction in the local tumor growth while treatment of melanoma-bearing CD44 KO mice with IL-2 was ineffective at controlling tumor growth. Furthermore, the ability of splenocytes from IL-2-treated CD44 KO mice to kill melanoma tumor targets was significantly reduced when compared to the anti-tumor activity of splenocytes from IL-2-treated CD44 WT mice. The importance of CD44 expression on the LAK cells was further confirmed by the observation that adoptively transferred CD44 WT LAK cells were significantly more effective than CD44 KO LAK cells at controlling tumor growth in vivo. Next, the significance of the increased expression of CD44 in tumor killing was examined and showed that following stimulation with IL-2, distinct populations of cells with low (CD44(lo)) or elevated (CD44(hi)) expression of CD44 are generated and that the CD44(hi) cells are responsible for killing of the melanoma cells. The reduced killing activity of the CD44 KO LAK cells did not result from reduced activation or expression of effector molecules but was due, at least in part, to a reduced ability to adhere to B16F10 tumor cells.

Heterologous expression of a rice metallothionein isoform (OsMTI-1b) in Saccharomyces cerevisiae enhances cadmium, hydrogen peroxide and ethanol tolerance.

PubMed

Ansarypour, Zahra; Shahpiri, Azar

Metallothioneins are a superfamily of low-molecular-weight, cysteine (Cys)-rich proteins that are believed to play important roles in protection against metal toxicity and oxidative stress. The main purpose of this study was to investigate the effect of heterologous expression of a rice metallothionein isoform (OsMTI-1b) on the tolerance of Saccharomyces cerevisiae to Cd 2+ , H 2 O 2 and ethanol stress. The gene encoding OsMTI-1b was cloned into p426GPD as a yeast expression vector. The new construct was transformed to competent cells of S. cerevisiae. After verification of heterologous expression of OsMTI-1b, the new strain and control were grown under stress conditions. In comparison to control strain, the transformed S. cerevisiae cells expressing OsMTI-1b showed more tolerance to Cd 2+ and accumulated more Cd 2+ ions when they were grown in the medium containing CdCl 2 . In addition, the heterologous expression of GST-OsMTI-1b conferred H 2 O 2 and ethanol tolerance to S. cerevisiae cells. The results indicate that heterologous expression of plant MT isoforms can enhance the tolerance of S. cerevisiae to multiple stresses. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

CD44v6 expression in patients with stage II or stage III sporadic colorectal cancer is superior to CD44 expression for predicting progression

PubMed Central

Zhao, LH; Lin, QL; Wei, J; Huai, YL; Wang, KJ; Yan, HY

2015-01-01

Background: Currently, it is difficult to predict the prognosis of patients exhibiting stage II or stage III colorectal cancer (CRC) and to identify those patients most likely to benefit from aggressive treatment. The current study was performed to examine the clinicopathological significance of CD44 and CD44v6 protein expression in these patients. Study design: We retrospectively investigated 187 consecutive patients who underwent surgery with curative intent for stage II to III CRC from 2007 to 2013 in the Beijing Civil Aviation Hospital. CD44 and CD44v6 protein expression levels were determined using immunohistochemistry and compared to the clinicopathological data. Results: Using immunohistochemical detection, CD44 expression was observed in 108 (57.75%) of the CRC patients; and its detection was significantly associated with greater invasion depth, lymph node metastasis, angiolymphatic invasion, and a more advanced pathological tumor-lymph node-metastasis (TNM) stage. CD44v6 expression was observed in 135 (72.19%) of the CRC patients; and its expression was significantly associated with a poorly differentiated histology, greater invasion depth, lymph node metastasis, angiolymphatic invasion, and a more advanced pathological TNM stage. Expression of CD44v6 was higher than that of CD44 in stage II and stage III sporadic CRC. Conclusion: CD44v6 is a more useful marker for predicting a poor prognosis in stage II and stage III sporadic CRC as compared to CD44. PMID:25755763

Expression and significance of CD44s, CD44v6, and nm23 mRNA in human cancer.

PubMed

Liu, Yong-Jun; Yan, Pei-Song; Li, Jun; Jia, Jing-Fen

2005-11-14

To investigate the relationship between the expression levels of nm23 mRNA, CD44s, and CD44v6, and oncogenesis, development and metastasis of human gastric adenocarcinoma, colorectal adenocarcinoma, intraductal carcinoma of breast, and lung cancer. Using tissue microarray by immuhistochemical (IHC) staining and in situ hybridization (ISH), we examined the expression levels of nm23 mRNA, CD44s, and CD44v6 in 62 specimens of human gastric adenocarcinoma and 62 specimens of colorectal adenocarcinoma; the expression of CD44s and CD44v6 in 120 specimens of intraductal carcinoma of breast and 20 specimens of normal breast tissue; the expression of nm23 mRNA in 72 specimens of human lung cancer and 23 specimens of normal tissue adjacent to cancer. The expression of nm23 mRNA in the tissues of gastric and colorectal adenocarcinoma was not significantly different from that in the normal tissues adjacent to cancer (P>0.05), and was not associated with the invasion of tumor and the pathology grade of adenocarcinoma (P>0.05). However, the expression of nm23 mRNA was correlated negatively to the lymph node metastasis of gastric and colorectal adenocarcinoma (r = -0.49, P<0.01; r = -4.93, P<0.01). The expression of CD44s in the tissues of gastric and colorectal adenocarcinoma was significantly different from that in the normal tissues adjacent to cancer (P<0.05; P<0.01). CD44v6 was expressed in the tissues of gastric and colorectal adenocarcinoma only, the expression of CD44v6 was significantly associated with the lymph node metastasis, invasion and pathological grade of the tumor (r = 0.47, P<0.01; r = 5.04, P<0.01). CD44s and CD44v6 were expressed in intraductal carcinoma of breast, the expression of CD44s and CD44v6 was significantly associated with lymph node metastases and invasion (P<0.01). However, neither of them was expressed in the normal breast tissue. In addition, the expression of CD44v6 was closely related to the degree of cell differentiation of intraductal carcinoma of breast (c2 = 5.68, P<0.05). The expressional level of nm23 mRNA was closely related to the degree of cell differentiation (P<0.05) and lymph node metastasis (P<0.01), but the expression of nm23 gene was not related to sex, age, and type of histological classification (P>0.05). Patients with overexpression of CD44s and CD44v6 and low expression of nm23 mRNA have a higher lymph node metastatic rate and invasion. In addition, overexpression of CD44v6 is closely related to the degree of cell differentiation. Detection of the three genes is able to provide a reliable index to evaluate the invasion and metastasis of tumor cells.

CD44 Splice Variants as Potential Players in Alzheimer's Disease Pathology.

PubMed

Pinner, Elhanan; Gruper, Yaron; Ben Zimra, Micha; Kristt, Don; Laudon, Moshe; Naor, David; Zisapel, Nava

2017-01-01

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive deficits, deposition of amyloid-β (Aβ) plaques, intracellular neurofibrillary tangles, and neuronal cell death. Neuroinflammation is commonly believed to participate in AD pathogenesis. CD44 is an inflammation-related gene encoding a widely-distributed family of alternatively spliced cell surface glycoproteins that have been implicated in inflammation, metastases, and inflammation-linked neuronal injuries. Here we investigated the expression patterns of CD44S (which does not contain any alternative exon) and CD44 splice variants in postmortem hippocampal samples from AD patients and matched non-AD controls. The expression of CD44S and CD44 splice variants CD44V3, CD44V6, and CD44V10 was significantly higher in AD patients compared to non-AD controls. Immunohistochemistry of human hippocampal sections revealed that CD44S differentially localized to neuritic plaques and astrocytes, whereas CD44V3, CD44V6, and CD44V10 expression was mostly neuronal. Consistent with these findings, we found that the expression of CD44V6 and CD44V10 was induced by Aβ peptide in neuroblastoma cells and primary neurons. Furthermore, in loss of function studies we found that both CD44V10-specific siRNA and CD44V10 antibody protected neuronal cells from Aβ-induced toxicity, suggesting a causal relationship between CD44V10 and neuronal cell death. These data indicate that certain CD44 splice variants contribute to AD pathology and that CD44V10 inhibition may serve as a new neuroprotective treatment strategy for this disease.

MicroRNA miR-328 Regulates Zonation Morphogenesis by Targeting CD44 Expression

PubMed Central

Wang, Chia-Hui; Lee, Daniel Y.; Deng, Zhaoqun; Jeyapalan, Zina; Lee, Shao-Chen; Kahai, Shireen; Lu, Wei-Yang; Zhang, Yaou; Yang, Burton B.

2008-01-01

Morphogenesis is crucial to initiate physiological development and tumor invasion. Here we show that a microRNA controls zonation morphogenesis by targeting hyaluronan receptor CD44. We have developed a novel system to study microRNA functions by generating constructs expressing pre-miRNAs and mature miRNAs. Using this system, we have demonstrated that expression of miR-328 reduced cell adhesion, aggregation, and migration, and regulated formation of capillary structure. Protein analysis indicated that miR-328 repressed CD44 expression. Activities of luciferase constructs harboring the target site in CD44, but not the one containing mutation, were repressed by miR-328. Zonation morphogenesis appeared in cells transfected by miR-328: miR-328-transfected cells were present on the surface of zonating structures while the control cells stayed in the middle. MiR-328-mediated CD44 actions was validated by anti-CD44 antibody, hyaluronidase, CD44 siRNA, and CD44 expression constructs. In vivo experiments showed that CD44-silencing cells appeared as layers on the surfaces of nodules or zonating structures. Immuno-histochemistry also exhibited CD44-negative cells on the surface layers of normal rat livers and the internal zones of Portal veins. Our results demonstrate that miR-328 targets CD44, which is essential in regulating zonation morphogenesis: silencing of CD44 expression is essential in sealing the zonation structures to facilitate their extension and to inhibit complex expansion. PMID:18560585

MicroRNA miR-328 regulates zonation morphogenesis by targeting CD44 expression.

PubMed

Wang, Chia-Hui; Lee, Daniel Y; Deng, Zhaoqun; Jeyapalan, Zina; Lee, Shao-Chen; Kahai, Shireen; Lu, Wei-Yang; Zhang, Yaou; Yang, Burton B

2008-06-18

Morphogenesis is crucial to initiate physiological development and tumor invasion. Here we show that a microRNA controls zonation morphogenesis by targeting hyaluronan receptor CD44. We have developed a novel system to study microRNA functions by generating constructs expressing pre-miRNAs and mature miRNAs. Using this system, we have demonstrated that expression of miR-328 reduced cell adhesion, aggregation, and migration, and regulated formation of capillary structure. Protein analysis indicated that miR-328 repressed CD44 expression. Activities of luciferase constructs harboring the target site in CD44, but not the one containing mutation, were repressed by miR-328. Zonation morphogenesis appeared in cells transfected by miR-328: miR-328-transfected cells were present on the surface of zonating structures while the control cells stayed in the middle. MiR-328-mediated CD44 actions was validated by anti-CD44 antibody, hyaluronidase, CD44 siRNA, and CD44 expression constructs. In vivo experiments showed that CD44-silencing cells appeared as layers on the surfaces of nodules or zonating structures. Immuno-histochemistry also exhibited CD44-negative cells on the surface layers of normal rat livers and the internal zones of Portal veins. Our results demonstrate that miR-328 targets CD44, which is essential in regulating zonation morphogenesis: silencing of CD44 expression is essential in sealing the zonation structures to facilitate their extension and to inhibit complex expansion.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoshida, Go J., E-mail: medical21go@yahoo.co.jp; Saya, Hideyuki

Highlights: •CD44 variant8–10 and c-Myc are inversely expressed in gastric cancer cells. •Redox-stress enhances c-Myc expression via canonical Wnt signal. •CD44v, but not CD44 standard, suppresses redox stress-induced Wnt activation. •CD44v expression promotes both transcription and proteasome degradation of c-Myc. •Inversed expression pattern between CD44v and c-Myc is often recognized in vivo. -- Abstract: Cancer stem-like cells express high amount of CD44 variant8-10 which protects cancer cells from redox stress. We have demonstrated by immunohistochemical analysis and Western blotting, and reverse-transcription polymerase chain reaction, that CD44 variant8-10 and c-Myc tend to show the inversed expression manner in gastric cancer cells.more » That is attributable to the oxidative stress-induced canonical Wnt activation, and furthermore, the up-regulation of the downstream molecules, one of which is oncogenic c-Myc, is not easily to occur in CD44 variant-positive cancer cells. We have also found out that CD44v8-10 expression is associated with the turn-over of the c-Myc with the experiments using gastric cancer cell lines. This cannot be simply explained by the model of oxidative stress-induced Wnt activation. CD44v8-10-positive cancer cells are enriched at the invasive front. Tumor tissue at the invasive area is considered to be composed of heterogeneous cellular population; dormant cancer stem-like cells with CD44v8-10 {sup high}/ Fbw7 {sup high}/ c-Myc {sup low} and proliferative cancer stem-like cells with CD44v8-10 {sup high}/ Fbw7 {sup low}/ c-Myc {sup high}.« less

Activation of p44/42 in Human Natural Killer Cells Decreases Cell-surface Protein Expression: Relationship to Tributyltin-induced alterations of protein expression

PubMed Central

Dudimah, Fred D.; Abraha, Abraham; Wang, Xiaofei; Whalen, Margaret M.

2010-01-01

Tributyltin (TBT) activates the mitogen activated protein kinase (MAPK), p44/42 in human natural killer (NK) cells. TBT also reduces NK cytotoxic function and decreases the expression of several NK-cell proteins. To understand the role that p44/42 activation plays in TBT-induced loss of NK cell function, we have investigated how selective activation of p44/42 by phorbol 12-myristate 13-acetate (PMA) affects NK cells. Previously we showed that PMA caused losses of lytic function similar to those seen with TBT exposures. Here we examined activation of p44/42 in the regulation of NK-cell protein expression and how this regulation may explain the protein expression changes seen with TBT exposures. NK cells exposed to PMA were examined for levels of cell-surface proteins, granzyme mRNA, and perforin mRNA expression. The expression of CD11a, CD16, CD18, and CD56 were reduced, perforin mRNA levels were unchanged and granzyme mRNA levels were increased. To verify that activation of p44/42 was responsible for the alterations seen in CD11a, CD16, CD18, and CD56 with PMA, NK cells were treated with the p44/42 pathway inhibitor (PD98059) prior to PMA exposures. In the presence of PD98059, PMA caused no decreases in the expression of the cell-surface proteins. Results of these studies indicate that the activation of p44/42 may lead to the loss of NK cell cytotoxic function by decreasing the expression of CD11a, CD16, CD18, and CD56. Further, activation of p44/42 appears to be at least in part responsible for the TBT-induced decreases in expression of CD16, CD18, and CD56. PMID:20883105

Tissue- and cell-specific expression of metallothionein genes in cadmium- and copper-exposed mussels analyzed by in situ hybridization and RT-PCR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zorita, I.; Bilbao, E.; Schad, A.

2007-04-15

Metallothioneins (MTs) are metal-inducible proteins that can be used as biomarkers of metal exposure. In mussels two families of MT isoforms (MT10 and MT20) have been characterized. In this study, mussels (Mytilus galloprovincialis) were exposed to 200 ppb Cd and 40 ppb Cu for 2 and 9 days to characterize the tissue and isoform specificity of metal-induced MT expression. Non-radioactive in situ hybridization demonstrated that both MT isoforms were mainly transcribed in digestive tubule epithelial cells, especially in basophilic cells. Weaker MT expression was detected in non-ciliated duct cells, stomach and gill epithelial cells, haemocytes, adipogranular cells, spermatic follicles andmore » oocytes. RT-PCR resulted in cloning of a novel M. galloprovincialis isoform homologous to recently cloned Mytilus edulis intron-less MT10B isoform. In gills, Cd only affected MT10 gene expression after 2 days of exposure while increases in MT protein levels occurred at day 9. In the digestive gland, a marked increase of both isoforms, but especially of MT20, was accompanied by increased levels of MT proteins and basophilic cell volume density (Vv{sub BAS}) after 2 and 9 days and of intralysosomal metal accumulation in digestive cells after 9 days. Conversely, although metal was accumulated in digestive cells lysosomes and the Vv{sub BAS} increased in Cu-exposed mussels, Cu exposure did not produce an increase of MT gene expression or MT protein levels. These data suggest that MTs are expressed in a tissue-, cell- and isoform-specific way in response to different metals.« less

JNK1 Mediates Lipopolysaccharide-Induced CD14 and SR-AI Expression and Macrophage Foam Cell Formation.

PubMed

An, Dong; Hao, Feng; Hu, Chen; Kong, Wei; Xu, Xuemin; Cui, Mei-Zhen

2017-01-01

Foam cell formation is the key process in the development of atherosclerosis. The uptake of oxidized low-density lipoprotein (oxLDL) converts macrophages into foam cells. We recently reported that lipopolysaccharide (LPS)-induced foam cell formation is regulated by CD14 and scavenger receptor AI (SR-AI). In this study, we employed pharmaceutical and gene knockdown approaches to determine the upstream molecular mediators, which control LPS-induced foam cell formation. Our results demonstrated that the specific c-Jun N-terminal kinase (JNK) pathway inhibitor, SP600125, but neither the specific inhibitor of extracellular signaling-regulated kinase (ERK) kinase MEK1/2, U0126, nor the specific inhibitor of p38 MAPK, SB203580, significantly blocks LPS-induced oxLDL uptake, suggesting that the JNK pathway is the upstream mediator of LPS-induced oxLDL uptake/foam cell formation. To address whether JNK pathway mediates LPS-induced oxLDL uptake is due to JNK pathway-regulated CD14 and SR-AI expression, we assessed whether the pharmaceutical inhibitor of JNK influences LPS-induced expression of CD14 and SR-AI. Our results indicate that JNK pathway mediates LPS-induced CD14 and SR-AI expression. To conclusively address the isoform role of JNK family, we depleted JNK isoforms using the JNK isoform-specific siRNA. Our data showed that the depletion of JNK1, but not JNK2 blocked LPS-induced CD14/SR-AI expression and foam cell formation. Taken together, our results reveal for the first time that JNK1 is the key mediator of LPS-induced CD14 and SR-AI expression in macrophages, leading to LPS-induced oxLDL uptake/foam cell formation. We conclude that the novel JNK1/CD14/SR-AI pathway controls macrophage oxLDL uptake/foam cell formation.

Up-regulation of CD44 in the development of metastasis, recurrence and drug resistance of ovarian cancer

PubMed Central

Gao, Yan; Foster, Rosemary; Yang, Xiaoqian; Feng, Yong; Shen, Jacson K.; Mankin, Henry J.; Hornicek, Francis J.; Amiji, Mansoor M.; Duan, Zhenfeng

2015-01-01

The clinical significance of Cluster of Differentiation 44 (CD44) remains controversial in human ovarian cancer. The aim of this study is to evaluate the clinical significance of CD44 expression by using a unique tissue microarray, and then to determine the biological functions of CD44 in ovarian cancer. In this study, a unique ovarian cancer tissue microarray (TMA) was constructed with paired primary, metastatic, and recurrent tumor tissues from 26 individual patients. CD44 expression in TMA was assessed by immunohistochemistry. Both the metastatic and recurrent ovarian cancer tissues expressed higher level of CD44 than the patient-matched primary tumor. A significant association has been shown between CD44 expression and both the disease free survival and overall survival. A strong increase of CD44 was found in the tumor recurrence of mouse model. Finally, when CD44 was knocked down, proliferation, migration/invasion activity, and spheroid formation were significantly suppressed, while drug sensitivity was enhanced. Thus, up-regulation of CD44 represents a crucial event in the development of metastasis, recurrence, and drug resistance to current treatments in ovarian cancer. Developing strategies to target CD44 may prevent metastasis, recurrence, and drug resistance in ovarian cancer. PMID:25823654

Up-regulation of CD44 in the development of metastasis, recurrence and drug resistance of ovarian cancer.

PubMed

Gao, Yan; Foster, Rosemary; Yang, Xiaoqian; Feng, Yong; Shen, Jacson K; Mankin, Henry J; Hornicek, Francis J; Amiji, Mansoor M; Duan, Zhenfeng

2015-04-20

The clinical significance of Cluster of Differentiation 44 (CD44) remains controversial in human ovarian cancer. The aim of this study is to evaluate the clinical significance of CD44 expression by using a unique tissue microarray, and then to determine the biological functions of CD44 in ovarian cancer. In this study, a unique ovarian cancer tissue microarray (TMA) was constructed with paired primary, metastatic, and recurrent tumor tissues from 26 individual patients. CD44 expression in TMA was assessed by immunohistochemistry. Both the metastatic and recurrent ovarian cancer tissues expressed higher level of CD44 than the patient-matched primary tumor. A significant association has been shown between CD44 expression and both the disease free survival and overall survival. A strong increase of CD44 was found in the tumor recurrence of mouse model. Finally, when CD44 was knocked down, proliferation, migration/invasion activity, and spheroid formation were significantly suppressed, while drug sensitivity was enhanced. Thus, up-regulation of CD44 represents a crucial event in the development of metastasis, recurrence, and drug resistance to current treatments in ovarian cancer. Developing strategies to target CD44 may prevent metastasis, recurrence, and drug resistance in ovarian cancer.

Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide Gene

PubMed Central

Casucci, Monica; Falcone, Laura; Camisa, Barbara; Norelli, Margherita; Porcellini, Simona; Stornaiuolo, Anna; Ciceri, Fabio; Traversari, Catia; Bordignon, Claudio; Bonini, Chiara; Bondanza, Attilio

2018-01-01

Chimeric antigen receptor (CAR)-T cell immunotherapy is at the forefront of innovative cancer therapeutics. However, lack of standardization of cellular products within the same clinical trial and lack of harmonization between different trials have hindered the clear identification of efficacy and safety determinants that should be unveiled in order to advance the field. With the aim of facilitating the isolation and in vivo tracking of CAR-T cells, we here propose the inclusion within the CAR molecule of a novel extracellular spacer based on the low-affinity nerve-growth-factor receptor (NGFR). We screened four different spacer designs using as target antigen the CD44 isoform variant 6 (CD44v6). We successfully generated NGFR-spaced CD44v6 CAR-T cells that could be efficiently enriched with clinical-grade immuno-magnetic beads without negative consequences on subsequent expansion, immuno-phenotype, in vitro antitumor reactivity, and conditional ablation when co-expressing a suicide gene. Most importantly, these cells could be tracked with anti-NGFR monoclonal antibodies in NSG mice, where they expanded, persisted, and exerted potent antitumor effects against both high leukemia and myeloma burdens. Similar results were obtained with NGFR-enriched CAR-T cells specific for CD19 or CEA, suggesting the universality of this strategy. In conclusion, we have demonstrated that the incorporation of the NGFR marker gene within the CAR sequence allows for a single molecule to simultaneously work as a therapeutic and selection/tracking gene. Looking ahead, NGFR spacer enrichment might allow good manufacturing procedures-manufacturing of standardized CAR-T cell products with high therapeutic potential, which could be harmonized in different clinical trials and used in combination with a suicide gene for future application in the allogeneic setting. PMID:29619024

Clinical significance of CD44 expression in children with hepatoblastoma.

PubMed

Cai, H-Y; Yu, B; Feng, Z-C; Qi, X; Wei, X-J

2015-10-27

The aim of this study was to investigate the expression of CD44 and its clinical significance in children suffering from hepatoblastoma (HB). CD44 expression was detected with immunohistochemistry staining in 30 samples from hepatoblastoma children and 10 normal liver tissue samples from normal children. The data obtained was statistically analyzed using the chi-square test, using the SPSS (v.11.0) software. The rate of CD44 expression was significantly higher (66.7%) in hepatoblastoma tissues than in normal liver tissues (χ(2) = 4.848, P < 0.05). The rate of CD44 expression was significantly higher in children with stage III or IV hepatoblastoma (83.3%) than that in children with stage I and II hepatoblastoma (χ(2) ＝ 5.625, P < 0.05) (41.7%). Therefore, CD44 expression might play an important role in the pathogenesis, progression, and prognosis of HB in children.

Expression of CD44v6 as matrix-associated ectodomain in the bone development.

PubMed

Nakajima, Kosei; Taniguchi, Kazumi; Mutoh, Ken-ichiro

2010-08-01

This study describes the expression of CD44v6 in the bone development and is the first study of its kind to the authors' best knowledge. The CD44 family is a family of transmembrane glycoproteins that acts as cell adhesion molecules binding cells to other cells as well as cells to the extracellular matrix. It has been suggested that the CD44v6, a family member of CD44, is closely related to the osteosarcoma metastasis. In general, when cancer cells metastasize, they revert to their immature forms. In the present study, therefore, we have investigated CD44v6 and the standard form of CD44 (CD44st) in two types of immature forms of bone tissues: developmentally immature stages from fetuses to adults as well as experimentally immature stages using fracture models. CD44st expression was identified in osteoblasts, osteocytes, and in the peripheral portion of the bone matrix from the fetal to young ages of rats. Many more intense reactions for CD44v6 were observed in the bone matrix than CD44st in fetal stages. In experimental fracture models, positive immunoreactions to CD44st were clearly observed in the osteoblasts and osteocytes. CD44v6-positive immunoreactivity, however, was not detected in either osteoblasts or the bone matrix. In conclusion, CD44v6 is expressed in the embryonic stages and may be involved in the bone matrix formation as a matrix-associated ectodomain during normal ontogenetic development but not involved in the process of fracture healing.

Co-Expression of Putative Cancer Stem Cell Markers CD44 and CD133 in Prostate Carcinomas.

PubMed

Kalantari, Elham; Asgari, Mojgan; Nikpanah, Seyedehmoozhan; Salarieh, Naghme; Asadi Lari, Mohammad Hossein; Madjd, Zahra

2017-10-01

Cancer stem cells (CSCs) are the main players of prostate tumorigenesis thus; characterization of CSCs can pave the way for understanding the early detection, drug resistance, metastasis and relapse. The current study was conducted to evaluate the expression level and clinical significance of the potential CSC markers CD44 and CD133 in a series of prostate tissues. One hundred and forty eight prostate tissues composed of prostate cancer (PCa), high-grade prostatic intraepithelial neoplasia (HGPIN), and benign prostate hyperplasia (BPH) were immunostained for the putative CSC markers CD44 and CD133. Subsequently, the correlation between the expression of these markers and the clinicopathological variables was examined. A higher level of CD44 expression was observed in 42% of PCa, 57% of HGPIN, and 42% BPH tissues. In the case of CD133 expression PCa, HGPIN, and BPH samples demonstrated high immunoreactivity in 46%, 43%, and 42% of cells, respectively. Statistical analysis showed an inverse significant correlation between CD44 expression with Gleason score of PCa (P = 0.02), while no significant correlation was observed between CD133 expression and clinicopathological parameters. A significant reciprocal correlation was observed between the expression of two putative CSC markers CD44 and CD133 in PCa specimens while not indicating clinical significance. Further clinical investigation is required to consider these markers as targets of new therapeutic strategies for PCa.

Increased expression of CD44 is associated with more aggressive behavior in clear cell renal cell carcinoma.

PubMed

Zanjani, Leili Saeednejad; Madjd, Zahra; Abolhasani, Maryam; Rasti, Arezoo; Fodstad, Oystein; Andersson, Yvonne; Asgari, Mojgan

2018-01-01

Although CD44 has been suggested as a prognostic marker in renal cell carcinoma (RCC), the prognostic significance of this marker in three main subtypes of RCC is still unclear. Thus, the present study was conducted to evaluate the expression and prognostic significance of CD44 as a cancer stem cell marker in different histological subtypes of RCC. Methodology & results: CD44 expression was evaluated in 206 well-defined renal tumor samples using immunohistochemistry on tissue microarrays. Higher CD44 expression was associated with more aggressive behavior, tumor progression and worse prognosis in clear cell RCC (ccRCC) but not in papillary and chromophobe RCC subtypes. Cancer stem cell marker CD44 may be a promising target for cancer treatment only in ccRCC.

CD44-mediated hyaluronan binding marks proliferating hematopoietic progenitor cells and promotes bone marrow engraftment

PubMed Central

Lee-Sayer, Sally S. M.; Dougan, Meghan N.; Cooper, Jesse; Sanderson, Leslie; Dosanjh, Manisha; Maxwell, Christopher A.

2018-01-01

CD44 is a widely expressed cell adhesion molecule that binds to the extracellular matrix component, hyaluronan. However, this interaction is not constitutive in most immune cells at steady state, as the ability of CD44 to engage hyaluronan is highly regulated. While activated T cells and macrophages gain the ability to bind hyaluronan by CD44, the status in other immune cells is less studied. Here we found a percentage of murine eosinophils, natural killer and natural killer T cells were capable of interacting with hyaluronan at steady state. To further investigate the consequences of hyaluronan binding by CD44 in the hematopoietic system, point mutations of CD44 that either cannot bind hyaluronan (LOF-CD44) or have an increased affinity for hyaluronan (GOF-CD44) were expressed in CD44-deficient bone marrow. Competitive bone marrow reconstitution of irradiated mice revealed an early preference for GOF-CD44 over WT-CD44 expressing cells, and for WT-CD44 over LOF-CD44 expressing cells, in the hematopoietic progenitor cell compartment. The advantage of the hyaluronan-binding cells was observed in the hematopoietic stem and progenitor populations, and was maintained throughout the immune system. Hematopoietic stem cells bound minimal hyaluronan at steady state, and this was increased when the cells were induced to proliferate whereas multipotent progenitors had an increased ability to bind hyaluronan at steady state. In vitro, the addition of hyaluronan promoted their proliferation. Thus, proliferating hematopoietic progenitors bind hyaluronan, and hyaluronan binding cells have a striking competitive advantage in bone marrow engraftment. PMID:29684048

Lung cancer tumorigenicity and drug resistance are maintained through ALDH(hi)CD44(hi) tumor initiating cells.

PubMed

Liu, Jing; Xiao, Zhijie; Wong, Sunny Kit-Man; Tin, Vicky Pui-Chi; Ho, Ka-Yan; Wang, Junwen; Sham, Mai-Har; Wong, Maria Pik

2013-10-01

Limited improvement in long term survival of lung cancer patients has been achieved by conventional chemotherapy or targeted therapy. To explore the potentials of tumor initiating cells (TIC)-directed therapy, it is essential to identify the cell targets and understand their maintenance mechanisms. We have analyzed the performance of ALDH/CD44 co-expression as TIC markers and treatment targets of lung cancer using well-validated in vitro and in vivo analyses in multiple established and patient-derived lung cancer cells. The ALDH(hi)CD44(hi) subset showed the highest enhancement of stem cell phenotypic properties compared to ALDH(hi)CD44(lo), ALDH(lo)CD44(hi), ALDH(lo)CD44(lo) cells and unsorted controls. They showed higher invasion capacities, pluripotency genes and epithelial-mesenchymal transition transcription factors expression, lower intercellular adhesion protein expression and higher G2/M phase cell cycle fraction. In immunosuppressed mice, the ALDH(hi)CD44(hi)xenografts showed the highest tumor induction frequency, serial transplantability, shortest latency, largest volume and highest growth rates. Inhibition of sonic Hedgehog and Notch developmental pathways reduced ALDH+CD44+ compartment. Chemotherapy and targeted therapy resulted in higher AALDH(hi)CD44(hi) subset viability and ALDH(lo)CD44(lo) subset apoptosis fraction. ALDH inhibition and CD44 knockdown led to reduced stemness gene expression and sensitization to drug treatment. In accordance, clinical lung cancers containing a higher abundance of ALDH and CD44-coexpressing cells was associated with lower recurrence-free survival. Together, results suggested theALDH(hi)CD44(hi)compartment was the cellular mediator of tumorigenicity and drug resistance. Further investigation of the regulatory mechanisms underlying ALDH(hi)CD44(hi)TIC maintenance would be beneficial for the development of long term lung cancer control.

Expression of Bmi-1, P16, and CD44v6 in Uterine Cervical Carcinoma and Its Clinical Significance.

PubMed

Weng, Mei-Ying; Li, Lin; Feng, Shu-Ying; Hong, Shun-Jia

2012-03-01

Bmi-1, a putative proto-oncogene, is a core member of the polycomb gene family, which is expressed in many human tumors. The p16 protein negatively regulated cell proliferation, whereas CD44v6 is associated with proliferation as an important protein. Additionally, CD44v6 is an important nuclear antigen closely correlated to tumor metastasis. The present study aims to investigate the expression and significance of Bmi-1, p16, and CD44v6 in uterine cervical carcinoma (UCC). A total of 62 UCC, 30 cervical neoplasic, and 20 normal cervical mucosal tissues were used in the current study. The expression of Bmi-1, p16, and CD44v6 in these tissues was determined using immunohistochemical assay. The relationships among the expression of these indices, the clinicopathologic features of UCC, and the survival rate of UCC patients were also discussed. The correlation between Bmi-1 protein expression and p16 or CD44v6 protein in UCC was analyzed. The expression of Bmi-1, p16, and CD44v6 was significantly high in cervical carcinoma compared with that in the cervical neoplasia and normal colorectal mucosa (P<0.05). The over-expression of Bmi-1 protein in UCC was apparently related to the distant metastasis (P<0.01) and the tumor, nodes and metastasis-classification, i.e. the TNM staging, World Health Organization (P<0.05). Nevertheless, the positive expression of p16 protein in UCC was not significantly associated with the clinicopathologic features (P>0.05). The Kaplan-Meier survival analysis showed that the over-expression of Bmi-1 significantly decreased the survival rate of UCC patients (P<0.05). A strong correlation indicated that there was statistical significance between the expression of Bmi-1 and CD44V6 proteins in UCC (r=0.419, P=0.001). The over-expression of Bmi-1 and CD44v6 protein closely correlate to the tumorigenesis, metastasis, and prognosis of UCC. Bmi-1 and CD44v6 may be used to predict the prognosis of cervical carcinoma. Bmi-1 may indirectly regulate the expression of CD44v6 in UCC patients. The positive expression of p16 protein is possibly associated with the tumorigenesis, but not with the metastasis or prognosis of UCC.

[Dectection of G3BP and CD44v6 in the tissues of laryngeal squamous cell carcinoma and their clinical significance].

PubMed

Luo, Dahu; Lou, Weihua

2017-07-01

Objective To study the expressions of RNA-binding Ras-GAP SH3 binding protein (G3BP) and tumor stem cell marker CD44v6 in laryngeal squamous cell carcinoma and their correlations with angiogenesis. Methods We collected the cancer tissues and corresponding paracancerous tissues from 56 patients with laryngeal squamous cell carcinoma. The expressions of G3BP and CD44v6 proteins were detected by Western blotting in cancer tissues and corresponding paracancerous tissues; the expressions of G3BP, CD44v6 and vascular endothelial growth factor A (VEGF-A) were tested by immunohistochemistry. Thereafter, we compared the positive expression rates of G3BP and CD44v6 between in cancer tissues and in normal tissues, analyzed the correlations between the expressions of G3BP, CD44v6 and the laryngeal squamous cell carcinoma features as well as their correlations with microvessel density (MVD) that was determined by FVIIIAg immunohistochemistry. Results Western blotting showed that the expressions of G3BP and CD44v6 proteins in the laryngeal squamous cell carcinoma were higher than those in the paracancerous tissues. Immunohistochemistry showed that compared with the paracancerous tissues, G3BP, CD44v6 and VEGF-A expressions (the positive rates are 58.9%, 53.6%, 46.4%, respectively) were higher in cancer tissues. The positive rates of G3BP and CD44v6 in cancer tissues were related with the clinical stage, recurrence or metastasis, and lymph node metastasis of laryngeal squamous cell carcinoma, but had nothing to do with patients' age and tumor size. Pearson correlation analysis showed the expressions of both G3BP and CD44v6 were positively correlated with VEGF-A (r=0.741, r=0.756). MVD values were significantly higher in the G3BP and CD44v6 positive cases than in paracancerous tissues, but there was no difference in MVD between those without G3BP and CD44v6 positive expressions and the paracancerous tissues. Conclusion The positive expression rates of G3BP and CD44v6 in laryngeal squamous cell carcinoma tissues are very high, and they have a close relationship with the clinical prognosis. They may raise the VEGF-A expression so as to promote angiogenesis, and then accelerate the development of the laryngeal squamous cell carcinoma.

СD44+/CD24- markers of cancer stem cells in patients with breast cancer of different molecular subtypes.

PubMed

Chekhun, S V; Zadvorny, T V; Tymovska, Yu O; Anikusko, M F; Novak, O E; Polishchuk, L Z

2015-03-01

To determine frequency of tumors with immunohistochemical markers of cancer stem cells (CSC) CD44+/CD24- in patients with breast cancer (BC) of different molecular subtype and to evaluate their prognostic value. Surgical material of 132 patients with BC stage I-II, age from 23 to 75 years, mean age - 50.2 ± 3.1 years was studied. Clinical, immunohistochemical (expression CD44+/CD24-), morphological, statistical. BC is characterized by heterogeneity of molecular subtypes and expression of markers (CD44+/CD24-). Immunohistochemical study of expression of CSC markers in surgical material has detected their expression in 34 (25.4%) patients with BC of different molecular subtypes. The highest frequency of cells with expression of CSC marker was observed in patients with basal molecular subtype (44.8% patients). Most of BC patients with phenotype CD44+/CD24 had stage I of tumor process (34.3%). Statistical processing of data has showen that Yule colligation coefficient equaled 0.28 (р > 0.05) that argues poor correlation between stage of tumor process and number of tumors with positive expression of CSC markers. Statistical processing of data has showen high correlation between presence of cells with expression of CSC markers and metastases of BC in regional lymph nodes (Yule colligation coefficient equals 0.943; р < 0.5). Difference in overall survival of patients with BC of basal molecular subtype depending on expression of CSC CD44+/CD24- markers was detected. Survival of patients with basal BC was reliably higher at lack in tumors of cells with CSC markers CD44+/CD24- and, correspondingly, lower at presence of such cells (р < 0.05). In patients with BC of luminal (A and B), HER-2-positive subtypes, significant change in survival of patients depending on expression of CSC markers was not determined (р > 0.05). Significance of tumor cells with markers CD44+/CD24- within the limits of molecular subtype of BC may be additional criterion for advanced biological characteristic of BC, and in patients with BC of basal molecular subtype - for predictive evaluation of individual potential of tumor to aggressive clinical course.

A negative regulatory role in mouse cardiac transplantation for a splice variant of CD80.

PubMed

Bugeon, Laurence; Wong, Kenneth K; Rankin, Alasdair M; Hargreaves, Roseanna E G; Dallman, Margaret J

2006-11-27

Members of the B7 costimulatory protein family (CD80 and CD86) play a determining role in allograft rejection. Both CD80 and CD86 have naturally occurring splice variants whose roles in transplantation are unknown. Full length CD80 has two immunoglobulin (Ig)-like domains in the extracellular portion, IgC and IgV. In mouse, the isoform IgV-CD80 lacks the IgC-like domain. Here we analyzed the role of mouse IgV-CD80 in heart allograft rejection and search for equivalent splice variants in human. Mice made deficient for full-length CD80 but which retain expression of the shorter IgV-CD80 (CD80 mice) were used as donor or recipient of a heart allograft. Recipient animals were untreated or pretreated with alloantigen expressing cells and/or treated with CD80 and CTLA4 monoclonal antibodies (mAbs). Recipients expressing IgV-CD80 but not full length CD80 exhibited a slight prolongation in survival of either wild-type (Wt) or CD80 grafts. More dramatically, CD80 animals pretreated with donor alloantigen exhibited permanent graft survival, whereas their Wt counterparts rejected their grafts with a median survival of 24 days. This prolonged survival was due to the expression of IgV-CD80 in recipients since treatment with CD80 mAb abrogated the beneficial effect observed. We identified and report here a similar isoform of CD80 from human cDNA encoding a putative soluble, IgV-containing protein. IgV-CD80 bearing recipients show enhanced allograft survival especially after donor alloantigen pretreatment. This together with data from other species suggests that regulation delivered by splice variants of CD80 significantly modulates immunity and may be common across the species.

CD44 expression in curettage and postoperative specimens of endometrial cancer.

PubMed

Wojciechowski, Michał; Krawczyk, Tomasz; Śmigielski, Janusz; Malinowski, Andrzej

2015-02-01

Adhesive molecules like CD44 are well defined key players in the metastatic cascade in many cancers, including endometrial cancer. They could play a role of markers of invasion, metastasis and prognostic factors. The aim of the study is to assess a possible role of the CD44 as a marker of invasion in endometrial cancer, both at the moment of preoperative workup and final staging. Available for analysis were archival specimens of 51 patients who had underwent curettage and surgery between 2002 and 2007. An immunohistochemical study for CD44 expression was performed in curettage and postoperative specimens. Normal endometrium of 20 randomly chosen patients was used as a control group. In endometrial cancer the expression of CD44 was significantly more intensive than in normal endometrium. In postoperative specimens, the CD44 expression was weaker in serous than in endometrioid cancer. There was no significant correlation between the adhesion molecule expression and clinicopathological features: grade,depth of invasion, cervical involvement, serosal and adnexal involvement, lymph-vascular space involvement, lymph node and distant metastases nor FIGO stage. An increased expression of CD44 in endometrial cancer suggests its possible role in pathogenesis of this disease, however, it doesn't seem to be crucial. Different expression of the CD44 in endometrioid and papillary-serous type may reflect different pathogenesis of these types of cancer. No statistically proved relation between the investigated molecule expression and clinicopathological parameters suggests scepticism about its use in diagnostic process of endometrial cancer.

CD44 functions in Wnt signaling by regulating LRP6 localization and activation

PubMed Central

Schmitt, M; Metzger, M; Gradl, D; Davidson, G; Orian-Rousseau, V

2015-01-01

Wnt reception at the membrane is complex and not fully understood. CD44 is a major Wnt target gene in the intestine and is essential for Wnt-induced tumor progression in colorectal cancer. Here we show that CD44 acts as a positive regulator of the Wnt receptor complex. Downregulation of CD44 expression decreases, whereas CD44 overexpression increases Wnt activity in a concentration-dependent manner. Epistasis experiments place CD44 function at the level of the Wnt receptor LRP6. Mechanistically, CD44 physically associates with LRP6 upon Wnt treatment and modulates LRP6 membrane localization. Moreover, CD44 regulates Wnt signaling in the developing brain of Xenopus laevis embryos as shown by a decreased expression of Wnt targets tcf-4 and en-2 in CD44 morphants. PMID:25301071

Genome-Wide Associations of CD46 and IFI44L Genetic Variants with Neutralizing Antibody Response to Measles Vaccine

PubMed Central

Haralambieva, Iana H.; Ovsyannikova, Inna G.; Kennedy, Richard B.; Larrabee, Beth R.; Zimmermann, Michael T.; Grill, Diane E.; Schaid, Daniel J.; Poland, Gregory A.

2017-01-01

Background Population-based studies have revealed 2 to 10% measles vaccine failure rate even after two vaccine doses. While the mechanisms behind this remain unknown, we hypothesized that host genetic factors are likely to be involved. Methods We performed a genome-wide association study of measles specific neutralizing antibody and IFNγ ELISPOT response in a combined sample of 2,872 subjects. Results We identified two distinct chromosome 1 regions (previously associated with MMR-related febrile seizures), associated with vaccine-induced measles neutralizing antibody titers. The 1q32 region contained 20 significant SNPs in/around the measles virus receptor-encoding CD46 gene, including the intronic rs2724384 (p-value = 2.64x10−09) and rs2724374 (p-value = 3.16x10−09) SNPs. The 1q31.1 region contained nine significant SNPs in/around IFI44L, including the intronic rs1333973 (p-value = 1.41x10−10) and the missense rs273259 (His73Arg, p-value = 2.87x10−10) SNPs. Analysis of differential exon usage with mRNA-Seq data and RT-PCR suggests the involvement of rs2724374 minor G allele in the CD46 STP region exon B skipping, resulting in shorter CD46 isoforms. Conclusions Our study reveals common CD46 and IFI44L SNPs associated with measles-specific humoral immunity, and highlights the importance of alternative splicing/virus cellular receptor isoform usage as a mechanism explaining inter-individual variation in immune response after live measles vaccine. PMID:28289848

Transforming growth factor-β-mediated CD44/STAT3 signaling contributes to the development of atrial fibrosis and fibrillation.

PubMed

Chang, Shang-Hung; Yeh, Yung-Hsin; Lee, Jia-Lin; Hsu, Yu-Juei; Kuo, Chi-Tai; Chen, Wei-Jan

2017-09-04

Atrial fibrillation (AF) is associated with atrial fibrosis. Inhibition of atrial fibrosis might be a plausible approach for AF prevention and therapy. This study is designed to evaluate the potential role of CD44, a membrane receptor known to regulate fibrosis, and its related signaling in the pathogenesis of atrial fibrosis and AF. Treatment of cultured rat atrial fibroblasts with transforming growth factor-β (TGF-β, a key mediator of atrial fibrosis) led to a higher expression of hyaluronan (HA), CD44, STAT3, and collagen (a principal marker of fibrosis) than that of ventricular fibroblasts. In vivo, TGF-β transgenic mice and AF patients exhibited a greater expression of HA, CD44, STAT3, and collagen in their atria than wild-type mice and sinus rhythm subjects, respectively. Treating TGF-β transgenic mice with an anti-CD44 blocking antibody resulted in a lower expression of STAT3 and collagen in their atria than those with control IgG antibody. Programmed stimulation triggered less AF episodes in TGF-β transgenic mice treated with anti-CD44 blocking antibody than in those with control IgG. Blocking CD44 signaling with anti-CD44 antibody and mutated CD44 plasmids attenuated TGF-β-induced STAT3 activation and collagen expression in cultured atrial fibroblasts. Deletion and mutational analysis of the collagen promoter along with chromatin immunoprecipitation demonstrated that STAT3 served as a vital transcription factor in collagen expression. TGF-β-mediated HA/CD44/STAT3 pathway plays a crucial role in the development of atrial fibrosis and AF. Blocking CD44-dependent signaling may be a feasible way for AF management.

Uncovering the dual role of RHAMM as an HA receptor and a regulator of CD44 expression in RHAMM-expressing mesenchymal progenitor cells.

PubMed

Veiseh, Mandana; Leith, Sean J; Tolg, Cornelia; Elhayek, Sallie S; Bahrami, S Bahram; Collis, Lisa; Hamilton, Sara; McCarthy, James B; Bissell, Mina J; Turley, Eva

2015-01-01

The interaction of hyaluronan (HA) with mesenchymal progenitor cells impacts trafficking and fate after tissue colonization during wound repair and these events contribute to diseases such as cancer. How this interaction occurs is poorly understood. Using 10T½ cells as a mesenchymal progenitor model and fluorescent (F-HA) or gold-labeled HA (G-HA) polymers, we studied the role of two HA receptors, RHAMM and CD44, in HA binding and uptake in non-adherent and adherent mesenchymal progenitor (10T½) cells to mimic aspects of cell trafficking and tissue colonization. We show that fluorescent labeled HA (F-HA) binding/uptake was high in non-adherent cells but dropped over time as cells became increasingly adherent. Non-adherent cells displayed both CD44 and RHAMM but only function-blocking anti-RHAMM and not anti-CD44 antibodies significantly reduced F-HA binding/uptake. Adherent cells, which also expressed CD44 and RHAMM, primarily utilized CD44 to bind to F-HA since anti-CD44 but not anti-RHAMM antibodies blocked F-HA uptake. RHAMM overexpression in adherent 10T½ cells led to increased F-HA uptake but this increased binding remained CD44 dependent. Further studies showed that RHAMM-transfection increased CD44 mRNA and protein expression while blocking RHAMM function reduced expression. Collectively, these results suggest that cellular microenvironments in which these receptors function as HA binding proteins differ significantly, and that RHAMM plays at least two roles in F-HA binding by acting as an HA receptor in non-attached cells and by regulating CD44 expression and display in attached cells. Our findings demonstrate adhesion-dependent mechanisms governing HA binding/ uptake that may impact development of new mesenchymal cell-based therapies.

Uncovering the dual role of RHAMM as an HA receptor and a regulator of CD44 expression in RHAMM-expressing mesenchymal progenitor cells

PubMed Central

Veiseh, Mandana; Leith, Sean J.; Tolg, Cornelia; Elhayek, Sallie S.; Bahrami, S. Bahram; Collis, Lisa; Hamilton, Sara; McCarthy, James B.; Bissell, Mina J.; Turley, Eva

2015-01-01

The interaction of hyaluronan (HA) with mesenchymal progenitor cells impacts trafficking and fate after tissue colonization during wound repair and these events contribute to diseases such as cancer. How this interaction occurs is poorly understood. Using 10T½ cells as a mesenchymal progenitor model and fluorescent (F-HA) or gold-labeled HA (G-HA) polymers, we studied the role of two HA receptors, RHAMM and CD44, in HA binding and uptake in non-adherent and adherent mesenchymal progenitor (10T½) cells to mimic aspects of cell trafficking and tissue colonization. We show that fluorescent labeled HA (F-HA) binding/uptake was high in non-adherent cells but dropped over time as cells became increasingly adherent. Non-adherent cells displayed both CD44 and RHAMM but only function-blocking anti-RHAMM and not anti-CD44 antibodies significantly reduced F-HA binding/uptake. Adherent cells, which also expressed CD44 and RHAMM, primarily utilized CD44 to bind to F-HA since anti-CD44 but not anti-RHAMM antibodies blocked F-HA uptake. RHAMM overexpression in adherent 10T½ cells led to increased F-HA uptake but this increased binding remained CD44 dependent. Further studies showed that RHAMM-transfection increased CD44 mRNA and protein expression while blocking RHAMM function reduced expression. Collectively, these results suggest that cellular microenvironments in which these receptors function as HA binding proteins differ significantly, and that RHAMM plays at least two roles in F-HA binding by acting as an HA receptor in non-attached cells and by regulating CD44 expression and display in attached cells. Our findings demonstrate adhesion-dependent mechanisms governing HA binding/ uptake that may impact development of new mesenchymal cell-based therapies. PMID:26528478

Visualization of CD44 and CD133 in Normal Pancreas and Pancreatic Ductal Adenocarcinomas

PubMed Central

Immervoll, Heike; Hoem, Dag; Steffensen, Ole Johnny; Miletic, Hrvoje; Molven, Anders

2011-01-01

Tumor-initiating cells of pancreatic ductal adenocarcinoma (PDAC) have been isolated based on expression of either CD133 or CD44. The authors aimed to visualize pancreatic cells simultaneously expressing both these cell surface markers by employing the same antibodies commonly used in cell-sorting studies. Normal and diseased pancreatic tissue, including 51 PDAC cases, were analyzed. CD44 and CD133 expression was determined by immunohistochemical double staining on formalin-fixed material and subcellular protein distribution evaluated by immunofluorescence/confocal microscopy. In the normal pancreas, CD44 and CD133 were coexpressed in the centroacinar regions but in non-overlapping subcellular compartments. As expected, CD44 was found mainly basolaterally, whereas CD133 was present on the apical/endoluminal membrane. This was also the case in chronically inflamed/atrophic pancreatic tissue and in PDAC. In some malignant ducts, CD44 was found at the apical cell membrane adjacent to but never overlapping with CD133 expression. CD44 level was significantly associated with the patient’s lymph node status. In conclusion, a CD44+/CD133+ cell population does exist in the normal and neoplastic pancreas. The preferentially centroacinar localization of the doubly positive cells in the normal parenchyma suggests that this population could be of particular interest in attempts to identify tumor-initiating cells in PDAC. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. PMID:21411814

Expression of Bmi-1, P16, and CD44v6 in Uterine Cervical Carcinoma and Its Clinical Significance

PubMed Central

Weng, Mei-ying; Li, Lin; Feng, Shu-ying; Hong, Shun-jia

2012-01-01

Objective Bmi-1, a putative proto-oncogene, is a core member of the polycomb gene family, which is expressed in many human tumors. The p16 protein negatively regulated cell proliferation, whereas CD44v6 is associated with proliferation as an important protein. Additionally, CD44v6 is an important nuclear antigen closely correlated to tumor metastasis. The present study aims to investigate the expression and significance of Bmi-1, p16, and CD44v6 in uterine cervical carcinoma (UCC). Methods A total of 62 UCC, 30 cervical neoplasic, and 20 normal cervical mucosal tissues were used in the current study. The expression of Bmi-1, p16, and CD44v6 in these tissues was determined using immunohistochemical assay. The relationships among the expression of these indices, the clinicopathologic features of UCC, and the survival rate of UCC patients were also discussed. The correlation between Bmi-1 protein expression and p16 or CD44v6 protein in UCC was analyzed. Results The expression of Bmi-1, p16, and CD44v6 was significantly high in cervical carcinoma compared with that in the cervical neoplasia and normal colorectal mucosa (P<0.05). The over-expression of Bmi-1 protein in UCC was apparently related to the distant metastasis (P<0.01) and the tumor, nodes and metastasis-classification, i.e. the TNM staging, World Health Organization (P<0.05). Nevertheless, the positive expression of p16 protein in UCC was not significantly associated with the clinicopathologic features (P>0.05). The Kaplan–Meier survival analysis showed that the over-expression of Bmi-1 significantly decreased the survival rate of UCC patients (P<0.05). A strong correlation indicated that there was statistical significance between the expression of Bmi-1 and CD44V6 proteins in UCC (r=0.419, P=0.001). Conclusions The over-expression of Bmi-1 and CD44v6 protein closely correlate to the tumorigenesis, metastasis, and prognosis of UCC. Bmi-1 and CD44v6 may be used to predict the prognosis of cervical carcinoma. Bmi-1 may indirectly regulate the expression of CD44v6 in UCC patients. The positive expression of p16 protein is possibly associated with the tumorigenesis, but not with the metastasis or prognosis of UCC. PMID:23691455

Aberrant expression of cancer stem cell markers (CD44, CD90, and CD133) contributes to disease progression and reduced survival in hepatoblastoma patients: 4-year survival data.

PubMed

Bahnassy, Abeer A; Fawzy, Mohamed; El-Wakil, Mohamed; Zekri, Abdel-Rahman N; Abdel-Sayed, Ahmed; Sheta, Marwa

2015-03-01

Hepatoblastoma (HB) is an embryonal tumor of the liver in children. Prognosis and response to treatment in HB are highly variable. Cancer stem cells (CSCs) constitute a population of cells, which contribute to the development and progression of many tumors. However, their role in HB is not well defined yet. We assessed the prognostic and predictive values of some CSC markers in HB patients. Protein and messenger RNA expressions of the CSC markers CD133, CD90, and CD44 were assessed in 43 HB patients and 20 normal hepatic tissues using immunohistochemistry and quantitative real-time polymerase chain reaction. The expression levels of these markers were correlated to standard prognostic factors, patients' response to treatment, overall survival (OS), and disease-free survival (DFS). CD44, CD90, and CD133 proteins were detected in 48.8%, 32.6%, and 48.8% compared with 46.5%, 41.7%, and 58.1% RNA, respectively (concordance, 77.8%-96%). None of the normal tissue samples was positive for any of the markers. Significant correlations were reported between α-fetoprotein and both CD44 and CD133 (P = 0.02) as well as between tumor types CD90 and CD133 (P = 0.009). Reduced OS correlated with CD44, CD90, and CD133 expressions (P < 0.001), advanced stage (P < 0.001), response to treatment (P < 0.001), and total excision of the tumor. Reduced DFS correlated with CD44 and CD133 expressions (P < 0.001) only. In conclusion, CD133, CD44, and CD90 could be used as prognostic and predictive markers in HB. High expression of these markers is significantly associated with poor response to treatment and reduced survival. Moreover, complete surgical resection and systemic chemotherapy are essential to achieve good response and prolonged survival, especially in early stage patients. Copyright © 2015 Elsevier Inc. All rights reserved.

CIITA promoter I CARD-deficient mice express functional MHC class II genes in myeloid and lymphoid compartments.

PubMed

Zinzow-Kramer, W M; Long, A B; Youngblood, B A; Rosenthal, K M; Butler, R; Mohammed, A-U-R; Skountzou, I; Ahmed, R; Evavold, B D; Boss, J M

2012-06-01

Three distinct promoters control the master regulator of major histocompatibility complex (MHC) class II expression, class II transactivator (CIITA), in a cell type-specific manner. Promoter I (pI) CIITA, expressed primarily by dendritic cells (DCs) and macrophages, expresses a unique isoform that contains a caspase-recruitment domain (CARD). The activity and function of this isoform are not understood, but are believed to enhance the function of CIITA in antigen-presenting cells. To determine whether isoform I of CIITA has specific functions, CIITA mutant mice were created in which isoform I was replaced with isoform III sequences. Mice in which pI and the CARD-encoding exon were deleted were also created. No defect in the formation of CD4 T cells, the ability to respond to a model antigen or bacterial or viral challenge was observed in mice lacking CIITA isoform I. Although CIITA and MHC-II expression was decreased in splenic DCs, pI knockout animals expressed CIITA from downstream promoters, suggesting that control of pI activity is mediated by unknown distal elements that could act at pIII, the B-cell promoter. Thus, no critical function is linked to the CARD domain of CIITA isoform I with respect to basic immune system development, function and challenge.

CD44v3 and VEGF-C expression and its relationship with lymph node metastasis in squamous cell carcinomas of the uterine cervix.

PubMed

Liu, Ye-Qing; Li, Hai-Feng; Han, Jing-Jing; Tang, Qiong-Lan; Sun, Qing; Huang, Zhi-Quan; Li, Hai-Gang

2014-01-01

To investigate the expression of CD44v3 and vascular endothelial growth factor-C (VEGF-C) and their relationship with lymph node metastasis in squamous cell carcinomas (SCC) of the uterine cervix. Expression of CD44v3 and VEGF-C was analyzed in 109 cases of cervical SCC by immunohistochemistry (IHC). The relationship was analyzed between expression and the patient age, histological differentiation, formation of tumor emboli in lymphoid vessels, lymph node metastasis, FIGO staging, and TNM classification. Expression rates for both CD44v3 and VEGF-C were 43.1% in cervical SCC. The cells with positive immunohistochemical staining of CD44v3 were distributed mainly around the keratin pearls in well differentiated carcinomas, but distributed diffusely in the moderately and poorly differentiated lesions. VEGF-C was found stained positively in most of the tumor cells. There were differences in expression between normal epithelium and atypical hyperplasia as well as carcinoma. Both CD44v3 and VEGF-C were found to be associated positively with lymph node metastasis and TNM classification (both p=0.000). Neither CD44v3 nor VEGF-C was found to be associated with patient age, histological differentiation, formation of tumor emboli in lymphoid vessels and FIGO staging. CD44v3 was found to be associated with VEGF-C positively (p=0.000). Abnormal expression of CD44v3 and VEGF-C is associated closely with the lymph node metastasis in cervical SCC, and these agents may cooperate in carcinogenesis and development of metastatic lesions.

A rational approach for cancer stem-like cell isolation and characterization using CD44 and prominin-1(CD133) as selection markers

PubMed Central

Lee, Yi-Jen; Wu, Chang-Cheng; Li, Jhy-Wei; Ou, Chien-Chih; Hsu, Shih-Chung; Tseng, Hsiu-Hsueh; Kao, Ming-Ching; Liu, Jah-Yao

2016-01-01

The availability of adequate cancer stem cells or cancer stem-like cell (CSC) is important in cancer study. From ovarian cancer cell lines, SKOV3 and OVCAR3, we induced peritoneal ascites tumors in immunodeficient mice. Among the cells (SKOV3.PX1 and OVCAR3.PX1) from those tumors, we sorted both CD44 and CD133 positive cells (SKOV3.PX1_133+44+, OVCAR3.PX1_133+44+), which manifest the characteristics of self-renewal, multi-lineage differentiation, chemoresistance and tumorigenicity, those of cancer stem-like cells (CSLC). Intraperitoneal transplantation of these CD44 and CD133 positive cells resulted in poorer survival in the engrafted animals. Clinically, increased CD133 expression was found in moderately and poorly differentiated (grade II and III) ovarian serous cystadenocarcinomas. The ascites tumor cells from human ovarian cancers demonstrated more CD133 and CD44 expressions than those from primary ovarian or metastatic tumors and confer tumorigenicity in immunodeficient mice. Compared to their parental cells, the SKOV3.PX1_133+44+ and OVCAR3.PX1_133+44+ cells uniquely expressed 5 CD markers (CD97, CD104, CD107a, CD121a, and CD125). Among these markers, CD97, CD104, CD107a, and CD121a are significantly more expressed in the CD133+ and CD44+ double positive cells of human ovarian ascites tumor cells (Ascites_133+44+) than those from primary ovarian or metastatic tumors. The cancer stem-like cells were enriched from 3% to more than 70% after this manipulation. This intraperitoneal enrichment of cancer stem-like cells, from ovarian cancer cell lines or primary ovarian tumor, potentially provides an adequate amount of ovarian cancer stem-like cells for the ovarian cancer study and possibly benefits cancer therapy. PMID:27655682

The influence of rat mesenchymal stem cell CD44 surface markers on cell growth, fibronectin expression, and cardiomyogenic differentiation on silk fibroin - Hyaluronic acid cardiac patches.

PubMed

Yang, Ming-Chia; Chi, Nai-Hsin; Chou, Nai-Kuan; Huang, Yi-You; Chung, Tze-Wen; Chang, Yu-Lin; Liu, Hwa-Chang; Shieh, Ming-Jium; Wang, Shoei-Shen

2010-02-01

Since MSCs contain an abundant of CD44 surface markers, it is of interesting to investigate whether CD44 on rat MSC (rMSCs) influenced cell growth, fibronectin expression and cardiomyogenic differentiation on new SF/HA cardiac patches. For this investigation, we examined the influences of rMSCs with or without a CD44-blockage treatment on the aforementioned issues after they were cultivated, and further induced by 5-aza on SF and SF/HA patches. The results showed that the relative growth rates of rMSCs cultured on cultural wells, SF/HA patches without or with a CD44-blockage treatment were 100%, 208.9+/-7.1 (%) or 48.4+/-6.0 (%) (n=3, for all), respectively, after five days of cultivations. Moreover, rMSCs cultivated on SF/HA patches highly promoted fibronectin expressions (e.g., 1.8x10(5)/cell, in fluorescent intensity) while cells with a CD44-blockage treatment markedly diminished the expressions (e.g., 1.1x10(4)/cell, in fluorescent intensity) on same patches. For investigating possible influences of CD44 surface markers of rMSCs on their cardiomyogenic differentiation, the expressions of specific cardiac genes of cells were examined by using real-time PCR analysis. The results indicated that 5-aza inducing rMSCs significantly promoted the expressions of Gata4, Nkx2.5, Tnnt2 and Actc1 genes (all, P<0.01 or better, n=3) on SF/HA patches compared with those expressions on SF patches and for cells with a CD44-blockage treatment on SF/HA patches. Furthermore, the intensity of the expressions of cardiotin and connexin 43 of 5-aza inducing rMSCs were markedly higher than those of cells with a CD44-blockage treatment after they were cultured on SF/HA patches. Through this study, we reported that CD44 surface markers of rMSCs highly influenced the proliferations, fibronectin expressions and cardiomyogenic differentiation of rMSCs cultivated on cardiac SF/HA patches.

Decreased expression of cell adhesion genes in cancer stem-like cells isolated from primary oral squamous cell carcinomas.

PubMed

Mishra, Amrendra; Sriram, Harshini; Chandarana, Pinal; Tanavde, Vivek; Kumar, Rekha V; Gopinath, Ashok; Govindarajan, Raman; Ramaswamy, S; Sadasivam, Subhashini

2018-05-01

The goal of this study was to isolate cancer stem-like cells marked by high expression of CD44, a putative cancer stem cell marker, from primary oral squamous cell carcinomas and identify distinctive gene expression patterns in these cells. From 1 October 2013 to 4 September 2015, 76 stage III-IV primary oral squamous cell carcinoma of the gingivobuccal sulcus were resected. In all, 13 tumours were analysed by immunohistochemistry to visualise CD44-expressing cells. Expression of CD44 within The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma RNA-sequencing data was also assessed. Seventy resected tumours were dissociated into single cells and stained with antibodies to CD44 as well as CD45 and CD31 (together referred as Lineage/Lin). From 45 of these, CD44 + Lin - and CD44 - Lin - subpopulations were successfully isolated using fluorescence-activated cell sorting, and good-quality RNA was obtained from 14 such sorted pairs. Libraries from five pairs were sequenced and the results analysed using bioinformatics tools. Reverse transcription quantitative polymerase chain reaction was performed to experimentally validate the differential expression of selected candidate genes identified from the transcriptome sequencing in the same 5 and an additional 9 tumours. CD44 was expressed on the surface of poorly differentiated tumour cells, and within the The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma samples, its messenger RNA levels were higher in tumours compared to normal. Transcriptomics revealed that 102 genes were upregulated and 85 genes were downregulated in CD44 + Lin - compared to CD44 - Lin - cells in at least 3 of the 5 tumours sequenced. The upregulated genes included those involved in immune regulation, while the downregulated genes were enriched for genes involved in cell adhesion. Decreased expression of PCDH18, MGP, SPARCL1 and KRTDAP was confirmed by reverse transcription quantitative polymerase chain reaction. Lower expression of the cell-cell adhesion molecule PCDH18 correlated with poorer overall survival in the The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma data highlighting it as a potential negative prognostic factor in this cancer.

ELK3 promotes the migration and invasion of liver cancer stem cells by targeting HIF-1α.

PubMed

Lee, Joon Ho; Hur, Wonhee; Hong, Sung Woo; Kim, Jung-Hee; Kim, Sung Min; Lee, Eun Byul; Yoon, Seung Kew

2017-02-01

Hepatocellular carcinoma (HCC) is the fifth most common solid cancer and the third most common cause of cancer-related mortality. HCC develops via a multistep process associated with genetic aberrations that facilitate HCC invasion and migration and promote metastasis. A growing body of evidence indicates that cancer stem cells (CSCs) are responsible for tumorigenesis, cancer cell invasion and metastasis. Despite the extremely small proportion of cancer cells represented by this subpopulation of HCC cells, CSCs play a key role in cancer metastasis and poor prognosis. ELK3 (Net/SAP-2/Erp) is a transcription factor that is activated by the Ras/extracellular signal-regulated kinase (ERK) signaling pathway. It plays several important roles in various physiological processes, including cell migration, invasion, wound healing, angiogenesis and tumorigenesis. In the present study, we investigated the role of ELK3 in cancer cell invasion and metastasis in CD133+/CD44+ liver cancer stem cells (LCSCs). We isolated LCSCs expressing CD133 and CD44 from Huh7 HCC cells and evaluated their metastatic potential using invasion and migration assays. We found that CD133+/CD44+ cells had increased metastatic potential compared with non-CD133+/CD44+ cells. We also demonstrated that ELK3 expression was upregulated in CD133+/CD44+ cells and that this aberration enhanced cell migration and invasion. In addition, we identified the molecular mechanism by which ELK3 promotes cancer cell migration and invasion. We found that silencing of ELK3 expression in CD133+/CD44+ LCSCs attenuated their metastatic potential by modulating the expression of heat shock-induced factor-1α (HIF-1α). Collectively, the results of the present study demonstrated that ELK3 overexpression promoted metastasis in CD133+/CD44+ cells by regulating HIF-1α expression and that silencing of ELK3 expression attenuated the metastatic potential of CD133+/CD44+ LCSCs. In conclusion, modulation of ELK3 expression may represent a novel therapeutic strategy for preventing HCC metastasis and invasion.

Differential induction of CD94 and NKG2 in CD4 helper T cells. A consequence of influenza virus infection and interferon-γ?

PubMed Central

Graham, Christine M; Christensen, Jillian R; Thomas, D Brian

2007-01-01

Influenza A virus causes worldwide epidemics and pandemics and the investigation of memory T helper (Th) cells that help maintain serological memory following infection is important for vaccine design. In this study we investigated CD94 and NKG2 gene expression in memory CD4 T-cell clones established from the spleens of C57BL/10 (H-2b) and BALB/c (H-2d) mice infected with influenza A virus (H3N2). CD94 and NKG2A/C/E proteins form heterodimeric membrane receptors that are involved in virus recognition. CD94 and NKG2 expression have been well characterized in natural killer (NK) and cytotoxic T cells. Despite CD94 being potentially an important marker for Th1 cells involved in virus infection, however, there has been little investigation of its expression or function in the CD4 T-cell lineage and no studies have looked at in-vivo-generated Th cells or memory cells. We show in this study that in-vivo-generated CD4 Th1 cells, but not Th2 cells, exhibited full-length CD94 and NKG2A gene expression following activation with viral peptide. For NKG2A, a novel ‘short’ (possibly redundant) truncated isoform was detectable in a Th2 cell clone. Another member of the NK receptor family, NKG2D, but not NKG2C or E, was also differentially expressed in Th1 cells. We show here that CD94 and NKG2A may exist as multiple isoforms with the potential to distinguish helper T-cell subsets. PMID:17462078

Structural characterization of NRAS isoform 5

PubMed Central

Mal, Tapas K.; Yuan, Chunhua; Courtney, Nicholas B.; Patel, Mitra; Stiff, Andrew R.; Blachly, James; Walker, Christopher; Eisfeld, Ann‐Kathrin; de la Chapelle, Albert

2016-01-01

Abstract It was recently discovered that the NRAS isoform 5 (20 amino acids) is expressed in melanoma and results in a more aggressive cell phenotype. This novel isoform is responsible for increased phosphorylation of downstream targets such as AKT, MEK, and ERK as well as increased cellular proliferation. This structure report describes the NMR solution structure of NRAS isoform 5 to be used as a starting point to understand its biophysical interactions. The isoform is highly flexible in aqueous solution, but forms a helix‐turn‐coil structure in the presence of trifluoroethanol as determined by NMR and CD spectroscopy. PMID:26947772

CD44 Promotes Inflammation and Extracellular Matrix Production During Arteriovenous Fistula Maturation.

PubMed

Kuwahara, Go; Hashimoto, Takuya; Tsuneki, Masayuki; Yamamoto, Kota; Assi, Roland; Foster, Trenton R; Hanisch, Jesse J; Bai, Hualong; Hu, Haidi; Protack, Clinton D; Hall, Michael R; Schardt, John S; Jay, Steven M; Madri, Joseph A; Kodama, Shohta; Dardik, Alan

2017-06-01

Arteriovenous fistulae (AVF) remain the optimal conduit for hemodialysis access but continue to demonstrate poor patency and poor rates of maturation. We hypothesized that CD44, a widely expressed cellular adhesion molecule that serves as a major receptor for extracellular matrix components, promotes wall thickening and extracellular matrix deposition during AVF maturation. AVF were created via needle puncture in wild-type C57BL/6J and CD44 knockout mice. CD44 mRNA and protein expression was increased in wild-type AVF. CD44 knockout mice showed no increase in AVF wall thickness (8.9 versus 26.8 μm; P =0.0114), collagen density, and hyaluronic acid density, but similar elastin density when compared with control AVF. CD44 knockout mice also showed no increase in vascular cell adhesion molecule-1 expression, intercellular adhesion molecule-1 expression, and monocyte chemoattractant protein-1 expression in the AVF compared with controls; there were also no increased M2 macrophage markers (transglutaminase-2: 81.5-fold, P =0.0015; interleukin-10: 7.6-fold, P =0.0450) in CD44 knockout mice. Delivery of monocyte chemoattractant protein-1 to CD44 knockout mice rescued the phenotype with thicker AVF walls (27.2 versus 14.7 μm; P =0.0306), increased collagen density (2.4-fold; P =0.0432), and increased number of M2 macrophages (2.1-fold; P =0.0335). CD44 promotes accumulation of M2 macrophages, extracellular matrix deposition, and wall thickening during AVF maturation. These data show the association of M2 macrophages with wall thickening during AVF maturation and suggest that enhancing CD44 activity may be a strategy to increase AVF maturation. © 2017 American Heart Association, Inc.

CD44 Promotes Inflammation and Extracellular Matrix Production During Arteriovenous Fistula Maturation

PubMed Central

Kuwahara, Go; Hashimoto, Takuya; Tsuneki, Masayuki; Yamamoto, Kota; Assi, Roland; Foster, Trenton R; Hanisch, Jesse J; Bai, Hualong; Hu, Haidi; Protack, Clinton D; Hall, Michael R; Schardt, John S; Jay, Steven M; Madri, Joseph A; Kodama, Shohta; Dardik, Alan

2017-01-01

Objective Arteriovenous fistulae (AVF) remain the optimal conduit for hemodialysis access but continue to demonstrate poor patency and poor rates of maturation. We hypothesized that CD44, a widely expressed cellular adhesion molecule that serves as a major receptor for extracellular matrix (ECM) components, promotes wall thickening and ECM deposition during AVF maturation. Approach and Results AVF were created via needle puncture in wild-type (WT) C57BL/6J and CD44 knockout (KO) mice. CD44 mRNA and protein expression was increased in WT AVF. CD44 KO mice showed no increase in AVF wall thickness (8.9 μm vs. 26.8 μm; P = 0.0114), collagen density, and hyaluronic acid density, but similar elastin density when compared to control AVF. CD44 KO mice also showed no increase in VCAM-1 expression, ICAM-1 expression and MCP-1 expression in the AVF compared to controls; there were also no increased M2 macrophage markers (TGM2: 81.5 fold, P = 0.0015; IL-10: 7.6 fold, P = 0.0450) in CD44 KO mice. Delivery of MCP-1 to CD44 KO mice rescued the phenotype with thicker AVF walls (27.2 μm vs. 14.7 μm; P = 0.0306), increased collagen density (2.4 fold; P = 0.0432), and increased number of M2 macrophages (2.1 fold; P = 0.0335). Conclusions CD44 promotes accumulation of M2 macrophages, ECM deposition and wall thickening during AVF maturation. These data show the association of M2 macrophages with wall thickening during AVF maturation and suggest that enhancing CD44 activity may be a strategy to increase AVF maturation. PMID:28450292

Activated ERK1/2 increases CD44 in glomerular parietal epithelial cells leading to matrix expansion

PubMed Central

Roeder, Sebastian S.; Barnes, Taylor J.; Lee, Jonathan S.; Kato, India; Eng, Diana G.; Kaverina, Natalya V.; Sunseri, Maria W.; Daniel, Christoph; Amann, Kerstin; Pippin, Jeffrey W.; Shankland, Stuart J.

2017-01-01

The glycoprotein CD44 is barely detected in normal mouse and human glomeruli, but is increased in glomerular parietal epithelial cells following podocyte injury in focal segmental glomerulosclerosis (FSGS). To determine the biological role and regulation of CD44 in these cells, we employed an in vivo and in vitro approach. Experimental FSGS was induced in CD44 knockout and wildtype mice with a cytotoxic podocyte antibody. Albuminuria, focal and global glomerulosclerosis (periodic acid-Schiff stain) and collagen IV staining were lower in CD44 knockout compared with wild type mice with FSGS. Parietal epithelial cells had lower migration from Bowman’s capsule to the glomerular tuft in CD44 knockout mice with disease compared with wild type mice. In cultured murine parietal epithelial cells, overexpressing CD44 with a retroviral vector encoding CD44 was accompanied by significantly increased collagen IV expression and parietal epithelial cells migration. Because our results showed de novo co-staining for activated ERK1/2 (pERK) in parietal epithelial cells in experimental FSGS, and also in biopsies from patients with FSGS, two in vitro strategies were employed to prove that pERK regulated CD44 levels. First, mouse parietal epithelial cells were infected with a retroviral vector for the upstream kinase MEK-DD to increase pERK, which was accompanied by increased CD44 levels. Second, in CD44 overexpressing parietal epithelial cells, decreasing pERK with U0126 was accompanied by reduced CD44. Finally, parietal epithelial cell migration was higher in cells with increased and reduced in cells with decreased pERK. Thus, pERK is a regulator of CD44 expression and increased CD44 expression leads to a pro-sclerotic and migratory parietal epithelial cells phenotype. PMID:27998643

CD1-Restricted T Cells at the Crossroad of Innate and Adaptive Immunity.

PubMed

Pereira, Catia S; Macedo, M Fatima

2016-01-01

Lipid-specific T cells comprise a group of T cells that recognize lipids bound to the MHC class I-like CD1 molecules. There are four isoforms of CD1 that are expressed at the surface of antigen presenting cells and therefore capable of presenting lipid antigens: CD1a, CD1b, CD1c, and CD1d. Each one of these isoforms has distinct structural features and cellular localizations, which promotes binding to a broad range of different types of lipids. Lipid antigens originate from either self-tissues or foreign sources, such as bacteria, fungus, or plants and their recognition by CD1-restricted T cells has important implications in infection but also in cancer and autoimmunity. In this review, we describe the characteristics of CD1 molecules and CD1-restricted lipid-specific T cells, highlighting the innate-like and adaptive-like features of different CD1-restricted T cell subtypes.

Heparan Sulfate Modification of the Transmembrane Receptor CD47 Is Necessary for Inhibition of T Cell Receptor Signaling by Thrombospondin-1*

PubMed Central

Kaur, Sukhbir; Kuznetsova, Svetlana A.; Pendrak, Michael L.; Sipes, John M.; Romeo, Martin J.; Li, Zhuqing; Zhang, Lijuan; Roberts, David D.

2011-01-01

Cell surface proteoglycans on T cells contribute to retroviral infection, binding of chemokines and other proteins, and are necessary for some T cell responses to the matricellular glycoprotein thrombospondin-1. The major cell surface proteoglycans expressed by primary T cells and Jurkat T cells have an apparent Mr > 200,000 and are modified with chondroitin sulfate and heparan sulfate chains. Thrombospondin-1 bound in a heparin-inhibitable manner to this proteoglycan and to a soluble form released into the medium. Based on mass spectrometry, knockdown, and immunochemical analyses, the proteoglycan contains two major core proteins as follows: amyloid precursor-like protein-2 (APLP2, apparent Mr 230,000) and CD47 (apparent Mr > 250,000). CD47 is a known thrombospondin-1 receptor but was not previously reported to be a proteoglycan. This proteoglycan isoform of CD47 is widely expressed on vascular cells. Mutagenesis identified glycosaminoglycan modification of CD47 at Ser64 and Ser79. Inhibition of T cell receptor signaling by thrombospondin-1 was lost in CD47-deficient T cells that express the proteoglycan isoform of APLP2, indicating that binding to APLP2 is not sufficient. Inhibition of CD69 induction was restored in CD47-deficient cells by re-expressing CD47 or an S79A mutant but not by the S64A mutant. Therefore, inhibition of T cell receptor signaling by thrombospondin-1 is mediated by CD47 and requires its modification at Ser64. PMID:21343308

Differential expression of CD44 and CD24 markers discriminates the epitheliod from the fibroblastoid subset in a sarcomatoid renal carcinoma cell line: evidence suggesting the existence of cancer stem cells in both subsets as studied with sorted cells.

PubMed

Hsieh, Chin-Hsuan; Hsiung, Shih-Chieh; Yeh, Chi-Tai; Yen, Chih-Feng; Chou, Yah-Huei Wu; Lei, Wei-Yi; Pang, See-Tong; Chuang, Cheng-Keng; Liao, Shuen-Kuei

2017-02-28

Epithelioid and fibroblastoid subsets coexist in the human sarcomatoid renal cell carcinoma (sRCC) cell line, RCC52, according to previous clonal studies. Herein, using monoclonal antibodies to CD44 and CD24 markers, we identified and isolated these two populations, and showed that CD44bright/CD24dim and CD44bright/CD24bright phenotypes correspond to epithelioid and fibroblastoid subsets, respectively. Both sorted subsets displayed different levels of tumorigenicity in xenotransplantation, indicating that each harbored its own cancer stem cells (CSCs). The CD44bright/CD24bright subset, associated with higher expression of MMP-7, -8 and TIMP-1 transcripts, showed greater migratory/invasive potential than the CD44bright/CD24dim subset, which was associated with higher expression of MMP-2, -9 and TIMP-2 transcripts. Both subsets differentially expressed stemness gene products c-Myc, Oct4A, Notch1, Notch2 and Notch3, and the RCC stem cell marker, CD105 in 4-5% of RCC52 cells. These results suggest the presence of CSCs in both sRCC subsets for the first time and should therefore be considered potential therapeutic targets for this aggressive malignancy.

CD44-mediated activation of α5β1-integrin, cortactin and paxillin signaling underpins adhesion of basal-like breast cancer cells to endothelium and Fibronectin-enriched matrices

PubMed Central

McFarlane, Suzanne; McFarlane, Cheryl; Montgomery, Nicola; Hill, Ashleigh; Waugh, David J.J.

2015-01-01

CD44 expression is elevated in basal-like breast cancer (BLBC) tissue, and correlates with increased efficiency of distant metastasis in patients and experimental models. We sought to characterize mechanisms underpinning CD44-promoted adhesion of BLBC cells to vascular endothelial monolayers and extracellular matrix (ECM) substrates. Stimulation with hyaluronan (HA), the native ligand for CD44, increased expression and activation of β1-integrin receptors, and increased α5-integrin subunit expression. Adhesion assays confirmed that CD44-signalling potentiated BLBC cell adhesion to endothelium and Fibronectin in an α5B1-integrin-dependent mechanism. Co-immunoprecipitation experiments confirmed HA-promoted association of CD44 with talin and the β1-integrin chain in BLBC cells. Knockdown of talin inhibited CD44 complexing with β1-integrin and repressed HA-induced, CD44-mediated activation of β1-integrin receptors. Immunoblotting confirmed that HA induced rapid phosphorylation of cortactin and paxillin, through a CD44-dependent and β1-integrin-dependent mechanism. Knockdown of CD44, cortactin or paxillin independently attenuated the adhesion of BL-BCa cells to endothelial monolayers and Fibronectin. Accordingly, we conclude that CD44 induced, integrin-mediated signaling not only underpins efficient adhesion of BLBC cells to BMECs to facilitate extravasation but initiates their adhesion to Fibronectin, enabling penetrant cancer cells to adhere more efficiently to underlying Fibronectin-enriched matrix present within the metastatic niche. PMID:26447611

CD44 as a receptor for colonization of the pharynx by group A Streptococcus

PubMed Central

Cywes, Colette; Stamenkovic, Ivan; Wessels, Michael R.

2000-01-01

The pharynx is the primary reservoir for strains of group A Streptococcus (GAS) associated both with pharyngitis (streptococcal sore throat) and with invasive or “flesh-eating” soft tissue infections. We now report that CD44, a hyaluronic acid-binding protein that mediates human cell-cell– and cell-extracellular matrix–binding interactions, functions as a receptor for GAS colonization of the pharynx in vivo. We found that attachment of GAS to murine epithelial keratinocytes was mediated by binding of the GAS hyaluronic acid capsular polysaccharide to CD44. In studies of transgenic mice with a selective defect in epithelial expression of CD44, GAS adherence to CD44-deficient keratinocytes in vitro was reduced compared with adherence to keratinocytes expressing normal levels of CD44. After intranasal inoculation, GAS colonized the oropharynx of wild-type mice but failed to colonize transgenic mice deficient in CD44 expression. GAS colonization of wild-type mice could be blocked by coadministration of mAb to CD44 or by pretreatment of the animals with exogenous hyaluronic acid. These results provide evidence that CD44 serves as a receptor for GAS colonization of the pharynx and support the potential efficacy of disrupting the interaction between the GAS hyaluronic acid capsule and CD44 as a novel approach to preventing pharyngeal infection. PMID:11032859

New pyrimido-indole compound CD-160130 preferentially inhibits the KV11.1B isoform and produces antileukemic effects without cardiotoxicity.

PubMed

Gasparoli, Luca; D'Amico, Massimo; Masselli, Marika; Pillozzi, Serena; Caves, Rachel; Khuwaileh, Rawan; Tiedke, Wolfgang; Mugridge, Kenneth; Pratesi, Alessandro; Mitcheson, John S; Basso, Giuseppe; Becchetti, Andrea; Arcangeli, Annarosa

2015-02-01

KV11.1 (hERG1) channels are often overexpressed in human cancers. In leukemias, KV11.1 regulates pro-survival signals that promote resistance to chemotherapy, raising the possibility that inhibitors of KV11.1 could be therapeutically beneficial. However, because of the role of KV11.1 in cardiac repolarization, blocking these channels may cause cardiac arrhythmias. We show that CD-160130, a novel pyrimido-indole compound, blocks KV11.1 channels with a higher efficacy for the KV11.1 isoform B, in which the IC50 (1.8 μM) was approximately 10-fold lower than observed in KV11.1 isoform A. At this concentration, CD-160130 also had minor effects on Kir2.1, KV 1.3, Kv1.5, and KCa3.1. In vitro, CD-160130 induced leukemia cell apoptosis, and could overcome bone marrow mesenchymal stromal cell (MSC)-induced chemoresistance. This effect was caused by interference with the survival signaling pathways triggered by MSCs. In vivo, CD-160130 produced an antileukemic activity, stronger than that caused by cytarabine. Consistent with its atypical target specificity, CD-160130 did not bind to the main binding site of the arrhythmogenic KV11.1 blockers (the Phe656 pore residue). Importantly, in guinea pigs CD-160130 produced neither alteration of the cardiac action potential shape in dissociated cardiomyocytes nor any lengthening of the QT interval in vivo. Moreover, CD-160130 had no myelotoxicity on human bone marrow-derived cells. Therefore, CD-160130 is a promising first-in-class compound to attempt oncologic therapy without cardiotoxicity, based on targeting KV11.1. Because leukemia and cardiac cells tend to express different ratios of the A and B KV11.1 isoforms, the pharmacological properties of CD-160130 may depend, at least in part, on isoform specificity. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

CD44 deficiency enhanced Streptococcus equi ssp. zooepidemicus dissemination and inflammation response in a mouse model.

PubMed

Fu, Qiang; Xiao, Pingping; Chen, Yaosheng; Wei, Zigong; Liu, Xiaohong

2017-12-01

Streptococcus equi ssp. zooepidemicus (S. zooepidemicus) is responsible for peritonitis, septicemia, meningitis, arthritis and several other serious diseases in various species. Recent studies have demonstrated that CD44 is implicated in the process of host defense against pathogenic microorganisms. In the present study, the role of CD44 in the host response to S. zooepidemicus infection was investigated in a mouse model. Upon intraperitoneal infection with S. zooepidemicus, the expression of CD44 on the peritoneal exudate cells from wild-type (WT) mice was increased. CD44 deficiency accelerated mortality, which was accompanied by increased peritoneal bacterial growth and dissemination to distant body sites. CD44 knock-out (KO) mice showed enhanced early inflammatory cell recruitment into the peritoneal fluid on S. zooepidemicus infection. In line with this, the expression of proinflammatory cytokines, chemokines in peritoneal exudate cells and peritoneal macrophages of CD44 KO mice were increased compared with those of WT mice. In addition, CD44 deficiency was associated with reduced expression of A20, a negative regulator in TLR signaling. Overall, the present study suggests that CD44 plays a protective role in antibacterial defense against S. zooepidemicus in mice. Copyright © 2017. Published by Elsevier Ltd.

Clinical relevance of TRKA expression on neuroblastoma: comparison with N-MYC amplification and CD44 expression.

PubMed Central

Combaret, V.; Gross, N.; Lasset, C.; Balmas, K.; Bouvier, R.; Frappaz, D.; Beretta-Brognara, C.; Philip, T.; Favrot, M. C.; Coll, J. L.

1997-01-01

TRKA expression was evaluated on 122 untreated neuroblastomas by immunohistochemistry using an antibody with predetermined specificity. This procedure is simple and reliable for protein detection at cellular level in a routine clinical setting. Fourteen tumours were classified as benign ganglioneuroma with a restricted expression of TRKA on ganglion cells; these patients were excluded from the following analysis. A total of 108 tumours were classified as neuroblastoma or ganglioneuroblastoma; 74 expressed TRKA protein, which strongly correlated with low stage, absence of N-MYC amplification, age (<1 year), CD44 expression and favourable clinical outcome. In a univariate analysis including tumour stage, age, histology, N-MYC amplification, CD44 and TRKA expression, all parameters had significant prognostic value. The absence of TRKA expression on CD44-positive or N-MYC non-amplified tumours permits the characterization of a subgroup of patients with intermediate prognosis. However, in a multivariate analysis taking into consideration the prognostic factors mentioned above, CD44 and tumour stage were the only independent prognostic factors for the prediction of patients' event-free survival. PMID:9099963

Na/K-ATPase/src complex mediates regulation of CD40 in renal parenchyma.

PubMed

Xie, Jeffrey X; Zhang, Shungang; Cui, Xiaoyu; Zhang, Jue; Yu, Hui; Khalaf, Fatimah K; Malhotra, Deepak; Kennedy, David J; Shapiro, Joseph I; Tian, Jiang; Haller, Steven T

2017-12-22

Recent studies have highlighted a critical role for CD40 in the pathogenesis of renal injury and fibrosis. However, little is currently understood about the regulation of CD40 in this setting. We use novel Na/K-ATPase cell lines and inhibitors in order to demonstrate the regulatory function of Na/K-ATPase with regards to CD40 expression and function. We utilize 5/6 partial nephrectomy as well as direct infusion of a Na/K-ATPase ligand to demonstrate this mechanism exists in vivo. We demonstrate that knockdown of the α1 isoform of Na/K-ATPase causes a reduction in CD40 while rescue of the α1 but not the α2 isoform restores CD40 expression in renal epithelial cells. Second, because the major functional difference between α1 and α2 is the ability of α1 to form a functional signaling complex with Src, we examined whether the Na/K-ATPase/Src complex is important for CD40 expression. We show that a gain-of-Src binding α2 mutant restores CD40 expression while loss-of-Src binding α1 reduces CD40 expression. Furthermore, loss of a functional Na/K-ATPase/Src complex also disrupts CD40 signaling. Importantly, we show that use of a specific Na/K-ATPase/Src complex antagonist, pNaKtide, can attenuate cardiotonic steroid (CTS)-induced induction of CD40 expression in vitro. Because the Na/K-ATPase/Src complex is also a key player in the pathogenesis of renal injury and fibrosis, our new findings suggest that Na/K-ATPase and CD40 may comprise a pro-fibrotic feed-forward loop in the kidney and that pharmacological inhibition of this loop may be useful in the treatment of renal fibrosis. © The Author 2017. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

Overexpression of molecular chaperons GRP78 and GRP94 in CD44(hi)/CD24(lo) breast cancer stem cells.

PubMed

Nami, Babak; Ghasemi-Dizgah, Armin; Vaseghi, Akbar

2016-01-01

Breast cancer stem cell with CD44(hi)/CD24(lo) phonotype is described having stem cell properties and represented as the main driving factor in breast cancer initiation, growth, metastasis and low response to anti-cancer agents. Glucoseregulated proteins (GRPs) are heat shock protein family chaperons that are charged with regulation of protein machinery and modulation of endoplasmic reticulum homeostasis whose important roles in stem cell development and invasion of various cancers have been demonstrated. Here, we investigated the expression levels of GRP78 and GRP94 in CD44(hi)/CD24(lo) phenotype breast cancer stem cells (BCSCs). MCF7, T-47D and MDA-MB-231 breast cancer cell lines were used. CD44(hi)/CD24(lo) phenotype cell population were analyzed and sorted by fluorescence-activated cell sorting (FACS). Transcriptional and translational expression of GRP78 and GRP94 were investigated by western blotting and quantitative real time PCR. RESULTS showed different proportion of CD44(hi)/CD24(lo) phenotype cell population in their original bulk cells. The ranking of the cell lines in terms of CD44(hi)/CD24(lo) phenotype cell population was as MCF7

The human CD94 gene encodes multiple, expressible transcripts including a new partner of NKG2A/B.

PubMed

Lieto, L D; Maasho, K; West, D; Borrego, F; Coligan, J E

2006-01-01

CD94/NKG2A is an inhibitory receptor expressed by natural killer (NK) cells and a subset of CD8+ T cells. Ligation of CD94/NKG2A by its ligand HLA-E results in tyrosine phosphorylation of the NKG2A immunoreceptor tyrosine-based inhibitory motifs, and recruitment and activation of the SH2 domain-bearing tyrosine phosphatase-1, which in turn suppresses activation signals. The nkg2a gene encodes two isoforms, NKG2A and NKG2B, with the latter lacking the stem region. We identified three new alternative transcripts of the cd94 gene in addition to the originally described canonical CD94Full. One of the transcripts, termed CD94-T4, lacks the portion that encodes the stem region. CD94-T4 associates with both NKG2A and NKG2B, but preferentially associates with the latter. This is probably due to the absence of a stem region in both CD94-T4 and NKG2B. CD94-T4/NKG2B is capable of binding HLA-E and, when expressed in E6-1 Jurkat T cells, inhibits TCR mediated signals, demonstrating that this heterodimer is functional. Coevolution of stemless isoforms of CD94 and NKG2A that preferentially pair with each other to produce a functional heterodimer indicates that this may be more than a serendipitous event. CD94-T4/NKG2B may contribute to the plasticity of the NK immunological synapse by insuring an adequate inhibitory signal.

Autophagy regulates cisplatin-induced stemness and chemoresistance via the upregulation of CD44, ABCB1 and ADAM17 in oral squamous cell carcinoma.

PubMed

Naik, Prajna Paramita; Mukhopadhyay, Subhadip; Panda, Prashanta Kumar; Sinha, Niharika; Das, Chandan Kanta; Mishra, Rajakishore; Patil, Shankargouda; Bhutia, Sujit Kumar

2018-02-01

We inspected the relevance of CD44, ABCB1 and ADAM17 in OSCC stemness and deciphered the role of autophagy/mitophagy in regulating stemness and chemoresistance. A retrospective analysis of CD44, ABCB1 and ADAM17 with respect to the various clinico-pathological factors and their correlation was analysed in sixty OSCC samples. Furthermore, the stemness and chemoresistance were studied in resistant oral cancer cells using sphere formation assay, flow cytometry and florescence microscopy. The role of autophagy/mitophagy was investigated by transient transfection of siATG14, GFP-LC3, tF-LC3, mKeima-Red-Mito7 and Western blot analysis of autophagic and mitochondrial proteins. In OSCC, high CD44, ABCB1 and ADAM17 expressions were correlated with higher tumour grades and poor differentiation and show significant correlation in their co-expression. In vitro and OSCC tissue double labelling confirmed that CD44 + cells co-expresses ABCB1 and ADAM17. Further, cisplatin (CDDP)-resistant FaDu cells displayed stem-like features and higher CD44, ABCB1 and ADAM17 expression. Higher autophagic flux and mitophagy were observed in resistant FaDu cells as compared to parental cells, and inhibition of autophagy led to the decrease in stemness, restoration of mitochondrial proteins and reduced expression of CD44, ABCB1 and ADAM17. The CD44 + /ABCB1 + /ADAM17 + expression in OSCC is associated with stemness and chemoresistance. Further, this study highlights the involvement of mitophagy in chemoresistance and autophagic regulation of stemness in OSCC. © 2017 John Wiley & Sons Ltd.

In Vitro and In Vivo Prostate Cancer Metastasis and Chemoresistance Can Be Modulated by Expression of either CD44 or CD147

PubMed Central

Hao, Jingli; Madigan, Michele C.; Khatri, Aparajita; Power, Carl A.; Hung, Tzong-Tyng; Beretov, Julia; Chang, Lei; Xiao, Weiwei; Cozzi, Paul J.; Graham, Peter H.; Kearsley, John H.; Li, Yong

2012-01-01

CD44 and CD147 are associated with cancer metastasis and progression. Our purpose in the study was to investigate the effects of down-regulation of CD44 or CD147 on the metastatic ability of prostate cancer (CaP) cells, their docetaxel (DTX) responsiveness and potential mechanisms involved in vitro and in vivo. CD44 and CD147 were knocked down (KD) in PC-3M-luc CaP cells using short hairpin RNA (shRNA). Expression of CD44, CD147, MRP2 (multi-drug resistance protein-2) and MCT4 (monocarboxylate tranporter-4) was evaluated using immunofluorescence and Western blotting. The DTX dose-response and proliferation was measured by MTT and colony assays, respectively. The invasive potential was assessed using a matrigel chamber assay. Signal transduction proteins in PI3K/Akt and MAPK/Erk pathways were assessed by Western blotting. An in vivo subcutaneous (s.c.) xenograft model was established to assess CaP tumorigenecity, lymph node metastases and DTX response. Our results indicated that KD of CD44 or CD147 decreased MCT4 and MRP2 expression, reduced CaP proliferation and invasive potential and enhanced DTX sensitivity; and KD of CD44 or CD147 down-regulated p-Akt and p-Erk, the main signal modulators associated with cell growth and survival. In vivo, CD44 or CD147-KD PC-3M-luc xenografts displayed suppressed tumor growth with increased DTX responsiveness compared to control xenografts. Both CD44 and CD147 enhance metastatic capacity and chemoresistance of CaP cells, potentially mediated by activation of the PI3K and MAPK pathways. Selective targeting of CD44/CD147 alone or combined with DTX may limit CaP metastasis and increase chemosensitivity, with promise for future CaP treatment. PMID:22870202

A mesenchymal-like phenotype and expression of CD44 predict lack of apoptotic response to sorafenib in liver tumor cells.

PubMed

Fernando, Joan; Malfettone, Andrea; Cepeda, Edgar B; Vilarrasa-Blasi, Roser; Bertran, Esther; Raimondi, Giulia; Fabra, Àngels; Alvarez-Barrientos, Alberto; Fernández-Salguero, Pedro; Fernández-Rodríguez, Conrado M; Giannelli, Gianluigi; Sancho, Patricia; Fabregat, Isabel

2015-02-15

The multikinase inhibitor sorafenib is the only effective drug in advanced cases of hepatocellular carcinoma (HCC). However, response differs among patients and effectiveness only implies a delay. We have recently described that sorafenib sensitizes HCC cells to apoptosis. In this work, we have explored the response to this drug of six different liver tumor cell lines to define a phenotypic signature that may predict lack of response in HCC patients. Results have indicated that liver tumor cells that show a mesenchymal-like phenotype, resistance to the suppressor effects of transforming growth factor beta (TGF-β) and high expression of the stem cell marker CD44 were refractory to sorafenib-induced cell death in in vitro studies, which correlated with lack of response to sorafenib in nude mice xenograft models of human HCC. In contrast, epithelial-like cells expressing the stem-related proteins EpCAM or CD133 were sensitive to sorafenib-induced apoptosis both in vitro and in vivo. A cross-talk between the TGF-β pathway and the acquisition of a mesenchymal-like phenotype with up-regulation of CD44 expression was found in the HCC cell lines. Targeted CD44 knock-down in the mesenchymal-like cells indicated that CD44 plays an active role in protecting HCC cells from sorafenib-induced apoptosis. However, CD44 effect requires a TGF-β-induced mesenchymal background, since the only overexpression of CD44 in epithelial-like HCC cells is not sufficient to impair sorafenib-induced cell death. In conclusion, a mesenchymal profile and expression of CD44, linked to activation of the TGF-β pathway, may predict lack of response to sorafenib in HCC patients. © 2014 UICC.

Alk5/Runx1 signaling mediated by extracellular vesicles promotes vascular repair in acute respiratory distress syndrome.

PubMed

Shah, Trushil; Qin, Shanshan; Vashi, Mona; Predescu, Dan N; Jeganathan, Niranjan; Bardita, Cristina; Ganesh, Balaji; diBartolo, Salvatore; Fogg, Louis F; Balk, Robert A; Predescu, Sanda A

2018-06-22

Pulmonary endothelial cells' (ECs) injury and apoptotic death are necessary and sufficient for the pathogenesis of the acute respiratory distress syndrome (ARDS), regardless of epithelial damage. Interaction of dysfunctional ECs with circulatory extracellular vesicles (EVs) holds therapeutic promise in ARDS. However, the presence in the blood of long-term ARDS survivors of EVs with a distinct phenotype compared to the EVs of non-surviving patients is not reported. With a multidisciplinary translational approach, we studied EVs from the blood of 33 patients with moderate-to-severe ARDS. The EVs were isolated from the blood of ARDS and control subjects. Immunoblotting and magnetic beads immunoisolation complemented by standardized flow cytometry and nanoparticles tracking analyses identified in the ARDS patients a subset of EVs with mesenchymal stem cell (MSC) origin (CD73 + CD105 + Cd34 - CD45 - ). These EVs have 4.7-fold greater counts compared to controls and comprise the transforming growth factor-beta receptor I (TβRI)/Alk5 and the Runx1 transcription factor. Time course analyses showed that the expression pattern of two Runx1 isoforms is critical for ARDS outcome: the p52 isoform shows a continuous expression, while the p66 is short-lived. A high ratio Runx1p66/p52 provided a survival advantage, regardless of age, sex, disease severity or length of stay in the intensive care unit. Moreover, the Runx1p66 isoform is transiently expressed by cultured human bone marrow-derived MSCs, it is released in the EVs recoverable from the conditioned media and stimulates the proliferation of lipopolysaccharide (LPS)-treated ECs. The findings are consistent with a causal effect of Runx1p66 expression on EC proliferation. Furthermore, morphological and functional assays showed that the EVs bearing the Runx1p66 enhanced junctional integrity of LPS-injured ECs and decreased lung histological severity in the LPS-treated mice. The expression pattern of Runx1 isoforms might be a reliable circulatory biomarker of ARDS activity and a novel determinant of the molecular mechanism for lung vascular/tissue repair and recovery after severe injury.

Comparative study of β-catenin and CD44 immunoexpression in oral lichen planus and squamous cell carcinoma.

PubMed

Zargaran, Massoumeh; Baghaei, Fahimeh; Moghimbeigi, Abbas

2018-04-24

Dysfunction of adhesion molecules is believed to play an early and important role in developing cancer. Accordingly, this study aims to compare beta-catenin (β-catenin) and CD44 expression in oral lichen planus (OLP) as a condition with malignant potential and oral squamous cell carcinoma (OSCC). β-Catenin and CD44 expression were evaluated in 15 patients with epithelial hyperplasia (group A), 20 OLP (group B), and 20 OSCC (group C) by immunohistochemistry. Quantitative and semi-quantitative evaluations revealed β-catenin, and CD44 membranous expression had significant differences among the three groups. Expression of these markers in the OSCC group decreased significantly compared to that of the OLP. Also, nuclear/cytoplasmic expression of β-catenin was significantly different among the three groups, considering that nuclear expression was not observed in any of the epithelial hyperplasia and OLP samples. According to the findings of this study, β-catenin and CD44 can differentiate between behavior of OLP and OSCC, while the precancerous nature of OLP and malignant transformation potential of it are not suggested. © 2018 The International Society of Dermatology.

CD44 is a direct target of miR-199a-3p and contributes to aggressive progression in osteosarcoma

PubMed Central

Gao, Yan; Feng, Yong; Shen, Jacson K.; Lin, Min; Choy, Edwin; Cote, Gregory M.; Harmon, David C.; Mankin, Henry J.; Hornicek, Francis J.; Duan, Zhenfeng

2015-01-01

Osteosarcoma is the most common primary bone malignancy in children and adolescents. Herein, we investigated the role of cluster of differentiation 44 (CD44), a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion, and migration in osteosarcoma. We constructed a human osteosarcoma tissue microarray with 114 patient tumor specimens, including tumor tissues from primary, metastatic, and recurrent stages, and determined the expression of CD44 by immunohistochemistry. Results showed that CD44 was overexpressed in metastatic and recurrent osteosarcoma as compared with primary tumors. Higher expression of CD44 was found in both patients with shorter survival and patients who exhibited unfavorable response to chemotherapy before surgical resection. Additionally, the 3′-untranslated region of CD44 mRNA was the direct target of microRNA-199a-3p (miR-199a-3p). Overexpression of miR-199a-3p significantly inhibited CD44 expression in osteosarcoma cells. miR-199a-3p is one of the most dramatically decreased miRs in osteosarcoma cells and tumor tissues as compared with normal osteoblast cells. Transfection of miR-199a-3p significantly increased the drug sensitivity through down-regulation of CD44 in osteosarcoma cells. Taken together, these results suggest that the CD44-miR-199a-3p axis plays an important role in the development of metastasis, recurrence, and drug resistance of osteosarcoma. Developing strategies to target CD44 may improve the clinical outcome of osteosarcoma. PMID:26079799

Cytoskeletal regulation of CD44 membrane organization and interactions with E-selectin.

PubMed

Wang, Ying; Yago, Tadayuki; Zhang, Nan; Abdisalaam, Salim; Alexandrakis, George; Rodgers, William; McEver, Rodger P

2014-12-19

Interactions of CD44 on neutrophils with E-selectin on activated endothelial cells mediate rolling under flow, a prerequisite for neutrophil arrest and migration into perivascular tissues. How CD44 functions as a rolling ligand despite its weak affinity for E-selectin is unknown. We examined the nanometer scale organization of CD44 on intact cells. CD44 on leukocytes and transfected K562 cells was cross-linked within a 1.14-nm spacer. Depolymerizing actin with latrunculin B reduced cross-linking. Fluorescence resonance energy transfer (FRET) revealed tight co-clustering between CD44 fused to yellow fluorescent protein (YFP) and CD44 fused to cyan fluorescent protein on K562 cells. Latrunculin B reduced FRET-reported co-clustering. Number and brightness analysis confirmed actin-dependent CD44-YFP clusters on living cells. CD44 lacking binding sites for ankyrin and for ezrin/radixin/moesin (ERM) proteins on its cytoplasmic domain (ΔANKΔERM) did not cluster. Unexpectedly, CD44 lacking only the ankyrin-binding site (ΔANK) formed larger but looser clusters. Fluorescence recovery after photobleaching demonstrated increased CD44 mobility by latrunculin B treatment or by deleting the cytoplasmic domain. ΔANKΔERM mobility increased only modestly, suggesting that the cytoplasmic domain engages the cytoskeleton by an additional mechanism. Ex vivo differentiated CD44-deficient neutrophils expressing exogenous CD44 rolled on E-selectin and activated Src kinases after binding anti-CD44 antibody. In contrast, differentiated neutrophils expressing ΔANK had impaired rolling and kinase activation. These data demonstrate that spectrin and actin networks regulate CD44 clustering and suggest that ankyrin enhances CD44-mediated neutrophil rolling and signaling. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Cytoskeletal Regulation of CD44 Membrane Organization and Interactions with E-selectin*

PubMed Central

Wang, Ying; Yago, Tadayuki; Zhang, Nan; Abdisalaam, Salim; Alexandrakis, George; Rodgers, William; McEver, Rodger P.

2014-01-01

Interactions of CD44 on neutrophils with E-selectin on activated endothelial cells mediate rolling under flow, a prerequisite for neutrophil arrest and migration into perivascular tissues. How CD44 functions as a rolling ligand despite its weak affinity for E-selectin is unknown. We examined the nanometer scale organization of CD44 on intact cells. CD44 on leukocytes and transfected K562 cells was cross-linked within a 1.14-nm spacer. Depolymerizing actin with latrunculin B reduced cross-linking. Fluorescence resonance energy transfer (FRET) revealed tight co-clustering between CD44 fused to yellow fluorescent protein (YFP) and CD44 fused to cyan fluorescent protein on K562 cells. Latrunculin B reduced FRET-reported co-clustering. Number and brightness analysis confirmed actin-dependent CD44-YFP clusters on living cells. CD44 lacking binding sites for ankyrin and for ezrin/radixin/moesin (ERM) proteins on its cytoplasmic domain (ΔANKΔERM) did not cluster. Unexpectedly, CD44 lacking only the ankyrin-binding site (ΔANK) formed larger but looser clusters. Fluorescence recovery after photobleaching demonstrated increased CD44 mobility by latrunculin B treatment or by deleting the cytoplasmic domain. ΔANKΔERM mobility increased only modestly, suggesting that the cytoplasmic domain engages the cytoskeleton by an additional mechanism. Ex vivo differentiated CD44-deficient neutrophils expressing exogenous CD44 rolled on E-selectin and activated Src kinases after binding anti-CD44 antibody. In contrast, differentiated neutrophils expressing ΔANK had impaired rolling and kinase activation. These data demonstrate that spectrin and actin networks regulate CD44 clustering and suggest that ankyrin enhances CD44-mediated neutrophil rolling and signaling. PMID:25359776

Smooth muscle myosin isoform expression and LC20 phosphorylation in innate rat airway hyperresponsiveness.

PubMed

Gil, Fulvio R; Zitouni, Nedjma B; Azoulay, Eric; Maghni, Karim; Lauzon, Anne-Marie

2006-11-01

Four smooth muscle myosin heavy chain (SMMHC) isoforms are generated by alternative mRNA splicing of a single gene. Two of these isoforms differ by the presence [(+)insert] or absence [(-)insert] of a 7-amino acid insert in the motor domain. The rate of actin filament propulsion of the (+)insert SMMHC isoform, as measured in the in vitro motility assay, is twofold greater than that of the (-)insert isoform. We hypothesized that a greater expression of the (+)insert SMMHC isoform and greater regulatory light chain (LC(20)) phosphorylation contribute to airway hyperresponsiveness. We measured airway responsiveness to methacholine in Fischer hyperresponsive and Lewis normoresponsive rats and determined SMMHC isoform mRNA and protein expression, as well as essential light chain (LC(17)) isoforms, h-caldesmon, and alpha-actin protein expression in their tracheae. We also measured tracheal muscle strip contractility in response to methacholine and corresponding LC(20) phosphorylation. We found Fischer rats have more (+)insert mRNA (69.4 +/- 2.0%) (mean +/- SE) than Lewis rats (53.0 +/- 2.4%; P < 0.05) and a 44% greater content of (+)insert isoform relative to total myosin protein. No difference was found for LC(17) isoform, h-caldesmon, and alpha-actin expression. The contractility experiments revealed a greater isometric force for Fischer trachealis segments (4.2 +/- 0.8 mN) than Lewis (1.9 +/- 0.4 mN; P < 0.05) and greater LC(20) phosphorylation level in Fischer (55.1 +/- 6.4) than in Lewis (41.4 +/- 6.1; P < 0.05) rats. These results further support the contention that innate airway hyperresponsiveness is a multifactorial disorder in which increased expression of the fast (+)insert SMMHC isoform and greater activation of LC(20) lead to smooth muscle hypercontractility.

Hyaluronan receptors in the human ocular surface: a descriptive and comparative study of RHAMM and CD44 in tissues, cell lines and freshly collected samples.

PubMed

García-Posadas, Laura; Contreras-Ruiz, Laura; López-García, Antonio; Villarón Álvarez, Sonia; Maldonado, Miguel J; Diebold, Yolanda

2012-02-01

The purpose of this study was to demonstrate the presence of the receptor for hyaluronan-mediated motility (RHAMM) in human conjunctival epithelium and in two widely used cell lines from human corneal (HCE) and conjunctival (IOBA-NHC) epithelia. We compared the distribution of RHAMM proteins and mRNAs in human ocular surface tissues (corneal, limbal and conjunctival), HCE and IOBA-NHC cell lines, and corneal and conjunctival epithelia primary samples from healthy donors with the previously identified hyaluronan receptor CD44. We also aimed to determine if soluble CD44 (sCD44) was present in human tears, as it could have a role in the interaction of the tear fluid with hyaluronan. Protein expression was evaluated by Western blots and immunofluorescence microscopy. mRNA expression was evaluated by RT-PCR and Q-PCR. sCD44 was analyzed by ELISA in culture supernatants and in human tears. We describe the expression of RHAMM in human healthy conjunctiva and in HCE and IOBA-NHC cells at both protein and mRNA levels, and the presence of sCD44 in human tears. Furthermore, we detected CD44 and sCD44 expression variations in in vitro inflammatory conditions. This study also focused on the necessary caution with which the conclusions extracted from cell lines should be made, and in the great value of using primary samples as often as possible.

Virtual screening-driven repositioning of etoposide as CD44 antagonist in breast cancer cells

PubMed Central

Aguirre-Alvarado, Charmina; Segura-Cabrera, Aldo; Velázquez-Quesada, Inés; Hernández-Esquivel, Miguel A.; García-Pérez, Carlos A.; Guerrero-Rodríguez, Sandra L.; Ruiz, Angel J.; Rodríguez-Moreno, Andrea; Pérez-Tapia, Sonia M.; Velasco-Velázquez, Marco A.

2016-01-01

CD44 is a receptor for hyaluronan (HA) that promotes epithelial-to-mesenchymal transition (EMT), induces cancer stem cell (CSC) expansion, and favors metastasis. Thus, CD44 is a target for the development of antineoplastic agents. In order to repurpose drugs as CD44 antagonists, we performed consensus-docking studies using the HA-binding domain of CD44 and 11,421 molecules. Drugs that performed best in docking were examined in molecular dynamics simulations, identifying etoposide as a potential CD44 antagonist. Ligand competition and cell adhesion assays in MDA-MB-231 cells demonstrated that etoposide decreased cell binding to HA as effectively as a blocking antibody. Etoposide-treated MDA-MB-231 cells developed an epithelial morphology; increased their expression of E-cadherin; and reduced their levels of EMT-associated genes and cell migration. By gene expression analysis, etoposide reverted an EMT signature similarly to CD44 knockdown, whereas other topoisomerase II (TOP2) inhibitors did not. Moreover, etoposide decreased the proportion of CD44+/CD24− cells, lowered chemoresistance, and blocked mammosphere formation. Our data indicate that etoposide blocks CD44 activation, impairing key cellular functions that drive malignancy, thus rendering it a candidate for further translational studies and a potential lead compound in the development of new CD44 antagonists. PMID:27009862

Virtual screening-driven repositioning of etoposide as CD44 antagonist in breast cancer cells.

PubMed

Aguirre-Alvarado, Charmina; Segura-Cabrera, Aldo; Velázquez-Quesada, Inés; Hernández-Esquivel, Miguel A; García-Pérez, Carlos A; Guerrero-Rodríguez, Sandra L; Ruiz-Moreno, Angel J; Rodríguez-Moreno, Andrea; Pérez-Tapia, Sonia M; Velasco-Velázquez, Marco A

2016-04-26

CD44 is a receptor for hyaluronan (HA) that promotes epithelial-to-mesenchymal transition (EMT), induces cancer stem cell (CSC) expansion, and favors metastasis. Thus, CD44 is a target for the development of antineoplastic agents. In order to repurpose drugs as CD44 antagonists, we performed consensus-docking studies using the HA-binding domain of CD44 and 11,421 molecules. Drugs that performed best in docking were examined in molecular dynamics simulations, identifying etoposide as a potential CD44 antagonist. Ligand competition and cell adhesion assays in MDA-MB-231 cells demonstrated that etoposide decreased cell binding to HA as effectively as a blocking antibody. Etoposide-treated MDA-MB-231 cells developed an epithelial morphology; increased their expression of E-cadherin; and reduced their levels of EMT-associated genes and cell migration. By gene expression analysis, etoposide reverted an EMT signature similarly to CD44 knockdown, whereas other topoisomerase II (TOP2) inhibitors did not. Moreover, etoposide decreased the proportion of CD44+/CD24- cells, lowered chemoresistance, and blocked mammosphere formation. Our data indicate that etoposide blocks CD44 activation, impairing key cellular functions that drive malignancy, thus rendering it a candidate for further translational studies and a potential lead compound in the development of new CD44 antagonists.

R-spondin1/Wnt-enhanced Ascl2 autoregulation controls the self-renewal of colorectal cancer progenitor cells.

PubMed

Ye, Jun; Liu, Shanxi; Shang, Yangyang; Chen, Haoyuan; Wang, Rongquan

2018-06-25

The Wnt signaling pathway controls stem cell identity in the intestinal epithelium and cancer stem cells (CSCs). The transcription factor Ascl2 (Wnt target gene) is fate decider of intestinal cryptic stem cells and colon cancer stem cells. It is unclear how Wnt signaling is translated into Ascl2 expression and keeping the self-renewal of CRC progenitor cells. We showed that the exogenous Ascl2 in colorectal cancer (CRC) cells activated the endogenous Ascl2 expression via a direct autoactivatory loop, including Ascl2 binding to its own promoter and further transcriptional activation. Higher Ascl2 expression in human CRC cancerous tissues led to greater enrichment in Ascl2 immunoprecipitated DNA within the Ascl2 promoter in the CRC cancerous sample than the peri-cancerous mucosa. Ascl2 binding to its own promoter and inducing further transcriptional activation of the Ascl2 gene was predominant in the CD133 + CD44 + CRC population. R-spondin1/Wnt activated Ascl2 expression dose-dependently in the CD133 + CD44 + CRC population, but not in the CD133 - CD44 - CRC population, which was caused by differences in Ascl2 autoregulation under R-spondin1/Wnt activation. R-spondin1/Wnt treatment in the CD133 + CD44 + or CRC CD133 - CD44 - populations exerted a different pattern of stemness maintenance, which was defined by alterations of the mRNA levels of stemness-associated genes, the protein expression levels (Bmi1, C-myc, Oct-4 and Nanog) and tumorsphere formation. The results indicated that Ascl2 autoregulation formed a transcriptional switch that was enhanced by Wnt signaling in the CD133 + CD44 + CRC population, thus conferring their self-renewal.

A Novel Bcl-x Isoform Connected to the T Cell Receptor Regulates Apoptosis in T Cells

PubMed Central

Yang, Xiao-Feng; Weber, Georg F.

2014-01-01

Summary We define a novel Bcl-x isoform, Bcl-xγ, that is generated by alternative splicing and characterized by a unique 47 amino acid C-terminus. Bcl-xγ is expressed primarily in thymocytes, where it may depend on an interaction between the TCR and host MHC products, and in mature T cells, where its expression is associated with ligation of the T cell receptor. Overexpression of Bcl-xγ in T cells inhibits activation-induced apoptosis; inhibition of Bcl-xγ, after stable expression of Bcl-xγ antisense cDNA, enhances activation-induced apoptosis. In contrast to other Bcl-x isoforms, cells that fail to express Bcl-xγ after CD3 ligation undergo programmed cell death, while activated T cells that express Bcl-xγ are spared. Identification of Bcl-xγ helps provide amolecular explanation of T cell activation and death after antigen engagement. PMID:9390687

Evaluation of CD44 and CD133 as markers of liver cancer stem cells in Egyptian patients with HCV-induced chronic liver diseases versus hepatocellular carcinoma

PubMed Central

Rozeik, Mohammed Saeed; Hammam, Olfat Ali; Ali, Ali Ibrahim; Magdy, Mona; Khalil, Heba; Anas, Amgad; Abo el Hassan, Ahmed Abdelaleem; Rahim, Ali Abdel; El-Shabasy, Ahmed Ibrahim

2017-01-01

Background Cancer stem cells (CSCs) play a critical role in tumor development, progression, metastasis and recurrence. Aim To evaluate hepatic expression of CD44 and CD133 in Egyptian patients with HCV-induced chronic liver diseases and hepatocellular carcinomas (HCCs), and to assess its correlation with inflammatory activity scores, stages of fibrosis (in chronic hepatitis with or without cirrhosis) and grades of HCC. Methods This prospective case-control study was conducted on eighty subjects who attended the Tropical Diseases Department, Al-Azhar University Hospital, and in collaboration with Theodor Bilharz Research Institute (2014–2016). They were divided as follows: A) Control healthy group: Ten individuals with serologically negative HCV-Ab and HBsAg, and histopathologically normal liver, B) Seventy patients subdivided into 3 groups; Twenty subjects each, as: HCV-Ab+ non-cirrhotic, HCV-Ab+ cirrhotic and HCC. Necroinflammatory activity and fibrosis in non-neoplastic liver biopsies were scored according to the METAVIR scoring system. CD44 and CD133 immunostaining was evaluated in all groups semi-quantitatively using H score. Statistical analysis was performed by SPSS version 22, using independent-samples t-test. Results Our study showed a significant increase of mean CD44 & CD133 expression values with disease progression among the groups (p<0.05). Their expressions increased significantly with the inflammatory activity scores and stages of fibrosis, reaching the highest values in A3F4 score compared to A1F1 (p<0.05). Moreover, there was a significant increase of their expressions across HCC grades (p<0.05), however with no significant correlation with focal lesions size. Conclusion CSCs clusters exhibiting CD133+ and/or CD44+ profiles were identified in chronic hepatitis, liver cirrhosis and HCC. CD133 and CD44 expressions significantly corresponded to the increased inflammatory activity, fibrosis stages and higher tumor grades. Therefore, evaluation of CD44 and CD133 expression profiles as CSCs markers in non-neoplastic liver and HCCs can help in development of novel therapeutic agents for HCC targeting and prevention. PMID:28894525

CD147 promotes the formation of functional osteoclasts through NFATc1 signalling.

PubMed

Nishioku, Tsuyoshi; Terasawa, Mariko; Baba, Misaki; Yamauchi, Atsushi; Kataoka, Yasufumi

2016-04-29

CD147, a membrane glycoprotein of the immunoglobulin superfamily, is highly upregulated during dynamic cellular events including tissue remodelling. Elevated CD147 expression is present in the joint of rheumatoid arthritis patients. However, the role of CD147 in bone destruction remains unclear. To determine whether CD147 is involved in osteoclastogenesis, we studied its expression in mouse osteoclasts and its role in osteoclast differentiation and function. CD147 expression was markedly upregulated during osteoclast differentiation. To investigate the role of CD147 in receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and bone resorption activity, osteoclast precursor cells were transfected with CD147 siRNA. Decreased CD147 expression inhibited osteoclast formation and bone resorption, inhibited RANKL-induced nuclear translocation of the nuclear factor of activated T cells (NFAT) c1 and decreased the expression of the d2 isoform of vacuolar ATPase Vo domain and cathepsin K. Therefore, CD147 plays a critical role in the differentiation and function of osteoclasts by upregulating NFATc1 through the autoamplification of its expression in osteoclastogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

Triple negative breast cancer initiating cell subsets differ in functional and molecular characteristics and in γ-secretase inhibitor drug responses

PubMed Central

Azzam, Diana J; Zhao, Dekuang; Sun, Jun; Minn, Andy J; Ranganathan, Prathibha; Drews-Elger, Katherine; Han, Xiaoqing; Picon-Ruiz, Manuel; Gilbert, Candace A; Wander, Seth A; Capobianco, Anthony J; El-Ashry, Dorraya; Slingerland, Joyce M

2013-01-01

Increasing evidence suggests that stem-like cells mediate cancer therapy resistance and metastasis. Breast tumour-initiating stem cells (T-ISC) are known to be enriched in CD44+CD24neg/low cells. Here, we identify two T-ISC subsets within this population in triple negative breast cancer (TNBC) lines and dissociated primary breast cancer cultures: CD44+CD24low+ subpopulation generates CD44+CD24neg progeny with reduced sphere formation and tumourigenicity. CD44+CD24low+ populations contain subsets of ALDH1+ and ESA+ cells, yield more frequent spheres and/or T-ISC in limiting dilution assays, preferentially express metastatic gene signatures and show greater motility, invasion and, in the MDA-MB-231 model, metastatic potential. CD44+CD24low+ but not CD44+CD24neg express activated Notch1 intracellular domain (N1-ICD) and Notch target genes. We show N1-ICD transactivates SOX2 to increase sphere formation, ALDH1+ and CD44+CD24low+cells. Gamma secretase inhibitors (GSI) reduced sphere formation and xenograft growth from CD44+CD24low+ cells, but CD44+CD24neg were resistant. While GSI hold promise for targeting T-ISC, stem cell heterogeneity as observed herein, could limit GSI efficacy. These data suggest a breast T-ISC hierarchy in which distinct pathways drive developmentally related subpopulations with different anti-cancer drug responsiveness. PMID:23982961

Differential Gene Expression Profiling of Functionally and Developmentally Distinct Human Prostate Epithelial Populations

PubMed Central

Liu, Haibo; Cadaneanu, Radu M; Lai, Kevin; Zhang, Baohui; Huo, Lihong; An, Dong Sun; Li, Xinmin; Lewis, Michael S; Garraway, Isla P

2015-01-01

BACKGROUND Human fetal prostate buds appear in the 10th gestational week as solid cords, which branch and form lumens in response to androgen 1. Previous in vivo analysis of prostate epithelia isolated from benign prostatectomy specimens indicated that Epcam+CD44−CD49fHi basal cells possess efficient tubule initiation capability relative to other subpopulations 2. Stromal interactions and branching morphogenesis displayed by adult tubule-initiating cells (TIC) are reminiscent of fetal prostate development. In the current study, we evaluated in vivo tubule initiation by human fetal prostate cells and determined expression profiles of fetal and adult epithelial subpopulations in an effort to identify pathways used by TIC. METHODS Immunostaining and FACS analysis based on Epcam, CD44, and CD49f expression demonstrated the majority (99.9%) of fetal prostate epithelial cells (FC) were Epcam+CD44− with variable levels of CD49f expression. Fetal populations isolated via cell sorting were implanted into immunocompromised mice. Total RNA isolation from Epcam+CD44−CD49fHi FC, adult Epcam+CD44−CD49fHi TIC, Epcam+CD44+CD49fHi basal cells (BC), and Epcam+CD44−CD49fLo luminal cells (LC) was performed, followed by microarray analysis of 19 samples using the Affymetrix Gene Chip Human U133 Plus 2.0 Array. Data was analyzed using Partek Genomics Suite Version 6.4. Genes selected showed >2-fold difference in expression and P < 5.00E-2. Results were validated with RT-PCR. RESULTS Grafts retrieved from Epcam+CD44− fetal cell implants displayed tubule formation with differentiation into basal and luminal compartments, while only stromal outgrowths were recovered from Epcam- fetal cell implants. Hierarchical clustering revealed four distinct groups determined by antigenic profile (TIC, BC, LC) and developmental stage (FC). TIC and BC displayed basal gene expression profiles, while LC expressed secretory genes. FC had a unique profile with the most similarities to adult TIC. Functional, network, and canonical pathway identification using Ingenuity Pathway Analysis Version 7.6 compiled genes with the highest differential expression (TIC relative to BC or LC). Many of these genes were found to be significantly associated with prostate tumorigenesis. CONCLUSIONS Our results demonstrate clustering gene expression profiles of FC and adult TIC. Pathways associated with TIC are known to be deregulated in cancer, suggesting a cell-of-origin role for TIC versus re-emergence of pathways common to these cells in tumorigenesis. Prostate 75: 764–776, 2015. © The Authors. The Prostate, published by Wiley Periodicals, Inc. PMID:25663004

Differential gene expression profiling of functionally and developmentally distinct human prostate epithelial populations.

PubMed

Liu, Haibo; Cadaneanu, Radu M; Lai, Kevin; Zhang, Baohui; Huo, Lihong; An, Dong Sun; Li, Xinmin; Lewis, Michael S; Garraway, Isla P

2015-05-01

Human fetal prostate buds appear in the 10th gestational week as solid cords, which branch and form lumens in response to androgen 1. Previous in vivo analysis of prostate epithelia isolated from benign prostatectomy specimens indicated that Epcam⁺ CD44⁻ CD49f(Hi) basal cells possess efficient tubule initiation capability relative to other subpopulations 2. Stromal interactions and branching morphogenesis displayed by adult tubule-initiating cells (TIC) are reminiscent of fetal prostate development. In the current study, we evaluated in vivo tubule initiation by human fetal prostate cells and determined expression profiles of fetal and adult epithelial subpopulations in an effort to identify pathways used by TIC. Immunostaining and FACS analysis based on Epcam, CD44, and CD49f expression demonstrated the majority (99.9%) of fetal prostate epithelial cells (FC) were Epcam⁺ CD44⁻ with variable levels of CD49f expression. Fetal populations isolated via cell sorting were implanted into immunocompromised mice. Total RNA isolation from Epcam⁺ CD44⁻ CD49f(Hi) FC, adult Epcam⁺ CD44⁻ CD49f(Hi) TIC, Epcam⁺ CD44⁺ CD49f(Hi) basal cells (BC), and Epcam⁺ CD44⁻ CD49f(Lo) luminal cells (LC) was performed, followed by microarray analysis of 19 samples using the Affymetrix Gene Chip Human U133 Plus 2.0 Array. Data was analyzed using Partek Genomics Suite Version 6.4. Genes selected showed >2-fold difference in expression and P < 5.00E-2. Results were validated with RT-PCR. Grafts retrieved from Epcam⁺ CD44⁻ fetal cell implants displayed tubule formation with differentiation into basal and luminal compartments, while only stromal outgrowths were recovered from Epcam- fetal cell implants. Hierarchical clustering revealed four distinct groups determined by antigenic profile (TIC, BC, LC) and developmental stage (FC). TIC and BC displayed basal gene expression profiles, while LC expressed secretory genes. FC had a unique profile with the most similarities to adult TIC. Functional, network, and canonical pathway identification using Ingenuity Pathway Analysis Version 7.6 compiled genes with the highest differential expression (TIC relative to BC or LC). Many of these genes were found to be significantly associated with prostate tumorigenesis. Our results demonstrate clustering gene expression profiles of FC and adult TIC. Pathways associated with TIC are known to be deregulated in cancer, suggesting a cell-of-origin role for TIC versus re-emergence of pathways common to these cells in tumorigenesis. © 2015 The Authors. The Prostate, published by Wiley Periodicals, Inc.

CD44 mediated hyaluronan adhesion of Toxoplasma gondii-infected leukocytes.

PubMed

Hayashi, Takeshi; Unno, Akihiro; Baba, Minami; Ohno, Tamio; Kitoh, Katsuya; Takashima, Yasuhiro

2014-04-01

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects humans and animals. Ingested parasites cross the intestinal epithelium, invade leukocytes and are then disseminated to peripheral organs. However, the mechanism of extravasation of the infected leukocytes remains poorly understood. In this study, we demonstrate that T. gondii-invaded human and mouse leukocytes express higher level of CD44, a ligand of hyaluronan (HA), and its expression on myeloid and non-myeloid leukocytes causes T. gondii-invaded human and mouse leukocyte to adhere to HA more effectively than non-invaded leukocytes. The specific adherence of parasite-invaded leukocytes was inhibited by anti CD44 antibody. Leukocytes of CD44 knockout mice did not show parasite-invaded leukocyte specific adhesion. Our results indicate that parasite-invaded leukocytes, regardless of whether myeloid or not, gain higher ability to adhere to HA than non-invaded leukocytes, via upregulation of CD44 expression and/or selective invasion to CD44 highly expressing cells. The difference in ability to adhere to HA between parasite-invaded cells and non-invaded neighboring cells might facilitate effective delivery of parasite-invaded leukocytes to the HA-producing endothelial cell surface and/or HA-rich extra cellular matrix. © 2013.

CD147 promotes the formation of functional osteoclasts through NFATc1 signalling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishioku, Tsuyoshi, E-mail: nishiokut@niu.ac.jp; Department of Pharmaceutical Care and Health Sciences, Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180; Terasawa, Mariko

CD147, a membrane glycoprotein of the immunoglobulin superfamily, is highly upregulated during dynamic cellular events including tissue remodelling. Elevated CD147 expression is present in the joint of rheumatoid arthritis patients. However, the role of CD147 in bone destruction remains unclear. To determine whether CD147 is involved in osteoclastogenesis, we studied its expression in mouse osteoclasts and its role in osteoclast differentiation and function. CD147 expression was markedly upregulated during osteoclast differentiation. To investigate the role of CD147 in receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and bone resorption activity, osteoclast precursor cells were transfected with CD147 siRNA. Decreasedmore » CD147 expression inhibited osteoclast formation and bone resorption, inhibited RANKL-induced nuclear translocation of the nuclear factor of activated T cells (NFAT) c1 and decreased the expression of the d2 isoform of vacuolar ATPase Vo domain and cathepsin K. Therefore, CD147 plays a critical role in the differentiation and function of osteoclasts by upregulating NFATc1 through the autoamplification of its expression in osteoclastogenesis. - Highlights: • CD147 expression was markedly upregulated during osteoclast differentiation. • Downregulation of CD147 expression inhibited osteoclastgenesis and bone resorption. • Decreased CD147 expression inhibited RANKL-induced nuclear translocation of NFATc1.« less

Challenging the roles of CD44 and lipolysis stimulated lipoprotein receptor in conveying Clostridium perfringens iota toxin cytotoxicity in breast cancer

PubMed Central

2014-01-01

Background Translational exploration of bacterial toxins has come to the forefront of research given their potential as a chemotherapeutic tool. Studies in select tissues have demonstrated that Clostridium perfringens iota toxin binds to CD44 and lipolysis stimulated lipoprotein receptor (LSR) cell-surface proteins. We recently demonstrated that LSR expression correlates with estrogen receptor positive breast cancers and that LSR signaling directs aggressive, tumor-initiating cell behaviors. Herein, we identify the mechanisms of iota toxin cytotoxicity in a tissue-specific, breast cancer model with the ultimate goal of laying the foundation for using iota toxin as a targeted breast cancer therapy. Methods In vitro model systems were used to determine the cytotoxic effect of iota toxin on breast cancer intrinsic subtypes. The use of overexpression and knockdown technologies confirmed the roles of LSR and CD44 in regulating iota toxin endocytosis and induction of cell death. Lastly, cytotoxicity assays were used to demonstrate the effect of iota toxin on a validated set of tamoxifen resistant breast cancer cell lines. Results Treatment of 14 breast cancer cell lines revealed that LSR+/CD44- lines were highly sensitive, LSR+/CD44+ lines were slightly sensitive, and LSR-/CD44+ lines were resistant to iota cytotoxicity. Reduction in LSR expression resulted in a significant decrease in toxin sensitivity; however, overexpression of CD44 conveyed toxin resistance. CD44 overexpression was correlated with decreased toxin-stimulated lysosome formation and decreased cytosolic levels of iota toxin. These findings indicated that expression of CD44 drives iota toxin resistance through inhibition of endocytosis in breast cancer cells, a role not previously defined for CD44. Moreover, tamoxifen-resistant breast cancer cells exhibited robust expression of LSR and were highly sensitive to iota-induced cytotoxicity. Conclusions Collectively, these data are the first to show that iota toxin has the potential to be an effective, targeted therapy for breast cancer. PMID:24990559

Challenging the roles of CD44 and lipolysis stimulated lipoprotein receptor in conveying Clostridium perfringens iota toxin cytotoxicity in breast cancer.

PubMed

Fagan-Solis, Katerina D; Reaves, Denise K; Rangel, M Cristina; Popoff, Michel R; Stiles, Bradley G; Fleming, Jodie M

2014-07-02

Translational exploration of bacterial toxins has come to the forefront of research given their potential as a chemotherapeutic tool. Studies in select tissues have demonstrated that Clostridium perfringens iota toxin binds to CD44 and lipolysis stimulated lipoprotein receptor (LSR) cell-surface proteins. We recently demonstrated that LSR expression correlates with estrogen receptor positive breast cancers and that LSR signaling directs aggressive, tumor-initiating cell behaviors. Herein, we identify the mechanisms of iota toxin cytotoxicity in a tissue-specific, breast cancer model with the ultimate goal of laying the foundation for using iota toxin as a targeted breast cancer therapy. In vitro model systems were used to determine the cytotoxic effect of iota toxin on breast cancer intrinsic subtypes. The use of overexpression and knockdown technologies confirmed the roles of LSR and CD44 in regulating iota toxin endocytosis and induction of cell death. Lastly, cytotoxicity assays were used to demonstrate the effect of iota toxin on a validated set of tamoxifen resistant breast cancer cell lines. Treatment of 14 breast cancer cell lines revealed that LSR+/CD44- lines were highly sensitive, LSR+/CD44+ lines were slightly sensitive, and LSR-/CD44+ lines were resistant to iota cytotoxicity. Reduction in LSR expression resulted in a significant decrease in toxin sensitivity; however, overexpression of CD44 conveyed toxin resistance. CD44 overexpression was correlated with decreased toxin-stimulated lysosome formation and decreased cytosolic levels of iota toxin. These findings indicated that expression of CD44 drives iota toxin resistance through inhibition of endocytosis in breast cancer cells, a role not previously defined for CD44. Moreover, tamoxifen-resistant breast cancer cells exhibited robust expression of LSR and were highly sensitive to iota-induced cytotoxicity. Collectively, these data are the first to show that iota toxin has the potential to be an effective, targeted therapy for breast cancer.

Prognostic relevance of miR-137 in patients with hepatocellular carcinoma.

PubMed

Sakabe, Tomohiko; Azumi, Junya; Umekita, Yoshihisa; Toriguchi, Kan; Hatano, Etsuro; Hirooka, Yasuaki; Shiota, Goshi

2017-02-01

Cancer stem cells (CSCs) play a pivotal role in progression, metastasis and recurrence of cancer. Therefore, it is clinically useful to identify the relevant CSC marker that is associated with prognosis of hepatocellular carcinoma (HCC) and clarify its genetic and biological characteristics. Expression of four CSC markers, CD13, EpCAM, CD44 and CD44v9, was examined in 99 HCC patients. Biological and cDNA/miRNA microarray data were compared among CD44-positive/-negative HCC cells and normal hepatic cells. The significance of the representative miRNAs was examined with regard to prognosis of additional 110 HCC patients. CD44-positive HuH7 cells proliferated faster and showed a greater sphere forming ability than CD44-negative HuH7 cells. CD44-positive HuH7 cells exhibited higher expression of specific genes involved in resistance to reactive oxygen species, anticancer drugs and tumour invasion than CD44-negative HCC cells. Higher expression of six miRNAs was observed in CD44-positive HuH7 cells, CD44-negative HuH7 cells, and human normal hepatic cells in that order. Of the six miRNAs, miR-137 was closely associated with overall and cancer-specific survivals, as well as with invasion of hepatic vein, hepatic artery, portal vein and bile duct, and alpha-foetoprotein in additional 110 HCC patients. miR-137 may serve as a prognostic marker in patients with HCC and may be a potential target for the elimination of liver CSCs. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

CD47 is an adverse prognostic factor and a therapeutic target in gastric cancer

PubMed Central

Yoshida, Kazumichi; Tsujimoto, Hironori; Matsumura, Kouji; Kinoshita, Manabu; Takahata, Risa; Matsumoto, Yusuke; Hiraki, Shuichi; Ono, Satoshi; Seki, Shuhji; Yamamoto, Junji; Hase, Kazuo

2015-01-01

CD47 is an antiphagocytic molecule that acts via ligation to signal regulatory protein alpha on phagocytes; its enhanced expression and therapeutic targeting have recently been reported for several malignancies. However, CD47 expression in gastric cancer is not well documented. Immunohistochemical expression of CD47 in surgical specimens was investigated. Expression of CD47 and CD44, a known gastric cancer stem cell marker, were investigated in gastric cancer cell lines by flow cytometry. MKN45 and MKN74 gastric cancer cells were sorted by fluorescence-activated cell sorting according to CD44 and CD47 expression levels, and their in vitro proliferation, spheroid-forming capacity, and in vivo tumorigenicity were studied. In vitro phagocytosis of cancer cells by human macrophages in the presence of a CD47 blocking monoclonal antibody (B6H12) and the survival of immunodeficient mice intraperitoneally engrafted with MKN45 cells and B6H12 were compared to experiments using control antibodies. Immunohistochemistry of the clinical specimens indicated that CD47 was positive in 57 out of 115 cases, and its positivity was an independent adverse prognostic factor. Approximately 90% of the MKN45 and MKN74 cells expressed CD47 and CD44. CD47hi gastric cancer cells showed significantly higher proliferation and spheroid colony formation than CD47lo, and CD44hiCD47hi cells showed the highest proliferation in vitro and tumorigenicity in vivo. B6H12 significantly enhanced in vitro phagocytosis of cancer cells by human macrophages and prolonged the survival of intraperitoneal cancer dissemination in mice compared to control antibodies. In conclusion, CD47 is an adverse prognostic factor and promising therapeutic target in gastric cancer. PMID:26077800

CD45RA and CD45RO isoforms in infected malnourished and infected well-nourished children

PubMed Central

Nájera, O; González, C; Toledo, G; López, L; Cortés, E; Betancourt, M; Ortiz, R

2001-01-01

The aim of this study was to determine if the distribution in vivo of CD4+CD45RA+/CD45RO− (naive), CD4+CD45RA+/CD45RO+ (Ddull) and CD4+CD45RO+ (memory) lymphocytes differs in malnourished infected and well-nourished infected children. The expression of CD45RA (naive) and CD45RO (memory) antigens on CD4+ lymphocytes was analysed by flow cytometry in a prospectively followed cohort of 15 malnourished infected, 12 well-nourished infected and 10 well-nourished uninfected children. Malnourished infected children showed higher fractions of Ddull cells (11·4 ± 0·7%) and lower fractions of memory cells (20·3 ± 1·7%) than the well-nourished infected group (8·8 ± 0·8 and 28·1 ± 1·8%, respectively). Well-nourished infected children showed increased percentages of memory cells, an expected response to infection. Impairment of the transition switch to the CD45 isoforms in malnourished children may explain these findings, and may be one of the mechanisms involved in immunodeficiency in these children. PMID:11737063

Adoptive cell therapy for lymphoma with CD4 T cells depleted of CD137-expressing regulatory T cells.

PubMed

Goldstein, Matthew J; Kohrt, Holbrook E; Houot, Roch; Varghese, Bindu; Lin, Jack T; Swanson, Erica; Levy, Ronald

2012-03-01

Adoptive immunotherapy with antitumor T cells is a promising novel approach for the treatment of cancer. However, T-cell therapy may be limited by the cotransfer of regulatory T cells (T(reg)). Here, we explored this hypothesis by using 2 cell surface markers, CD44 and CD137, to isolate antitumor CD4 T cells while excluding T(regs). In a murine model of B-cell lymphoma, only CD137(neg)CD44(hi) CD4 T cells infiltrated tumor sites and provided protection. Conversely, the population of CD137(pos)CD44hi CD4 T cells consisted primarily of activated T(regs). Notably, this CD137(pos) T(reg) population persisted following adoptive transfer and maintained expression of FoxP3 as well as CD137. Moreover, in vitro these CD137(pos) cells suppressed the proliferation of effector cells in a contact-dependent manner, and in vivo adding the CD137(pos)CD44(hi) CD4 cells to CD137(neg)CD44(hi) CD4 cells suppressed the antitumor immune response. Thus, CD137 expression on CD4 T cells defined a population of activated T(regs) that greatly limited antitumor immune responses. Consistent with observations in the murine model, human lymphoma biopsies also contained a population of CD137(pos) CD4 T cells that were predominantly CD25(pos)FoxP3(pos) T(regs). In conclusion, our findings identify 2 surface markers that can be used to facilitate the enrichment of antitumor CD4 T cells while depleting an inhibitory T(reg) population.

The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44.

PubMed

Liu, Can; Kelnar, Kevin; Liu, Bigang; Chen, Xin; Calhoun-Davis, Tammy; Li, Hangwen; Patrawala, Lubna; Yan, Hong; Jeter, Collene; Honorio, Sofia; Wiggins, Jason F; Bader, Andreas G; Fagin, Randy; Brown, David; Tang, Dean G

2011-02-01

Cancer stem cells (CSCs), or tumor-initiating cells, are involved in tumor progression and metastasis. MicroRNAs (miRNAs) regulate both normal stem cells and CSCs, and dysregulation of miRNAs has been implicated in tumorigenesis. CSCs in many tumors--including cancers of the breast, pancreas, head and neck, colon, small intestine, liver, stomach, bladder and ovary--have been identified using the adhesion molecule CD44, either individually or in combination with other marker(s). Prostate CSCs with enhanced clonogenic and tumor-initiating and metastatic capacities are enriched in the CD44(+) cell population, but whether miRNAs regulate CD44(+) prostate cancer cells and prostate cancer metastasis remains unclear. Here we show, through expression analysis, that miR-34a, a p53 target, was underexpressed in CD44(+) prostate cancer cells purified from xenograft and primary tumors. Enforced expression of miR-34a in bulk or purified CD44(+) prostate cancer cells inhibited clonogenic expansion, tumor regeneration, and metastasis. In contrast, expression of miR-34a antagomirs in CD44(-) prostate cancer cells promoted tumor development and metastasis. Systemically delivered miR-34a inhibited prostate cancer metastasis and extended survival of tumor-bearing mice. We identified and validated CD44 as a direct and functional target of miR-34a and found that CD44 knockdown phenocopied miR-34a overexpression in inhibiting prostate cancer regeneration and metastasis. Our study shows that miR-34a is a key negative regulator of CD44(+) prostate cancer cells and establishes a strong rationale for developing miR-34a as a novel therapeutic agent against prostate CSCs.

sCD44 overexpression increases intraocular pressure and aqueous outflow resistance

PubMed Central

Giovingo, Michael; Nolan, Michael; McCarty, Ryan; Pang, Iok-Hou; Clark, Abbot F.; Beverley, Rachel M.; Schwartz, Steven; Stamer, W. Daniel; Walker, Loyal; Grybauskas, Algis; Skuran, Kevin; Kuprys, Paulius V.; Yue, Beatrice Y.J.T.

2013-01-01

Purpose CD44 plays major roles in multiple physiologic processes. The ectodomain concentration of the CD44 receptor, soluble CD44 (sCD44), is significantly increased in the aqueous humor of primary open-angle glaucoma (POAG). The purpose of this study was to determine if adenoviral constructs of CD44 and isolated 32-kDa sCD44 change intraocular pressure (IOP) in vivo and aqueous outflow resistance in vitro. Methods Adenoviral constructs of human standard CD44 (Ad-CD44S), soluble CD44 (Ad-sCD44), and empty viral cDNA were injected into the vitreous of BALB/cJ mice, followed by serial IOP measurements. Overexpression of CD44S and sCD44 was verified in vitro by enzyme-linked immunosorbent assay (ELISA) and western blot analysis. Anterior segments of porcine eyes were perfused with the isolated sCD44. sCD44-treated human trabecular meshwork (TM) cells and microdissected porcine TM were examined by confocal microscopy and Optiprep density gradient with western blot analysis to determine changes in lipid raft components. Results Intravitreous injection of adenoviral constructs with either Ad-CD44S or Ad-sCD44 vectors caused prolonged ocular hypertension in mice. Eight days after vector injection, Ad-CD44S significantly elevated IOP to 28.3±1.2 mmHg (mean±SEM, n=8; p<0.001); Ad-sCD44 increased IOP to 18.5±2.6 mmHg (n=8; p<0.01), whereas the IOP of uninjected eyes was 12.7±0.2 mmHg (n=16). The IOP elevation lasted more than 50 days. Topical administration of a γ-secretase inhibitor normalized Ad-sCD44-induced elevated IOP. sCD44 levels were significantly elevated in the aqueous humor of Ad-CD44S and Ad-sCD44 eyes versus contralateral uninjected eyes (p<0.01). Anterior segment perfusion of isolated 32-kDa sCD44 significantly decreased aqueous outflow rates. Co-administration of isolated sCD44 and CD44 neutralizing antibody or of γ-secretase inhibitor significantly enhanced flow rates. sCD44-treated human TM cells displayed cross-linked actin network formation. Optiprep density gradient and western blot analysis of human TM cells treated with sCD44 showed decreased annexin 2 expression and increased phosphorylated annexin 2 and caveolin 1 expression. Conclusions Our data suggest that sCD44 increases outflow resistance in vivo and in vitro. Viral overexpression of both CD44S and sCD44 is sufficient to cause ocular hypertension. Infusion of sCD44 in porcine anterior segment eyes significantly decreased flow rates. Notably, sCD44 enhanced cross-linked actin network formation. The elevated sCD44 levels seen in POAG aqueous humor may play an important causative role in POAG pathogenesis. PMID:24194636

Dose-dependent modulation of CD8 and functional avidity as a result of peptide encounter

PubMed Central

Kroger, Charles J; Alexander-Miller, Martha A

2007-01-01

The generation of an optimal CD8+ cytotoxic T lymphocyte (CTL) response is critical for the clearance of many intracellular pathogens. Previous studies suggest that one contributor to an optimal immune response is the presence of CD8+ cells exhibiting high functional avidity. In this regard, CD8 expression has been shown to contribute to peptide sensitivity. Here, we investigated the ability of naive splenocytes to modulate CD8 expression according to the concentration of stimulatory peptide antigen. Our results showed that the level of CD8 expressed was inversely correlated with the amount of peptide used for the primary stimulation, with higher concentrations of antigen resulting in lower expression of both CD8α and CD8β. Importantly the ensuing CD8low and CD8high CTL populations were not the result of the selective outgrowth of naive CD8+ T-cell subpopulations expressing distinct levels of CD8. Subsequent encounter with peptide antigen resulted in continued modulation of both the absolute level and the isoform of CD8 expressed and in the functional avidity of the responding cells. We propose that CD8 cell surface expression is not a static property, but can be modulated to ‘fine tune’ the sensitivity of responding CTL to a defined concentration of antigen. PMID:17484768

Monoamine Oxidase Deficiency Causes Prostate Atrophy and Reduces Prostate Progenitor Cell Activity.

PubMed

Yin, Lijuan; Li, Jingjing; Liao, Chun-Peng; Jason Wu, Boyang

2018-04-10

Monoamine oxidases (MAOs) degrade a number of biogenic and dietary amines, including monoamine neurotransmitters, and play an essential role in many biological processes. Neurotransmitters and related neural events have been shown to participate in the development, differentiation, and maintenance of diverse tissues and organs by regulating the specialized cellular function and morphological structures of innervated organs such as the prostate. Here we show that mice lacking both MAO isoforms, MAOA and MAOB, exhibit smaller prostate mass and develop epithelial atrophy in the ventral and dorsolateral prostates. The cellular composition of prostate epithelium showed reduced CK5 + or p63 + basal cells, accompanied by lower Sca-1 expression in p63 + basal cells, but intact differentiated CK8 + luminal cells in MAOA/B-deficient mouse prostates. MAOA/B ablation also decreased epithelial cell proliferation without affecting cell apoptosis in mouse prostates. Using a human prostate epithelial cell line, we found that stable knockdown of MAOA and MAOB impaired the capacity of prostate stem cells to form spheres, coinciding with a reduced CD133 + /CD44 + /CD24 - stem cell population and less expression of CK5 and select stem cell markers, including ALDH1A1, TROP2, and CD166. Alternative pharmacological inhibition of MAOs also repressed prostate cell stemness. In addition, we found elevated expression of MAOA and MAOB in epithelial and/or stromal components of human prostate hyperplasia samples compared with normal prostate tissues. Taken together, our findings reveal critical roles for MAOs in the regulation of prostate basal progenitor cells and prostate maintenance. Stem Cells 2018. © AlphaMed Press 2018.

The High and Low Molecular Weight Forms of Hyaluronan Have Distinct Effects on CD44 Clustering*

PubMed Central

Yang, Cuixia; Cao, Manlin; Liu, Hua; He, Yiqing; Xu, Jing; Du, Yan; Liu, Yiwen; Wang, Wenjuan; Cui, Lian; Hu, Jiajie; Gao, Feng

2012-01-01

CD44 is a major cell surface receptor for the glycosaminoglycan hyaluronan (HA). Native high molecular weight hyaluronan (nHA) and oligosaccharides of hyaluronan (oHA) provoke distinct biological effects upon binding to CD44. Despite the importance of such interactions, however, the feature of binding with CD44 at the cell surface and the molecular basis for functional distinction between different sizes of HA is still unclear. In this study we investigated the effects of high and low molecular weight hyaluronan on CD44 clustering. For the first time, we provided direct evidence for a strong relationship between HA size and CD44 clustering in vivo. In CD44-transfected COS-7 cells, we showed that exogenous nHA stimulated CD44 clustering, which was disrupted by oHA. Moreover, naturally expressed CD44 was distributed into clusters due to abundantly expressed nHA in HK-2 cells (human renal proximal tubule cells) and BT549 cells (human breast cancer cell line) without exogenous stimulation. Our results suggest that native HA binding to CD44 selectively induces CD44 clustering, which could be inhibited by oHA. Finally, we demonstrated that HA regulates cell adhesion in a manner specifically dependent on its size. oHA promoted cell adhesion while nHA showed no effects. Our results might elucidate a molecular- and/or cellular-based mechanism for the diverse biological activities of nHA and oHA. PMID:23118219

STAT3 as a potential therapeutic target in ALDH+ and CD44+/CD24+ stem cell-like pancreatic cancer cells.

PubMed

Lin, Li; Jou, David; Wang, Yina; Ma, Haiyan; Liu, Tianshu; Fuchs, James; Li, Pui-Kai; Lü, Jiagao; Li, Chenglong; Lin, Jiayuh

2016-12-01

Persistent activation of signal transducers and activators of transcription 3 (STAT3) is commonly detected in many types of cancer including pancreatic cancer. Whether STAT3 is activated in stem cell-like pancreatic cancer cells and the effect of STAT3 inhibition, is still unknown. Flow cytometry was used to isolate pancreatic cancer stem-like cells which are identified by both aldehyde dehydrogenase (ALDH)-positive (ALDH+) as well as cluster of differentiation (CD) 44-positive/CD24-positive subpopulations (CD44+/CD24+). STAT3 activation and the effects of STAT3 inhibition by STAT3 inhibitors, LLL12, FLLL32, and Stattic in ALDH+ and CD44+/CD24+ cells were examined. Our results showed that ALDH+ and CD44+/CD24+ pancreatic cancer stem-like cells expressed higher levels of phosphorylated STAT3, an active form of STAT3, compared to ALDH-negative (ALDH-) and CD44-negative/CD24-negative (CD44-/CD24-) pancreatic cancer cells, suggesting that STAT3 is activated in pancreatic cancer stem-like cells. Small molecular STAT3 inhibitors inhibited STAT3 phosphorylation, STAT3 downstream target gene expression, cell viability, and tumorsphere formation in ALDH+ and CD44+/CD24+ cells. Our results indicate that STAT3 is a novel therapeutic target in pancreatic cancer stem-like cells and inhibition of activated STAT3 in these cells by STAT3 inhibitors may offer an effective treatment for pancreatic cancer.

Kaempferol regulates OPN-CD44 pathway to inhibit the atherogenesis of apolipoprotein E deficient mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiao, Hong-Bo, E-mail: xhbzhb@yahoo.com; Lu, Xiang-Yang; Sun, Zhi-Liang

Recent studies show that osteopontin (OPN) and its receptor cluster of differentiation 44 (CD44) are two pro-inflammatory cytokines contributing to the development of atherosclerosis. The objective of this study was to explore the inhibitory effect of kaempferol, a naturally occurring flavonoid compound, on atherogenesis and the mechanisms involved. The experiments were performed in aorta and plasma from C57BL/6J control and apolipoprotein E-deficient (ApoE{sup -/-}) mice treated or not with kaempferol (50 or 100 mg/kg, intragastrically) for 4 weeks. Kaempferol treatment decreased atherosclerotic lesion area, improved endothelium-dependent vasorelaxation, and increased the maximal relaxation value concomitantly with decrease in the half-maximum effectivemore » concentration, plasma OPN level, aortic OPN expression, and aortic CD44 expression in ApoE{sup -/-} mice. In addition, treatment with kaempferol also significantly decreased reactive oxygen species production in mice aorta. The present results suggest that kaempferol regulates OPN-CD44 pathway to inhibit the atherogenesis of ApoE{sup -/-} mice. -- Graphical abstract: Kaempferol regulates OPN-CD44 pathway to inhibit the atherogenesis of ApoE{sup -/-} mice. Highlights: Black-Right-Pointing-Pointer OPN-CD44 pathway plays a critical role in the development of atherosclerosis. Black-Right-Pointing-Pointer We examine lesion area, OPN and CD44 changes after kaempferol treatment. Black-Right-Pointing-Pointer Kaempferol treatment decreased atherosclerotic lesion area in ApoE{sup -/-} mice. Black-Right-Pointing-Pointer Kaempferol treatment decreased aortic OPN and CD44 expressions in ApoE{sup -/-} mice. Black-Right-Pointing-Pointer Kaempferol regulates OPN-CD44 pathway to inhibit the atherogenesis.« less

Identification and Characterization of Cells with Cancer Stem Cell Properties in Human Primary Lung Cancer Cell Lines

PubMed Central

Suo, Zhenhe; Munthe, Else; Solberg, Steinar; Ma, Liwei; Wang, Mengyu; Westerdaal, Nomdo Anton Christiaan; Kvalheim, Gunnar; Gaudernack, Gustav

2013-01-01

Lung cancer (LC) with its different subtypes is generally known as a therapy resistant cancer with the highest morbidity rate worldwide. Therapy resistance of a tumor is thought to be related to cancer stem cells (CSCs) within the tumors. There have been indications that the lung cancer is propagated and maintained by a small population of CSCs. To study this question we established a panel of 15 primary lung cancer cell lines (PLCCLs) from 20 fresh primary tumors using a robust serum-free culture system. We subsequently focused on identification of lung CSCs by studying these cell lines derived from 4 representative lung cancer subtypes such as small cell lung cancer (SCLC), large cell carcinoma (LCC), squamous cell carcinoma (SCC) and adenocarcinoma (AC). We identified a small population of cells strongly positive for CD44 (CD44high) and a main population which was either weakly positive or negative for CD44 (CD44low/−). Co-expression of CD90 further narrowed down the putative stem cell population in PLCCLs from SCLC and LCC as spheroid-forming cells were mainly found within the CD44highCD90+ sub-population. Moreover, these CD44highCD90+ cells revealed mesenchymal morphology, increased expression of mesenchymal markers N-Cadherin and Vimentin, increased mRNA levels of the embryonic stem cell related genes Nanog and Oct4 and increased resistance to irradiation compared to other sub-populations studied, suggesting the CD44highCD90+ population a good candidate for the lung CSCs. Both CD44highCD90+ and CD44highCD90− cells in the PLCCL derived from SCC formed spheroids, whereas the CD44low/− cells were lacking this potential. These results indicate that CD44highCD90+ sub-population may represent CSCs in SCLC and LCC, whereas in SCC lung cancer subtype, CSC potentials were found within the CD44high sub-population. PMID:23469181

Combined overexpression of cadherin 6, cadherin 11 and cluster of differentiation 44 is associated with lymph node metastasis and poor prognosis in oral squamous cell carcinoma.

PubMed

Ma, Chao; Zhao, Ji-Zhi; Lin, Run-Tai; Zhou, Lian; Chen, Yong-Ning; Yu, Li-Jiang; Shi, Tian-Yin; Wang, Mu; Liu, Man-Man; Liu, Yao-Ran; Zhang, Tao

2018-06-01

Oral squamous cell carcinoma (OSCC) is a highly invasive lesion that frequently metastasizes to the cervical lymph nodes and is associated with a poor prognosis. Several adhesion factors, including cadherin 6 (CDH6), cadherin 11 (CDH11) and cluster of differentiation 44 (CD44), have been reported to be involved in the invasion and metastasis of multiple types of cancer. Therefore, the aim of the present study was to determine the expression of CDH6, CDH11 and CD44 in tumor tissues from patients with OSCC, and whether this was associated with the metastasis and survival of OSCC. The mRNA expression of the human tumor metastasis-related cytokines was examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in OSCC tumors with or without lymph node metastasis (n=10/group). The expression of CDH6, CDH11 and CD44 in 101 OSCC and 10 normal oral mucosa samples was examined by immunohistochemical staining. The association between overall and disease-specific survival times of patients with OSCC and the expression of these three proteins was evaluated using Kaplan-Meier curves and the log-rank test. RT-qPCR results indicated that the mRNA expression of CDH6, CDH11 and CD44 was increased in OSCC patients with lymph node metastasis (2.93-, 2.01- and 1.92-fold; P<0.05). Overexpression of CDH6, CDH11 and CD44 was observed in 31/35 (89%), 25/35 (71%) and 31/35 (89%) patients, respectively. The number of OSCC patients with lymph node metastasis exhibiting CDH6, CDH11 and CD44 overexpression was significantly higher than the number of patients without lymph node metastasis exhibiting overexpression of these proteins (P=0.017, P=0.038 and P=0.007, respectively). OSCC patients with high co-expression of CDH6, CDH11 and CD44 exhibited lower disease-specific survival times (P=0.047; χ 2 =3.933) when compared with OSCC patients with low co-expression of these adhesion factors. CDH6, CDH11 and CD44 serve important roles in OSCC metastasis and the combined use of these factors as biomarkers may improve the accuracy of the prediction of cancer metastases and prognosis.

The herpes simplex virus-induced demise of keratinocytes is associated with a dysregulated pattern of p63 expression.

PubMed

Megyeri, Klára; Orosz, László; Kormos, Bernadett; Pásztor, Katalin; Seprényi, György; Ocsovszki, Imre; Mándi, Yvette; Bata-Csörgo, Zsuzsanna; Kemény, Lajos

2009-01-01

p63 plays a pivotal role in the development and maintenance of stratified epithelial tissues. In an effort to gain insight into the pathogenic mechanisms of skin infections caused by HSV-1 and HSV-2, we determined the patterns of p63 expression in primary keratinocytes and in the HaCaT cell line. The levels of DeltaNp63alpha and a 50kDa p73 isoform were decreased, Bax-alpha remained unaffected, while the expressions of the Bax-beta, TAp63gamma and a 44.5kDa p73 isoform were highly increased in both HSV-1-infected HaCaT cells and primary keratinocytes. In contrast, in response to HSV-2 infection the levels of DeltaNp63alpha, a 50kDa p73 isoform and a 44.5kDa p73 protein were decreased, Bax-alpha and TAp63gamma remained unaffected, while the expression of Bax-beta was slightly increased. The knockdown of TAp63 expression enhanced the viability of HSV-1-infected cells. Thus, HSV-1 and HSV-2 modulate the patterns of p63 and Bax expression in a serotype-specific manner. The dysregulated pattern of p63 expression observed in HSV-infected keratinocytes may comprise part of a mechanism by which these viruses perturb the functions of keratinocytes and lead to their demise.

Exosomes Promote Ovarian Cancer Cell Invasion through Transfer of CD44 to Peritoneal Mesothelial Cells.

PubMed

Nakamura, Koji; Sawada, Kenjiro; Kinose, Yasuto; Yoshimura, Akihiko; Toda, Aska; Nakatsuka, Erika; Hashimoto, Kae; Mabuchi, Seiji; Morishige, Ken-Ichirou; Kurachi, Hirohisa; Lengyel, Ernst; Kimura, Tadashi

2017-01-01

Epithelial ovarian cancer (EOC) cells metastasize within the peritoneal cavity and directly encounter human peritoneal mesothelial cells (HPMC) as the initial step of metastasis. The contact between ovarian cancer cells and the single layer of mesothelial cells involves direct communications that modulate cancer progression but the mechanisms are unclear. One candidate mediating cell-cell communications is exosomes, 30-100 nm membrane vesicles of endocytic origin, through the cell-cell transfer of proteins, mRNAs, or microRNAs. Therefore, the goal was to mechanistically characterize how EOC-derived exosomes modulate metastasis. Exosomes from ovarian cancer cells were fluorescently labeled and cocultured with HPMCs which internalized the exosomes. Upon exosome uptake, HPMCs underwent a change in cellular morphology to a mesenchymal, spindle phenotype. CD44, a cell surface glycoprotein, was found to be enriched in the cancer cell-derived exosomes, transferred, and internalized to HPMCs, leading to high levels of CD44 in HPMCs. This increased CD44 expression in HPMCs promoted cancer invasion by inducing the HPMCs to secrete MMP9 and by cleaning the mesothelial barrier for improved cancer cell invasion. When CD44 expression was knocked down in cancer cells, exosomes had fewer effects on HPMCs. The inhibition of exosome release from cancer cells blocked CD44 internalization in HPMCs and suppressed ovarian cancer invasion. In ovarian cancer omental metastasis, positive CD44 expression was observed in those mesothelial cells that directly interacted with cancer cells, whereas CD44 expression was negative in the mesothelial cells remote from the invading edge. This study indicates that ovarian cancer-derived exosomes transfer CD44 to HPMCs, facilitating cancer invasion. Mechanistic insight from the current study suggests that therapeutic targeting of exosomes may be beneficial in treating ovarian cancer. Mol Cancer Res; 15(1); 78-92. ©2016 AACR. ©2016 American Association for Cancer Research.

CD44+CD24+ subset of PANC-1 cells exhibits radiation resistance via decreased levels of reactive oxygen species.

PubMed

Wang, Lei; Li, Pengping; Hu, Wei; Xia, Youyou; Hu, Chenxi; Liu, Liang; Jiang, Xiaodong

2017-08-01

Emerging evidence has suggested that pancreatic adenocarcinoma is sustained by pancreatic cancer stem cells. The present study aimed to investigate the expression patterns of the pancreatic cancer stem cell surface markers cluster of differentiation CD44 and CD24 in a pancreatic adenocarcinoma cell line, and to investigate the possible mechanisms for their radiation resistance. Flow cytometry was used to analyze the expression patterns of CD44 and CD24 in the pancreatic adenocarcinoma PANC-1 cell line. In addition, a multi-target click model was used to fit cell survival curves and determine the sensitizer enhancement ratio. The apoptosis and cycle distribution of the four cell subsets was determined using flow cytometry, and the level of reactive oxygen species (ROS) was determined using the 2',7'-dichlorofluorescin diacetate probe. The present results identified that the ratios of CD44 + and CD24 + in the sorted PANC-1 cell line were 92.0 and 4.7%, respectively. Prior to radiation, no statistically significant differences were observed among the four groups. Following treatment with 6 MV of X-rays, the rate of apoptosis was decreased in the CD44 + CD24 + group compared with other subsets. The percentage of G0/G1 cells was highest in the CD44 + CD24 + group compared with the three other groups, which exhibited increased radiosensitivity. In addition, the level of ROS in the CD44 + CD24 + group was reduced compared with the other groups. In summary, the results of the present study indicated that CD44 + CD24 + exhibited stem cell properties. The lower level of ROS and apoptosis in CD44 + CD24 + cells may contribute to their resistance to radiation in pancreatic adenocarcinoma.

Breast Cancer Translational Research Center of Excellence FY12-14

DTIC Science & Technology

2014-09-01

DNA aptamers against CD44 that inhibit breast cancer invasion and metastasis. CD44 adhesion molecules are expressed in many breast cancer cells and...Somiari RI, Somiari S, Cutler ML, Mural RJ, Shriver CD. “DNA aptamers against exon v10 of CD44 inhibit breast cancer cell migration.” PLoS One

Preoperative chemoradiotherapy alters the expression and prognostic significance of adhesion molecules in Barrett's-associated adenocarcinoma.

PubMed

Turner, J R; Torres, C M; Wang, H H; Shahsafaei, A; Richards, W G; Sugarbaker, D; Odze, R D

2000-03-01

A variety of prognostic markers have been related to decreased patient survival in patients with epithelial malignancies. These include expression of the homotypic adhesion molecule E-cadherin (ECAD) and the hyaluronic acid receptor CD44. Expression of ECAD and CD44 was evaluated in Barrett's-associated adenocarcinoma (BAd) from 67 patients. Expression was determined by immunoperoxidase staining and graded semiquantitatively based on the proportion of positively stained cells. These data were then correlated with clinical and pathological parameters, including the presence or absence of chemoradiotherapy (chemrad) and patient survival. There were 56 men and 11 women (mean age, 62 years). Thirty-nine (58%) patients received preoperative chemrad. ECAD expression was detected in all (100%) tumors. The ECAD staining grade did not correlate with other pathological features of the tumors. However, ECAD staining was significantly increased in BAd of patients who received chemrad (P = .003), in comparison with those who did not, and in individual patients when prechemrad biopsies and postchemrad resection specimens were compared (P = .04). In terms of prognosis, increased ECAD expression was associated with shortened patient survival only in BAd patients who had received chemrad (univariate analysis of chemrad patients with stage I and II BAd, P = .02). ECAD expression was not significantly associated with survival in BAd patients who did not receive chemrad. CD44 expression was detected in 88% of cases. CD44 expression did not correlate with any of the pathological features of the tumors or with chemrad status. Increased expression of CD44 was significantly associated with shortened patient survival in chemrad patients only (univariate analysis P = .03, multivariate analysis P = .04), although a strong trend was observed when all patients were analyzed regardless of chemrad status (P = .07). The results of this study indicate that chemrad alters the expression of ECAD in BAd. Thus, the prognostic utility of ECAD expression must be evaluated in the context of chemrad status. CD44 also may be a valuable prognostic marker in BAd.

Predictive markers of chemoresistance in advanced stages epithelial ovarian carcinoma.

PubMed

Bonneau, Claire; Rouzier, Roman; Geyl, Caroline; Cortez, Annie; Castela, Mathieu; Lis, Raphael; Daraï, Emile; Touboul, Cyril

2015-01-01

DNA repair mechanisms, environment-mediated drug resistance and cancer initiating cells (CIC) are three major research concepts that can explain the chemoresistance of epithelial ovarian cancer (EOC). The objective was to test if changes in the expression of potential markers associated with drug resistance before and after chemotherapy would correlate with platinum resistance, defined as a recurrence within the first year after chemotherapy cessation, and with survival, in advanced EOC. We included 32 patients with stage IIIC-IV EOC who underwent laparoscopy to evaluate the extent of carcinomatosis, neoadjuvant chemotherapy (carboplatin/taxol) and interval surgery. Biopsies taken during the initial laparoscopies and interval surgeries were evaluated using immunohistochemistry for the expression of 7 proteins: CD117, CD44 and ALDH1 to evaluate CIC; IL-6, IL-8 and BMP2 to evaluate environment-mediated drug resistance; and ERCC1 to evaluate DNA repair. Expression measurements were correlated with platin resistance and survival. The markers' relevance was confirmed in vitro using chemoresistance tests and flow cytometric measurements of the proportion of CD44+ cells. 17 patients were chemoresistant and 15 patients were chemosensitive. We observed increases in CD44, IL-6 and ERCC1 expression and stable ALDH1, CD117, IL-8, and BMP2 expression. Reduced expression of cancer initiating cell markers and increased expression of environment-mediated drug resistance markers were associated with poor prognosis. We also demonstrated that CD44+ cells had survival advantages in vitro. Changes in CD44 and IL-8 expression on tumor cells appeared to correlate with overall survival and should be further tested as predictors of chemoresistance using larger cohort. Copyright © 2014 Elsevier Inc. All rights reserved.

HAb18G/CD147 Promotes pSTAT3-Mediated Pancreatic Cancer Development via CD44s †, ‡

PubMed Central

Li, Ling; Tang, Wenhua; Wu, Xiaoqing; Karnak, David; Meng, Xiaojie; Thompson, Rachel; Hao, Xinbao; Li, Yongmin; Qiao, Xiaotan T.; Lin, Jiayuh; Fuchs, James; Simeone, Diane M.; Chen, Zhi-Nan; Lawrence, Theodore S.; Xu, Liang

2013-01-01

Purpose STAT3 plays a critical role in initiation and progression of pancreatic cancer. However, therapeutically targeting STAT3 is failure in clinic. We previously identified HAb18G/CD147 as an effective target for cancer treatment. In this study, we aimed to investigate potential role of HAb18G/CD147 in STAT3-involved pancreatic tumorigenesis in vitro and in vivo. Experimental Design The expression of HAb18G/CD147, pSTAT3 and CD44s were determined in tissue microarrays. The tumorigenic function and molecular signaling mechanism of HAb18G/CD147 was assessed by in vitro cellular and clonogenic growth, reporter assay, immunoblot, immunofluorescence staining, immunoprecipitation, and in vivo tumor formationusing loss or gain-of-function strategies. Results Highly expressed HAb18G/CD147 promoted cellular and clonogenic growth in vitro and tumorigenicity in vivo. CyPA, a ligand of CD147, stimulated STAT3 phosphorylation and its downstream genes cyclin D1/survivin through HAb18G/CD147 dependent mechanisms. HAb18G/CD147 was associated and co-localized with cancer stem cell marker CD44s in lipid rafts. The inhibitors of STAT3 and survivin, as well as CD44s neutralizing antibodies suppressed the HAb18G/CD147-induced cell growth. High HAb18G/CD147 expression in pancreatic cancer was significantly correlated with the poor tumor differentiation, and the high co-expression of HAb18G/CD147-CD44s-STAT3 associated with poor survival of patients with pancreatic cancer. Conclusions We identified HAb18G/CD147 as a novel upstream activator of STAT3 via interacts with CD44s and plays a critical role in the development of pancreatic cancer. The data suggest HAb18G/CD147 could be a promising therapeutic target for highly aggressive pancreatic cancer and a surrogate marker in the STAT3-targeted molecular therapies. PMID:24132924

Immunohistochemical expression of CD44s in human neuroblastic tumors: Moroccan experience and highlights on current data

PubMed Central

2013-01-01

Background Peripheral neuroblastic tumors (pNTs), including neuroblastoma (NB), ganglioneuroblastoma (GNB) and ganglioneuroma (GN), are extremely heterogeneous pediatric tumors responsible for 15 % of childhood cancer death. The aim of the study was to evaluate the expression of CD44s (‘s’: standard form) cell adhesion molecule by comparison with other specific prognostic markers. Methods An immunohistochemical profile of 32 formalin-fixed paraffin-embedded pNTs tissues, diagnosed between January 2007 and December 2010, was carried out. Results Our results have demonstrated the association of CD44s negative pNTs cells to lack of differentiation and tumour progression. A significant association between absence of CD44s expression and metastasis in human pNTs has been reported. We also found that expression of CD44s defines subgroups of patients without MYCN amplification as evidenced by its association with low INSS stages, absence of metastasis and favorable Shimada histology. Discussion These findings support the thesis of the role of CD44s glycoprotein in the invasive growth potential of neoplastic cells and suggest that its expression could be taken into consideration in the therapeutic approaches targeting metastases. Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1034403150888863 Résumé Introduction les tumeurs neuroblastiques périphériques (TNPs), comprenant le neuroblastome (NB), le ganglioneuroblastome (GNB) et le ganglioneurome (GN), sont des tumeurs pédiatriques extrêmement hétérogènes responsables de 15% des décès par cancer chez les enfants. Le but de cette étude était d’évaluer l’expression de la molécule d’adhésion cellulaire CD44s (‘s’: pour standard) par rapport à d’autres facteurs pronostiques spécifiques. Méthodes Un profil immunohistochimique de 32 TNPs fixées au formol et incluses en paraffine, diagnostiquées entre Janvier 2007 et Décembre 2010, a été réalisé. Résultats Nos résultats ont mis en évidence l’association des TNPs n’exprimant pas le CD44s avec une perte de différenciation et une progression tumorale et nous avons rapporté une association significative entre l’absence d’expression du CD44s et la présence de métastases. Nous avons également constaté que l’expression du CD44s définit des sous-groupes de patients dans les tumeurs n’amplifiant pas le MYCN, comme en témoigne son association avec les stades INSS bas, l’absence de métastases et l’histologie favorable de Shimada. Discussion Ces résultats appuient l’hypothèse du rôle de la glycoprotéine CD44s dans le potentiel de croissance invasive des cellules néoplasiques et suggèrent que son expression pourrait être prise en considération dans des voies thérapeutiques ciblant les métastases. PMID:23445749

Generation and evaluation of antibody agents for molecular imaging of CD44v6-expressing cancers

PubMed Central

Haylock, Anna-Karin; Nilvebrant, Johan; Mortensen, Anja; Velikyan, Irina; Nestor, Marika; Falk, Ronny

2017-01-01

Aim The aim of this study was to generate and characterize scFv antibodies directed to human CD44v6, as well as to radiolabel and evaluate top candidates in vitro and in vivo for their potential use in CD44v6-targeted molecular imaging in cancer patients. Materials and methods Phage display selections were used to isolate CD44v6-specific scFvs. A chain shuffling strategy was employed for affinity maturation based on a set of CD44v6-specific first-generation clones. Two second-generation scFv clones were then chosen for labeling with 111In or 125I and assessed for CD44v6-specific binding on cultured tumor cells. In vivo uptake and distribution was evaluated in tumor-bearing mice using a dual tumor model. Finally, a proof-of-concept small animal PET-CT study was performed on one of the candidates labeled with 124I. Results Two affinity-matured clones, CD44v6-scFv-A11 and CD44v6-scFv-H12, displayed promising binding kinetics. Seven out of eight radiolabeled conjugates demonstrated CD44v6-specific binding. In vivo studies on selected candidates demonstrated very advantageous tumor-to-organ ratios, in particular for iodinated conjugates, where 125I-labeled scFvs exhibited favorable kinetics and tumor-to-blood ratios above five already at 24 hours p.i.. The small animal PET-CT study using 124I-labeled CD44v6-scFv-H12 was in line with the biodistribution data, clearly visualizing the high CD44v6-expressing tumor. Conclusion The single chain fragments, CD44v6-scFv-A11 and CD44v6-scFv-H12 specifically bind to CD44v6, and the radiolabeled counterparts provide high tumor-to-blood ratios and fast clearance from organs and blood. We conclude that radioiodinated CD44v6-scFv-A11 and CD44v6-scFv-H12 possess features highly suitable for stringent molecular imaging. PMID:29029420

Prognostic implications of adhesion molecule expression in colorectal cancer.

PubMed

Seo, Kyung-Jin; Kim, Maru; Kim, Jeana

2015-01-01

Research on the expression of adhesion molecules, E-cadherin (ECAD), CD24, CD44 and osteopontin (OPN) in colorectal cancer (CRC) has been limited, even though CRC is one of the leading causes of cancer-related deaths. This study was conducted to evaluate the expression of adhesion molecules in CRC and to determine their relationships with clinicopathologic variables, and the prognostic significance. The expression of ECAD, CD24, CD44 and OPN was examined in 174 stage II and III CRC specimens by immunohistochemistry of TMA. Negative ECAD expression was significantly correlated with advanced nodal stage and poor tumor differentiation. Multivariate analysis showed that both negative expression of ECAD and positive expression of CD24 were independent prognostic factors for disease-free survival (DFS) in CRC patients (P<0.001, relative risk [RR] = 5.596, 95% CI = 2.712-11.549; P = 0.038, RR = 3.768, 95% CI = 1.077-13.185, respectively). However, for overall survival (OS), only ECAD negativity showed statistically significant results in multivariate analysis (P<0.001, RR = 4.819, 95% CI = 2.515-9.234). Positive expression of CD24 was associated with poor OS in univariate analysis but was of no prognostic value in multivariate analysis. In conclusion, our study suggests that among these four adhesion molecules, ECAD and CD24 expression can be considered independent prognostic factors. The role of CD44 and OPN may need further evaluation.

Prognostic implications of adhesion molecule expression in colorectal cancer

PubMed Central

Seo, Kyung-Jin; Kim, Maru; Kim, Jeana

2015-01-01

Research on the expression of adhesion molecules, E-cadherin (ECAD), CD24, CD44 and osteopontin (OPN) in colorectal cancer (CRC) has been limited, even though CRC is one of the leading causes of cancer-related deaths. This study was conducted to evaluate the expression of adhesion molecules in CRC and to determine their relationships with clinicopathologic variables, and the prognostic significance. The expression of ECAD, CD24, CD44 and OPN was examined in 174 stage II and III CRC specimens by immunohistochemistry of TMA. Negative ECAD expression was significantly correlated with advanced nodal stage and poor tumor differentiation. Multivariate analysis showed that both negative expression of ECAD and positive expression of CD24 were independent prognostic factors for disease-free survival (DFS) in CRC patients (P<0.001, relative risk [RR] = 5.596, 95% CI = 2.712-11.549; P = 0.038, RR = 3.768, 95% CI = 1.077-13.185, respectively). However, for overall survival (OS), only ECAD negativity showed statistically significant results in multivariate analysis (P<0.001, RR = 4.819, 95% CI = 2.515-9.234). Positive expression of CD24 was associated with poor OS in univariate analysis but was of no prognostic value in multivariate analysis. In conclusion, our study suggests that among these four adhesion molecules, ECAD and CD24 expression can be considered independent prognostic factors. The role of CD44 and OPN may need further evaluation. PMID:26097606

[Analysis of expression of cancer stem cell-related markers in orbital adenoid cystic carcinoma].

PubMed

Lin, Ting-ting; Zhu, Li-min; He, Yan-jin; Zhang, Hong

2011-08-01

To observe the expression and distribution of CD44, CD133, and ABCG2 in orbital adenoid cystic carcinoma (ACC) and investigate their correlations with pathological type and prognosis. Two steps method of immunohistochemical staining was employed in 33 cases of paraffin embedded surgical specimens of human orbital ACC, 5 cases of recurrence samples, 3 cases of an excised lacrimal gland caused by neither inflammation nor tumor diseases, and 6 cases of xenograft tumors in nude mice. A retrospective analysis was performed on the clinical material of these patients, which were collected from Jan. 1991 to Mar. 2009. The positive rate of CD44 was 54.5% (18/33), with 76.9% (10/13) in solid type and 40.0% (8/20) in adeno-tubiform type. There was no statistically significant difference between them (P = 0.072). In solid type the positive expression cells were often located at the marginal part of the cancer nest. In the adeno-tubiform type, positive cells were often located at the outer layer of the tubiform structure (myoepithelial cells). CD44 was also expressed in normal tissues. The positive rate of CD133 was 57.6% (19/33), with 76.9% (10/13) in solid type and 45.0% (9/20) in adeno-tubiform type. There was no significant difference between them (P = 0.087). CD133 antigen was expressed in either the cytoplasm or nucleus, or expressed in both the cytoplasm and nucleus. The positive rate of ABCG2 was 21.2% (7/33), with 30.77% (4/13) in solid type and 15.0% (3/20) in adeno-tubiform type. There was no significant difference between them (P = 0.393). Many positive cells surrounded the vessels in tumor tissues. There were no significant differences between different prognosis groups of these surface phenotypes. The correlative analysis results of three surface phenotypes showed that CD44(+) cells have positive correlation with CD133(+) cells (Spearman, r(s) = 0.416, P = 0.016). In six transplanted tumors of nude mice, the number of positive cases for CD44(+), CD133(+) and ABCG2(+) was 1, 1 and 4 cases, respectively. The expression of CD44, CD133 and ABCG2 in ACC may influence the progress of ACC. However, they cannot be used as the markers for the evaluation of the prognosis of this tumor.

HIERARCHICAL ORGANIZATION OF OSTEOBLASTS REVEALS THE SIGNIFICANT ROLE OF CD166 IN HEMATOPOIETIC STEM CELL MAINTANANCE AND FUNCTION

PubMed Central

Chitteti, Brahmananda R.; Cheng, Ying-Hua; Kacena, Melissa A.; Srour, Edward F.

2013-01-01

The role of osteoblasts (OB) in maintaining hematopoietic stem cells (HSC) in their niche is well elucidated, but the exact definition, both phenotypically and hierarchically of OB responsible for these functions is not clearly known. We previously demonstrated that OB maturational status influences HSC function whereby immature OB with high Runx2 expression promote hematopoietic expansion. Here, we show that Activated Leukocyte Cell Adhesion Molecule (ALCAM) or CD166 expression on OB is directly correlated with Runx2 expression and high hematopoiesis enhancing activity (HEA). Fractionation of OB with lineage markers: Sca1, osteopontin (OPN), CD166, CD44, and CD90 revealed that Lin-Sca1-OPN+CD166+ cells (CD166+) and their subpopulations fractionated with CD44 and CD90 expressed high levels of Runx2 and low levels of osteocalcin (OC) demonstrating the relatively immature status of these cells. Conversely, the majority of the Lin-Sca1-OPN+CD166− cells (CD166−) expressed high OC levels suggesting that CD166− OB are more mature. In vitro hematopoietic potential of LSK cells co-cultured for 7 days with fresh OB or OB pre-cultured for 1, 2, or 3 weeks declined precipitously with increasing culture duration concomitant with loss of CD166 expression. Importantly, LSK cells co-cultured with CD166+CD44+CD90+ OB maintained their in vivo repopulating potential through primary and secondary transplantation, suggesting that robust HEA activity is best mediated by immature CD166+ OB with high Runx2 and low OC expression. These studies begin to define the hierarchical organization of osteoblastic cells and provide a more refined definition of OB that can mediate HEA. PMID:23369988

Regulation of Asymmetric Division by Atypical Protein Kinase C Influences Early Specification of CD8+ T Lymphocyte Fates

PubMed Central

Metz, Patrick J.; Lopez, Justine; Kim, Stephanie H.; Akimoto, Kazunori; Ohno, Shigeo; Chang, John T.

2016-01-01

Naïve CD8+ T lymphocytes responding to microbial pathogens give rise to effector T cells that provide acute defense and memory T cells that provide long-lived immunity. Upon activation, CD8+ T lymphocytes can undergo asymmetric division, unequally distributing factors to the nascent daughter cells that influence their eventual fate towards the effector or memory lineages. Individual loss of either atypical protein kinase C (aPKC) isoform, PKCζ or PKCλ/ι, partially impairs asymmetric divisions and increases CD8+ T lymphocyte differentiation toward a long-lived effector fate at the expense of memory T cell formation. Here, we show that deletion of both aPKC isoforms resulted in a deficit in asymmetric divisions, increasing the proportion of daughter cells that inherit high amounts of effector fate-associated molecules, IL-2Rα, T-bet, IFNγR, and interferon regulatory factor 4 (IRF4). However, unlike CD8+ T cells deficient in only one aPKC isoform, complete loss of aPKC unexpectedly increased CD8+ T cell differentiation toward a short-lived, terminal effector fate, as evidenced by increased rates of apoptosis and decreased expression of Eomes and Bcl2 early during the immune response. Together, these results provide evidence for an important role for asymmetric division in CD8+ T lymphocyte fate specification by regulating the balance between effector and memory precursors at the initiation of the adaptive immune response. PMID:26765121

CD133 expression is not restricted to stem cells, and both CD133+ and CD133– metastatic colon cancer cells initiate tumors

PubMed Central

Shmelkov, Sergey V.; Butler, Jason M.; Hooper, Andrea T.; Hormigo, Adilia; Kushner, Jared; Milde, Till; St. Clair, Ryan; Baljevic, Muhamed; White, Ian; Jin, David K.; Chadburn, Amy; Murphy, Andrew J.; Valenzuela, David M.; Gale, Nicholas W.; Thurston, Gavin; Yancopoulos, George D.; D’Angelica, Michael; Kemeny, Nancy; Lyden, David; Rafii, Shahin

2008-01-01

Colon cancer stem cells are believed to originate from a rare population of putative CD133+ intestinal stem cells. Recent publications suggest that a small subset of colon cancer cells expresses CD133, and that only these CD133+ cancer cells are capable of tumor initiation. However, the precise contribution of CD133+ tumor-initiating cells in mediating colon cancer metastasis remains unknown. Therefore, to temporally and spatially track the expression of CD133 in adult mice and during tumorigenesis, we generated a knockin lacZ reporter mouse (CD133lacZ/+), in which the expression of lacZ is driven by the endogenous CD133 promoters. Using this model and immunostaining, we discovered that CD133 expression in colon is not restricted to stem cells; on the contrary, CD133 is ubiquitously expressed on differentiated colonic epithelium in both adult mice and humans. Using Il10–/–CD133lacZ mice, in which chronic inflammation in colon leads to adenocarcinomas, we demonstrated that CD133 is expressed on a full gamut of colonic tumor cells, which express epithelial cell adhesion molecule (EpCAM). Similarly, CD133 is widely expressed by human primary colon cancer epithelial cells, whereas the CD133– population is composed mostly of stromal and inflammatory cells. Conversely, CD133 expression does not identify the entire population of epithelial and tumor-initiating cells in human metastatic colon cancer. Indeed, both CD133+ and CD133– metastatic tumor subpopulations formed colonospheres in in vitro cultures and were capable of long-term tumorigenesis in a NOD/SCID serial xenotransplantation model. Moreover, metastatic CD133– cells form more aggressive tumors and express typical phenotypic markers of cancer-initiating cells, including CD44 (CD44+CD24–), whereas the CD133+ fraction is composed of CD44lowCD24+ cells. Collectively, our data suggest that CD133 expression is not restricted to intestinal stem or cancer-initiating cells, and during the metastatic transition, CD133+ tumor cells might give rise to the more aggressive CD133– subset, which is also capable of tumor initiation in NOD/SCID mice. PMID:18497886

Expression profiles of cancer stem cell markers: CD133, CD44, Musashi-1 and EpCAM in the cardiac mucosa-Barrett's esophagus-early esophageal adenocarcinoma-advanced esophageal adenocarcinoma sequence.

PubMed

Mokrowiecka, Anna; Veits, Lothar; Falkeis, Christina; Musial, Jacek; Kordek, Radzislaw; Lochowski, Mariusz; Kozak, Jozef; Wierzchniewska-Lawska, Agnieszka; Vieth, Michael; Malecka-Panas, Ewa

2017-03-01

Barrett's esophagus (BE), which develops as a result of gastroesophageal reflux disease, is a preneoplastic condition for esophageal adenocarcinoma (EAC). A new hypothesis suggests that cancer is a disease of stem cells, however, their expression and pathways in BE - EAC sequence are not fully elucidated yet. We used a panel of putative cancer stem cells markers to identify stem cells in consecutive steps of BE-related cancer progression. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded blocks from 58 patients with normal cardiac mucosa (n=5), BE (n=14), early EAC (pT1) from mucosal resection (n=17) and advanced EAC (pT1-T4) from postoperative specimens (n=22). Expression of the CD133, CD44, Musashi-1 and EpCAM was analyzed using respective monoclonal antibodies. All markers showed a heterogeneous expression pattern, mainly at the base of the crypts of Barrett's epithelium and EAC, with positive stromal cells in metaplastic and dysplastic lesions. Immuno-expression of EpCAM, CD44 and CD133 in cardiac mucosa was significantly lower (mean immunoreactivity score (IRS)=1.2; 0.0; 0.4; respectively) compared to their expression in Barrett's metaplasia (mean IRS=4.3; 0.14; 0.7; respectively), in early adenocarcinoma (mean IRS=4.4; 0.29; 1.3; respectively) and in advanced adenocarcinoma (mean IRS=6.6; 0.7; 2.7; respectively) (p<0.05). On the contrary, Musashi-1 expression was higher in BE and early ADC compared to GM and advanced ADC (NS). Our results suggest that the stem cells could be present in premalignant lesions. EpCAM, CD44 and CD133 expression could be candidate markers for BE progression, whereas Musashi-1 may be a marker of the small intestinal features of Barrett's mucosa. Copyright © 2016 Elsevier GmbH. All rights reserved.

Investigating Cell Surface Markers on Normal Hematopoietic Stem Cells in Three Different Niche Conditions

PubMed Central

Garg, Swati; Madkaikar, Manisha

2013-01-01

Hematopoietic stem cells are of therapeutic interest to the clinicians and researchers due to their promising assistance in management of malignant and inherited hematological conditions. Evaluation of cell surface markers using multiparametric flow cytometry is a well adapted qualitative measure of cells in question for many years. An artillery of these markers has been studied in hematological malignancies and related disorders. However, their role and differential expression on normal hematopoietic stem cells from clinically available sources is not always described carefully. In the present study, we attempted to evaluate expression of CD44, CD90, CD96 and CD123 in three clinically available sources of normal HSCs (Hematopoietic stem cells). Sources of HSCs in the present study involved umbilical cord blood (UCB), normal bone marrow (NBM) and bone marrow from idiopathic thrombocytopenic purpura (ITP) patients (IBM). CD44 is an important homing receptor while CD90 is involved in maintaining stem cell quiescent. CD96 is known to be leukemia specific marker and CD123 is involved in stem cell differentiation and survival. We observed a significant difference in expression CD44, CD90 and CD123 on normal HSCs derived from umbilical cord and ITP marrow. CD96 was highly expressed on HSCs obtained from ITP marrow. Investigating expression of these markers on normal HSCs in different niches will be helpful in correlating their function with niche condition and delineating their ‘abnormal’ expression from the normal. PMID:24386557

Investigating cell surface markers on normal hematopoietic stem cells in three different niche conditions.

PubMed

Garg, Swati; Madkaikar, Manisha; Ghosh, Kanjaksha

2013-11-01

Hematopoietic stem cells are of therapeutic interest to the clinicians and researchers due to their promising assistance in management of malignant and inherited hematological conditions. Evaluation of cell surface markers using multiparametric flow cytometry is a well adapted qualitative measure of cells in question for many years. An artillery of these markers has been studied in hematological malignancies and related disorders. However, their role and differential expression on normal hematopoietic stem cells from clinically available sources is not always described carefully. In the present study, we attempted to evaluate expression of CD44, CD90, CD96 and CD123 in three clinically available sources of normal HSCs (Hematopoietic stem cells). Sources of HSCs in the present study involved umbilical cord blood (UCB), normal bone marrow (NBM) and bone marrow from idiopathic thrombocytopenic purpura (ITP) patients (IBM). CD44 is an important homing receptor while CD90 is involved in maintaining stem cell quiescent. CD96 is known to be leukemia specific marker and CD123 is involved in stem cell differentiation and survival. We observed a significant difference in expression CD44, CD90 and CD123 on normal HSCs derived from umbilical cord and ITP marrow. CD96 was highly expressed on HSCs obtained from ITP marrow. Investigating expression of these markers on normal HSCs in different niches will be helpful in correlating their function with niche condition and delineating their 'abnormal' expression from the normal.

[LincRNA-ROR functions as a ceRNA to regulate Oct4, Sox2, and Nanog expression by sponging miR-145 and its effect on biologic characteristics of colonic cancer stem cells].

PubMed

Yan, Z Y; Sun, X C

2018-04-08

Objective: To investigate the impact of lincRNA-ROR, a ceRNA by binding miR-145 on the expression of the downstream genes Oct4, Sox2 and Nanog, and related biological characteristics of colon cancer stem cells, and to elucidate the clinical significance of this molecular regulatory network. Methods: Fifty-two cases of colorectal cancer tissue and adjacent tissue were collected at Nanyang City Central Hospital and Nanyang Second Hospital, Henan Province, from 2014 to 2016. Real-time quantitative polymerase chain reaction (qPCR) was used to detect the expression of lincRNA-ROR and miR-145 in colorectal cancer tissue and isolated colon cancer cells. The correlation between the expression of lincRNA-ROR, miR-145 and the clinicopathologic features of colon cancer was performed. CD44(-)CD133(-) and CD44(+) CD133(+) cells were isolated from SW1116 by using flow cytometry. The expression of CD44, CD133, Oct4, Sox2, Nanog, lincRNA-ROR and miR-145 in cells were detected by qPCR. The relationship between lincRNA-ROR, miR-145, Oct4, Sox2 and Nanog was analyzed by bioinformatics, dual luciferase reporter assay, qPCR and Western blot. The effects of silencing lincRNA-ROR on the proliferation and chemosensitivity of colon cancer stem cells were detected by MTT, colony formation. Results: LincRNA-ROR was frequently up-regulated and inversely correlated with miR-145 down-regulation in the colon cancer specimens( P <0.05). LincRNA-ROR was related to tumor size, lymph node involvement and distant metastasis( P <0.05), and miR-145 was found related to tumor size and tumor location( P <0.05). CD44(+) CD133(+) cells were successfully isolated from SW1116 by flow cytometry. The levels of CD44, CD133, Oct4, Sox2, Nanog, lincRNA-ROR in CD44(+) CD133(+) cells were significantly increased, while miR-145 was decreased compared with CD44(-)CD133(-)cells( P <0.05). The levels of CD44, CD133, lnc-ROR in CD44(+) CD133(+) cells were significantly reduced upon cell adherence, while miR-145 was significantly increased( P <0.05). Bioinformatics analysis revealed that lincRNA-ROR shared miRNA response elements with core transcription factors Oct4, Sox2 and Nanog. MiR-145 significantly inhibited the expression of lincRNA-ROR, Oct4, Sox2 and Nanog. Silencing lincRNA-ROR significantly inhibited colon cancer stem cells proliferation and increased the sensitivity to chemotherapy. Conclusions: Linc-ROR functions as a key ceRNA to prevent core TFs, e. g., Oct4, Sox2, Nanog, from miR-145-mediated suppression in colon cancer stem cells and regulates cell proliferation and chemosensitivity.The data may provide insights into the pathophysiological interactions of the components of genetic networks in the development of colon cancer and may lead to new therapies in the future.

Characterization of mouse CD53: epitope mapping, cellular distribution and induction by T cell receptor engagement during repertoire selection.

PubMed

Tomlinson, M G; Hanke, T; Hughes, D A; Barclay, A N; Scholl, E; Hünig, T; Wright, M D

1995-08-01

The pan-leukocyte antigen CD53 is a member of the poorly understood transmembrane 4 superfamily (TM4SF) of cell membrane glycoproteins. CD53 is proposed to play a role in thymopoiesis, since rat CD53 is expressed on immature CD4-8-thymocytes and the functionally mature single-positive subset, but is largely absent from the intermediate CD4+8+ cells. We have characterized CD53 in the mouse through the production of two new monoclonal antibodies, MRC OX-79 and OX-80, which were raised against the RAW 264 cell line and screened on recombinant CD53 fusion proteins. The epitopes recognized by both antibodies are dependent on disulfide bonding and map to the major extracellular region of CD53, requiring the presence of a single threonine residue at position 154. Mouse CD53 has a molecular mass of 35-45 kDa and is expressed on virtually all peripheral leukocytes, but not on cells outside the lymphoid or myeloid lineages. CD53 expression distinguishes subpopulations of thymocytes in the mouse and resembles the expression pattern of rat CD53. Amongst the immature CD4-8-thymocytes, mouse CD53 is clearly detectable on the earliest CD44high25- subset, but down-regulated on the later CD44high25+, CD44low25+ and CD44low25- stages. Also, the subsequent transient TcR-/low CD4-8+ cells and most CD4+8+ thymocytes express little or no CD53. This is consistent with the idea that cells which are committed to enter the selectable CD4+8+ compartment switch off CD53. The effect of T cell receptor (TcR) engagement on the re-expression of CD53 on CD4+8+ thymocytes was studied both ex vivo and in vitro using F5 mice, transgenic for the H-2b/influenza nucleoprotein-peptide-specific TcR, back-crossed onto an H-2q or H-2b background of RAG-2-deficient mice. CD4+8+ thymocytes from non-selecting H-2q F5 mice are CD53 negative, but in vitro stimulation through the TcR dramatically induces CD53 expression. In contrast, a fraction of CD4+8+ thymocytes from positively selecting H-2b F5 transgenic mice express CD53. Therefore TcR engagement by selecting major histocompatibility complex peptide complexes, or surrogate ligands, induces CD53 expression on otherwise CD53-negative, non-selected CD4+8+ thymocytes. Whether CD53 itself participates as a signaling molecule in further stages of thymic selection is still a matter of speculation.

CD147, CD44, and the epidermal growth factor receptor (EGFR) signaling pathway cooperate to regulate breast epithelial cell invasiveness.

PubMed

Grass, G Daniel; Tolliver, Lauren B; Bratoeva, Momka; Toole, Bryan P

2013-09-06

The immunoglobulin superfamily glycoprotein CD147 (emmprin; basigin) is associated with an invasive phenotype in various types of cancers, including malignant breast cancer. We showed recently that up-regulation of CD147 in non-transformed, non-invasive breast epithelial cells is sufficient to induce an invasive phenotype characterized by membrane type-1 matrix metalloproteinase (MT1-MMP)-dependent invadopodia activity (Grass, G. D., Bratoeva, M., and Toole, B. P. (2012) Regulation of invadopodia formation and activity by CD147. J. Cell Sci. 125, 777-788). Here we found that CD147 induces breast epithelial cell invasiveness by promoting epidermal growth factor receptor (EGFR)-Ras-ERK signaling in a manner dependent on hyaluronan-CD44 interaction. Furthermore, CD147 promotes assembly of signaling complexes containing CD147, CD44, and EGFR in lipid raftlike domains. We also found that oncogenic Ras regulates CD147 expression, hyaluronan synthesis, and formation of CD147-CD44-EGFR complexes, thus forming a positive feedback loop that may amplify invasiveness. Last, we showed that malignant breast cancer cells are heterogeneous in their expression of surface-associated CD147 and that high levels of membrane CD147 correlate with cell surface EGFR and CD44 levels, activated EGFR and ERK1, and activated invadopodia. Future studies should evaluate CD147 as a potential therapeutic target and disease stratification marker in breast cancer.

Shear Stress Regulates Adhesion and Rolling of CD44+ Leukemic and Hematopoietic Progenitor Cells on Hyaluronan

PubMed Central

Christophis, Christof; Taubert, Isabel; Meseck, Georg R.; Schubert, Mario; Grunze, Michael; Ho, Anthony D.; Rosenhahn, Axel

2011-01-01

Leukemic cells and human hematopoietic progenitor cells expressing CD44 receptors have the ability to attach and roll on hyaluronan. We investigated quantitatively the adhesion behavior of leukemic cell lines and hematopoietic progenitor cells on thin films of the polysaccharides hyaluronan and alginate in a microfluidic system. An applied flow enhances the interaction between CD44-positive cells and hyaluronan if a threshold shear stress of 0.2 dyn/cm2 is exceeded. At shear stress ∼1 dyn/cm2, the cell rolling speed reaches a maximum of 15 μm/s. Leukemic Jurkat and Kasumi-1 cells lacking CD44-expression showed no adhesion or rolling on the polysaccharides whereas the CD44-expressing leukemic cells KG-1a, HL-60, K-562, and hematopoietic progenitor cells attached and rolled on hyaluronan. Interestingly, the observations of flow-induced cell rolling are related to those found in the recruitment of leukocytes to inflammatory sites and the mechanisms of stem-cell homing into the bone marrow. PMID:21806926

Novel Single-Cell Analysis Platform Based on a Solid-State Zinc-Coadsorbed Carbon Quantum Dots Electrochemiluminescence Probe for the Evaluation of CD44 Expression on Breast Cancer Cells.

PubMed

Qiu, Youyi; Zhou, Bin; Yang, Xiaojuan; Long, Dongping; Hao, Yan; Yang, Peihui

2017-05-24

A novel single-cell analysis platform was fabricated using solid-state zinc-coadsorbed carbon quantum dot (ZnCQDs) nanocomposites as an electrochemiluminescence (ECL) probe for the detection of breast cancer cells and evaluation of the CD44 expression level. Solid-state ZnCQDs nanocomposite probes were constructed through the attachment of ZnCQDs to gold nanoparticles and then the loading of magnetic beads to amplify the ECL signal, exhibiting a remarkable 120-fold enhancement of the ECL intensity. Hyaluronic acid (HA)-functionalized solid-state probes were used to label a single breast cancer cell by the specific recognition of HA with CD44 on the cell surface, revealing more stable, sensitive, and effective tagging in comparison with the water-soluble CQDs. This strategy exhibited a good analytical performance for the analysis of MDA-MB-231 and MCF-7 single cells with linear range from 1 to 18 and from 1 to 12 cells, respectively. Furthermore, this single-cell analysis platform was used for evaluation of the CD44 expression level of these two cell lines, in which the MDA-MB-231 cells revealed a 2.8-5.2-fold higher CD44 expression level. A total of 20 single cells were analyzed individually, and the distributions of the ECL intensity revealed larger variations, indicating the high cellular heterogeneity of the CD44 expression level on the same cell line. The as-proposed single-cell analysis platform might provide a novel protocol to effectively study the individual cellular function and cellular heterogeneity.

Engineered Metal-Phenolic Capsules Show Tunable Targeted Delivery to Cancer Cells.

PubMed

Ju, Yi; Cui, Jiwei; Sun, Huanli; Müllner, Markus; Dai, Yunlu; Guo, Junling; Bertleff-Zieschang, Nadja; Suma, Tomoya; Richardson, Joseph J; Caruso, Frank

2016-06-13

We engineered metal-phenolic capsules with both high targeting and low nonspecific cell binding properties. The capsules were prepared by coating phenolic-functionalized hyaluronic acid (HA) and poly(ethylene glycol) (PEG) on calcium carbonate templates, followed by cross-linking the phenolic groups with metal ions and removing the templates. The incorporation of HA significantly enhanced binding and association with a CD44 overexpressing (CD44+) cancer cell line, while the incorporation of PEG reduced nonspecific interactions with a CD44 minimal-expressing (CD44-) cell line. Moreover, high specific targeting to CD44+ cells can be balanced with low nonspecific binding to CD44- cells simply by using an optimized feed-ratio of HA and PEG to vary the content of HA and PEG incorporated into the capsules. Loading an anticancer drug (i.e., doxorubicin) into the obtained capsules resulted in significantly higher cytotoxicity to CD44+ cells but lower cytotoxicity to CD44- cells.

CD44 deficiency leads to decreased proinflammatory cytokine production in lung induced by PCV2 in mice.

PubMed

Fu, Qiang; Hou, Linbing; Xiao, Pingping; Guo, Chunhe; Chen, Yaosheng; Liu, Xiaohong

2014-12-01

Porcine circovirus type 2 (PCV2) is the primary etiological agent of postweaning multisystemic wasting syndrome (PMWS). CD44 is a widely expressed class I transmembrane glycoprotein implicated in immunological and inflammatory responses. In previous studies, the role of CD44 in host defense against microorganism infection remains controversial. The role of CD44 in host defense against PCV2 infection has never been studied before. In this study, we investigated the role of CD44 in the development of pneumonia induced by PCV2 in mice model. Upon infection, CD44 mRNA level in lung tissue was upregulated, and we confirmed a detrimental role of CD44 in host defense against PCV2 infection. The results demonstrated that CD44 deficiency could result in decreased proinflammatory cytokine production in lung induced by PCV2 in mice, suggesting a previously unrecognized role for CD44 in the development of pneumonia response to PCV2 infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

The Pluripotent Stem-Cell Marker Alkaline Phosphatase is Highly Expressed in Refractory Glioblastoma with DNA Hypomethylation.

PubMed

Iwadate, Yasuo; Suganami, Akiko; Tamura, Yutaka; Matsutani, Tomoo; Hirono, Seiichiro; Shinozaki, Natsuki; Hiwasa, Takaki; Takiguchi, Masaki; Saeki, Naokatsu

2017-02-01

Hypomethylation of genomic DNA induces stem-cell properties in cancer cells and contributes to the treatment resistance of various malignancies. To examine the correlation between the methylation status of stem-cell-related genes and the treatment outcomes in patients with glioblastoma (GBM). The genome-wide DNA methylation status was determined using HumanMethylation450 BeadChips, and the methylation status was compared between a group of patients with good prognosis (survival > 4 yr) and a group with poor prognosis (survival < 1 yr). Immunohistochemistry for proteins translated from hypomethylated genes, including alkaline phosphatase (ALPL), CD133, and CD44, was performed in 70 GBMs and 60 oligodendroglial tumors. The genomic DNA in refractory GBM was more hypomethylated than in GBM from patients with relatively long survival (P = .0111). Stem-cell-related genes including ALPL, CD133, and CD44 were also significantly hypomethylated. A validation study using immunohistochemistry showed that DNA hypomethylation was strongly correlated with high protein expression of ALPL, CD133, and CD44. GBM patients with short survival showed high expression of these stem-cell markers. Multivariate analysis confirmed that co-expression of ALPL + CD133 or ALPL + CD44 was a strong predictor of short survival. Anaplastic oligodendroglial tumors without isocitrate dehydrogenase 1 mutation were significantly correlated with high ALPL expression and poor survival. Accumulation of stem-cell properties due to aberrant DNA hypomethylation is associated with the refractory nature of GBM. Copyright © 2017 by the Congress of Neurological Surgeons

Identification of a Sjögren's syndrome susceptibility locus at OAS1 that influences isoform switching, protein expression, and responsiveness to type I interferons

PubMed Central

Li, He; Reksten, Tove Ragna; Ice, John A.; Kelly, Jennifer A.; Adrianto, Indra; Wang, Shaofeng; He, Bo; Grundahl, Kiely M.; Glenn, Stuart B.; Miceli-Richard, Corinne; Bowman, Simon; Lester, Sue; Eriksson, Per; Brun, Johan G.; Gøransson, Lasse G.; Harboe, Erna; Guthridge, Joel M.; Patel, Ketan; Adler, Adam J.; Farris, A. Darise; Brennan, Michael T.; Chodosh, James; Gopalakrishnan, Rajaram; Weisman, Michael H.; Venuturupalli, Swamy; Wallace, Daniel J.; Hefner, Kimberly S.; Houston, Glen D.; Hughes, Pamela J.; Lewis, David M.; Radfar, Lida; Vista, Evan S.; Rohrer, Michael D.; Stone, Donald U.; Vyse, Timothy J.; Harley, John B.; James, Judith A.; Turner, Sean; Alevizos, Ilias; Anaya, Juan-Manuel; Rhodus, Nelson L.; Segal, Barbara M.; Montgomery, Courtney G.; Scofield, R. Hal; Kovats, Susan; Mariette, Xavier; Witte, Torsten; Rischmueller, Maureen; Omdal, Roald; Lessard, Christopher J.; Sivils, Kathy L.

2017-01-01

Sjögren’s syndrome (SS) is a common, autoimmune exocrinopathy distinguished by keratoconjunctivitis sicca and xerostomia. Patients frequently develop serious complications including lymphoma, pulmonary dysfunction, neuropathy, vasculitis, and debilitating fatigue. Dysregulation of type I interferon (IFN) pathway is a prominent feature of SS and is correlated with increased autoantibody titers and disease severity. To identify genetic determinants of IFN pathway dysregulation in SS, we performed cis-expression quantitative trait locus (eQTL) analyses focusing on differentially expressed type I IFN-inducible transcripts identified through a transcriptome profiling study. Multiple cis-eQTLs were associated with transcript levels of 2'-5'-oligoadenylate synthetase 1 (OAS1) peaking at rs10774671 (PeQTL = 6.05 × 10−14). Association of rs10774671 with SS susceptibility was identified and confirmed through meta-analysis of two independent cohorts (Pmeta = 2.59 × 10−9; odds ratio = 0.75; 95% confidence interval = 0.66–0.86). The risk allele of rs10774671 shifts splicing of OAS1 from production of the p46 isoform to multiple alternative transcripts, including p42, p48, and p44. We found that the isoforms were differentially expressed within each genotype in controls and patients with and without autoantibodies. Furthermore, our results showed that the three alternatively spliced isoforms lacked translational response to type I IFN stimulation. The p48 and p44 isoforms also had impaired protein expression governed by the 3' end of the transcripts. The SS risk allele of rs10774671 has been shown by others to be associated with reduced OAS1 enzymatic activity and ability to clear viral infections, as well as reduced responsiveness to IFN treatment. Our results establish OAS1 as a risk locus for SS and support a potential role for defective viral clearance due to altered IFN response as a genetic pathophysiological basis of this complex autoimmune disease. PMID:28640813

CD44v6 expression in human skin keratinocytes as a possible mechanism for carcinogenesis associated with chronic arsenic exposure.

PubMed

Huang, S; Guo, S; Guo, F; Yang, Q; Xiao, X; Murata, M; Ohnishi, S; Kawanishi, S; Ma, N

2013-01-14

Inorganic arsenic is a well-known human skin carcinogen. Chronic arsenic exposure results in various types of human skin lesions, including squamous cell carcinoma (SCC). To investigate whether mutant stem cells participate in arsenic-associated carcinogenesis, we repeatedly exposed the HaCaT cells line to an environmentally relevant level of arsenic (0.05 ppm) in vitro for 18 weeks. Following sodium arsenic arsenite administration, cell cycle, colony-forming efficiency (CFE), cell tumorigenicity, and expression of CD44v6, NF-κB and p53, were analyzed at different time points (0, 5, 10, 15, 20, 25 and 30 passages). We found that a chronic exposure of HaCaT cells to a low level of arsenic induced a cancer stem- like phenotype. Furthermore, arsenic-treated HaCaT cells also became tumorigenic in nude mice, their growth cycle was predominantly in G2/M and S phases. Relative to nontreated cells, they exhibited a higher growth rate and a significant increase in CFE. Western blot analysis found that arsenic was capable of increasing cell proliferation and sprouting of cancer stem-like phenotype. Additionally, immunohistochemical analysis demonstrated that CD44v6 expression was up-regulated in HaCaT cells exposed to a low level of arsenic during early stages of induction. The expression of CD44v6 in arsenic-treated cells was positively correlated with their cloning efficiency in soft agar (r=0.949, P=0.01). Likewise, the expressions of activating transcription factor NF-κB and p53 genes in the arsenic-treated HaCaT cells were significantly higher than that in non-treated cells. Higher expressions of CD44v6, NF-κB and p53 were also observed in tumor tissues isolated from Balb/c nude mice. The present results suggest that CD44v6 may be a biomarker of arsenic-induced neoplastic transformation in human skin cells, and that arsenic promotes malignant transformation in human skin lesions through a NF-κB signaling pathway-stimulated expression of CD44v6.

Aberrant and alternative splicing in skeletal system disease.

PubMed

Fan, Xin; Tang, Liling

2013-10-01

The main function of skeletal system is to support the body and help movement. A variety of factors can lead to skeletal system disease, including age, exercise, and of course genetic makeup and expression. Pre-mRNA splicing plays a crucial role in gene expression, by creating multiple protein variants with different biological functions. The recent studies show that several skeletal system diseases are related to pre-mRNA splicing. This review focuses on the relationship between pre-mRNA splicing and skeletal system disease. On the one hand, splice site mutation that leads to aberrant splicing often causes genetic skeletal system disease, like COL1A1, SEDL and LRP5. On the other hand, alternative splicing without genomic mutation may generate some marker protein isoforms, for example, FN, VEGF and CD44. Therefore, understanding the relationship between pre-mRNA splicing and skeletal system disease will aid in uncovering the mechanism of disease and contribute to the future development of gene therapy. © 2013 Elsevier B.V. All rights reserved.

CD2-associated Protein (CD2AP) Enhances Casitas B Lineage Lymphoma-3/c (Cbl-3/c)-mediated Ret Isoform-specific Ubiquitination and Degradation via Its Amino-terminal Src Homology 3 Domains*

PubMed Central

Calco, Gina N.; Stephens, Olivia R.; Donahue, Laura M.; Tsui, Cynthia C.; Pierchala, Brian A.

2014-01-01

Ret is the receptor tyrosine kinase for the glial cell line-derived neurotrophic factor (GDNF) family of neuronal growth factors. Upon activation by GDNF, Ret is rapidly polyubiquitinated and degraded. This degradation process is isoform-selective, with the longer Ret51 isoform exhibiting different degradation kinetics than the shorter isoform, Ret9. In sympathetic neurons, Ret degradation is induced, at least in part, by a complex consisting of the adaptor protein CD2AP and the E3-ligase Cbl-3/c. Knockdown of Cbl-3/c using siRNA reduced the GDNF-induced ubiquitination and degradation of Ret51 in neurons and podocytes, suggesting that Cbl-3/c was a predominant E3 ligase for Ret. Coexpression of CD2AP with Cbl-3/c augmented the ubiquitination of Ret51 as compared with the expression of Cbl-3/c alone. Ret51 ubiquitination by the CD2AP·Cbl-3/c complex required a functional ring finger and TKB domain in Cbl-3/c. The SH3 domains of CD2AP were sufficient to drive the Cbl-3/c-dependent ubiquitination of Ret51, whereas the carboxyl-terminal coiled-coil domain of CD2AP was dispensable. Interestingly, activated Ret induced the degradation of CD2AP, but not Cbl-3/c, suggesting a potential inhibitory feedback mechanism. There were only two major ubiquitination sites in Ret51, Lys1060 and Lys1107, and the combined mutation of these lysines almost completely eliminated both the ubiquitination and degradation of Ret51. Ret9 was not ubiquitinated by the CD2AP·Cbl-3/c complex, suggesting that Ret9 was down-regulated by a fundamentally different mechanism. Taken together, these results suggest that only the SH3 domains of CD2AP were necessary to enhance the E3 ligase activity of Cbl-3/c toward Ret51. PMID:24425877

The Expression and Characterization of Functionally Active Soluble CD83 by Pichia pastoris Using High-Density Fermentation

PubMed Central

Song, Xiaoping; Zhong, Yongjun; Wang, Chenguang; Jia, Hao; Wu, Lidan; Wang, Dong; Fang, Fang; Ma, Jiajia; Kang, Wenyao; Sun, Jie; Tian, Zhigang; Xiao, Weihua

2014-01-01

CD83 is a highly glycosylated type I transmembrane glycoprotein that belongs to the immunoglobulin superfamily. CD83 is upregulated during dendritic cell (DC) maturation, which is critical for the initiation of adaptive immune responses. The soluble isoform of CD83 (sCD83) is encoded by alternative splicing from full-length CD83 mRNA and inhibits DC maturation, which suggests that sCD83 acts as a potential immune suppressor. In this study, we developed a sound strategy to express functional sCD83 from Pichia pastoris in extremely high-density fermentation. Purified sCD83 was expressed as a monomer at a yield of more than 200 mg/L and contained N-linked glycosylation sites that were characterized by PNGase F digestion. In vitro tests indicated that recombinant sCD83 bound to its putative counterpart on monocytes and specifically blocked the binding of anti-CD83 antibodies to cell surface CD83 on DCs. Moreover, sCD83 from yeast significantly suppressed ConA-stimulated PBMC proliferation. Therefore, sCD83 that was expressed from the P. pastoris was functionally active and may be used for in vivo and in vitro studies as well as future clinical applications. PMID:24586642

DOE Office of Scientific and Technical Information (OSTI.GOV)

Motegi, Atsushi, E-mail: amotegi@east.ncc.go.jp; Fujii, Satoshi; Zenda, Sadamoto

Purpose: To investigate the significance of CD44 protein expression on the treatment outcomes of radiation therapy in patients with oropharyngeal squamous cell carcinoma (OPSCC) with or without p16 protein expression in the tumor tissue. Methods and Materials: We reviewed the medical records of 58 OPSCC patients who had undergone radiation therapy and examined the tumor tissue expressions of CD44 and p16 protein by immunohistochemical staining. The correlations between the expressions of these proteins and the patients' treatment outcomes were analyzed. Results: The data of 58 consecutive OPSCC patients who had undergone definitive intensity modulated radiation therapy were analyzed. The male/femalemore » ratio was 55:3, and the median age was 64 years. The clinical stage of the disease was stage II in 7 patients, stage III in 5 patients, stage IVA in 35 patients, and stage IVB in 11 patients. Of the patients, 79% received additional induction and/or concurrent chemotherapy. The median follow-up duration was 34 months. The 3-year overall survival, progression-free survival (PFS) and locoregional control (LRC) rates of all the patients, regardless of the results of immunohistochemistry, were 73%, 64% and 76%, respectively. The PFS and LRC rates in the CD44{sup −} patients (86% and 93%, respectively) were significantly higher than those in the CD44{sup +} patients (57% and 70%, respectively). The PFS and LRC rates in the p16{sup +} patients (83% and 90%, respectively) were significantly higher than those in the p16{sup −} patients (45% and 61%, respectively). Patients who were CD44{sup −}/p16{sup +} showed the best LRC rates, and those who were CD44{sup +}/p16{sup −} showed the worst PFS and LRC rates among all the groups. Conclusions: Profiling of CD44 and p16 protein expressions by immunohistochemical staining is useful for predicting the treatment outcomes in patients with OPSCC undergoing definitive intensity modulated radiation therapy.« less

Isolation and characterization of porcine adipose tissue-derived adult stem cells.

PubMed

Williams, Kellie J; Picou, Alicia A; Kish, Sharon L; Giraldo, Angelica M; Godke, Robert A; Bondioli, Kenneth R

2008-01-01

Stem cell characteristics such as self-renewal, differentiation and expression of CD34 and CD44 stem cell markers have not been identified in porcine adipose tissue-derived adult stem (ADAS) cells. The objective of this study was to develop a protocol for the isolation and culture of porcine adipose tissue-derived cells and to determine stem cell-like characteristics. Primary cultures were established and cell cultures were maintained. Cloning capacity was determined using a ring cloning procedure. Primary cultures and clones were differentiated and stained for multiple differentiated phenotypes. CD34 and CD44 messenger ribonucleic acid (mRNA) was isolated and reverse transcriptase polymerase chain reaction was used to compare expression profiles. An average of 2,700,000 nucleated cells/ml was isolated; 26% were adherent, and cells completed a cell cycle approximately every 3.3 days. Ring cloning identified 19 colonies. Primary cultures and clones were determined to differentiate along osteogenic, adipogenic and chondrogenic tissue lineages. The mRNA expression profiles showed CD34 expression was higher for undifferentiated ADAS cells versus differentiated cell types and the CD34 expression level was lower than that of CD44 among differentiated cells. Improved culture conditions and defined cellular characteristics of these porcine ADAS cells have been identified. Porcine ADAS can self-renew, can differentiate into multiple tissue lineages and they express CD34. Copyright 2008 S. Karger AG, Basel.

CD133+CD54+CD44+ circulating tumor cells as a biomarker of treatment selection and liver metastasis in patients with colorectal cancer

PubMed Central

Wang, Cun; Huang, Qiaorong; Meng, Wentong; Yu, Yongyang; Yang, Lie; Peng, Zhihai; Hu, Jiankun; Li, Yuan; Mo, Xianming; Zhou, Zongguang

2016-01-01

Introduction Liver is the most common site of distant metastasis in colorectal cancer (CRC). Early diagnosis and appropriate treatment selection decides overall prognosis of patients. However, current diagnostic measures were basically imaging but not functional. Circulating tumor cells (CTCs) known as hold the key to understand the biology of metastatic mechanism provide a novel and auxiliary diagnostic strategy for CRC with liver metastasis (CRC-LM). Results The expression of CD133+ and CD133+CD54+CD44+ cellular subpopulations were higher in the peripheral blood of CRC-LM patients when compared with those without metastasis (P<0.001). Multivariate analysis proved the association between the expression of CD133+CD44+CD54+ cellular subpopulation and the existence of CRC-LM (P<0.001). The combination of abdominal CT/MRI, CEA and the CD133+CD44+CD54+ cellular subpopulation showed increased detection and discrimination rate for liver metastasis, with a sensitivity of 88.2% and a specificity of 92.4%. Meanwhile, it also show accurate predictive value for liver metastasis (OR=2.898, 95% C.I.1.374–6.110). Materials and Method Flow cytometry and multivariate analysis was performed to detect the expression of cancer initiating cells the correlation between cellular subpopulations and liver metastasis in patients with CRC. The receiver operating characteristic curves combined with the area under the curve were generated to compare the predictive ability of the cellular subpopulation for liver metastasis with current CT and MRI images. Conclusions The identification, expression and application of CTC subpopulations will provide an ideal cellular predictive marker for CRC liver metastasis and a potential marker for further investigation. PMID:27764803

Self-renewal and circulating capacities of metastatic hepatocarcinoma cells required for collaboration between TM4SF5 and CD44

PubMed Central

Lee, Doohyung; Lee, Jung Weon

2015-01-01

Tumor metastasis involves circulating and tumor-initiating capacities of metastatic cancer cells. Hepatic TM4SF5 promotes EMT for malignant growth and migration. Hepatocellular carcinoma (HCC) biomarkers remain unexplored for metastatic potential throughout metastasis. Here, novel TM4SF5/CD44 interaction-mediated self-renewal and circulating tumor cell (CTC) capacities were mechanistically explored. TM4SF5-dependent sphere growth was correlated with CD133+, CD24-, ALDH activity, and a physical association between CD44 and TM4SF5. The TM4SF5/CD44 interaction activated c-Src/STAT3/ Twist1/ B mi1 signaling for spheroid formation, while disturbing the interaction, expression, or activity of any component in this signaling pathway inhibited spheroid formation. In serial xenografts of less than 5,000 cells/injection, TM4SF5-positive tumors exhibited locally-increased CD44 expression, suggesting tumor cell differentiation. TM4SF5-positive cells were identified circulating in blood 4 to 6 weeks after orthotopic liver-injection. Anti-TM4SF reagents blocked their metastasis to distal intestinal organs. Altogether, our results provide evidence that TM4SF5 promotes self-renewal and CTC properties supported by CD133+/TM4SF5+/CD44+(TM4SF5-bound)/ALDH+/ CD24- markers during HCC metastasis. [BMB Reports 2015; 48(3): 127-128] PMID:25772760

CD147, CD44, and the Epidermal Growth Factor Receptor (EGFR) Signaling Pathway Cooperate to Regulate Breast Epithelial Cell Invasiveness*

PubMed Central

Grass, G. Daniel; Tolliver, Lauren B.; Bratoeva, Momka; Toole, Bryan P.

2013-01-01

The immunoglobulin superfamily glycoprotein CD147 (emmprin; basigin) is associated with an invasive phenotype in various types of cancers, including malignant breast cancer. We showed recently that up-regulation of CD147 in non-transformed, non-invasive breast epithelial cells is sufficient to induce an invasive phenotype characterized by membrane type-1 matrix metalloproteinase (MT1-MMP)-dependent invadopodia activity (Grass, G. D., Bratoeva, M., and Toole, B. P. (2012) Regulation of invadopodia formation and activity by CD147. J. Cell Sci. 125, 777–788). Here we found that CD147 induces breast epithelial cell invasiveness by promoting epidermal growth factor receptor (EGFR)-Ras-ERK signaling in a manner dependent on hyaluronan-CD44 interaction. Furthermore, CD147 promotes assembly of signaling complexes containing CD147, CD44, and EGFR in lipid raftlike domains. We also found that oncogenic Ras regulates CD147 expression, hyaluronan synthesis, and formation of CD147-CD44-EGFR complexes, thus forming a positive feedback loop that may amplify invasiveness. Last, we showed that malignant breast cancer cells are heterogeneous in their expression of surface-associated CD147 and that high levels of membrane CD147 correlate with cell surface EGFR and CD44 levels, activated EGFR and ERK1, and activated invadopodia. Future studies should evaluate CD147 as a potential therapeutic target and disease stratification marker in breast cancer. PMID:23888049

Hyaluronan functionalizing QDs as turn-on fluorescent probe for targeted recognition CD44 receptor

NASA Astrophysics Data System (ADS)

Zhou, Shang; Huo, Danqun; Hou, Changjun; Yang, Mei; Fa, Huanbao

2017-09-01

The recognition of tumor markers in living cancer cells has attracted increasing interest. In the present study, the turn-on fluorescence probe was designed based on the fluorescence of thiolated chitosan-coated CdTe QDs (CdTe/TCS QDs) quenched by hyaluronan, which could provide the low background signal for sensitive cellular imaging. This system is expected to offer specific recognition of CD44 receptor over other substances owing to the specific affinity of hyaluronan and CD44 receptor ( 8-9 kcal/mol). The probe is stable in aqueous and has little toxicity to living cells; thus, it can be utilized for targeted cancer cell imaging. The living lung cancer cell imaging experiments further demonstrate its value in recognizing cell-surface CD44 receptor with turn-on mode. In addition, the probe can be used to recognize and differentiate the subtypes of lung cancer cells based on the difference of CD44 expression on the surface of lung cancer cells. And, the western blot test further confirmed that the expression level of the CD44 receptor in lung cancer cells is different. Therefore, this probe may be potentially applied in recognizing lung cancer cells with higher contrast and sensitivity and provide new tools for cancer prognosis and therapy. [Figure not available: see fulltext.

Expression of CD44 and CD29 by PEComa cells suggests their possible origin of mesenchymal stem cells.

PubMed

Liu, Ruixue; Jia, Wei; Zou, Hong; Wang, Xinhua; Ren, Yan; Zhao, Jin; Wang, Lianghai; Li, Man; Qi, Yan; Shen, Yaoyuan; Liang, Weihua; Jiang, Jinfang; Sun, Zhenzhu; Pang, Lijuan; Li, Feng

2015-01-01

Perivascular epithelioid cell tumor (PEComa) is a rare mesenchymal tumor composed of histologically and immunohistochemically distinctive perivascular epithelioid cells. The perivascular epithelioid cell (PEC) co-expresses melanocytic and muscle markers. Since no normal counterpart to the PEC has ever been identified in any normal tissue, the cell origin of these tumors is still uncertain. Although, several hypotheses have recently been advanced to explain the histogenesis of PEComa, it remains unclear. The aim of this study was to discuss whether differential expression of stem cell-associated proteins could be used to aid in determining the histogenesis of PEComa. For this purpose, we detected the immunoexpression of 5 kinds of stem cell markers on PEComas, including CD29, CD44, CD133, ALDH1, and nestin. In addition to observed histopathologic morphology, we also performed PEComa relevant clinical diagnostic markers (HMB-45, SMA, melan-A, Desmin, Ki-67, S-100 and TFE3) to identify whether they belonged to PEComas. Our study included 13 PEComa samples, and we obtained positive immunoexpression results as follows: CD29 (13/13), CD44 (8/13), ALDH1 (10/13), nestin (1/13), and CD133 (0/13). Since CD44 and CD29 are surface proteins associated with MSCs, these results suggest that PEComa might arise from MSCs. However, whether MSCs are the origin of PEComa needs to be further explored in the future.

Prognostic value of CD44 expression in non-small cell lung cancer: a systematic review.

PubMed

Luo, Zhuang; Wu, Rong-Rong; Lv, Liang; Li, Peng; Zhang, Li-Yan; Hao, Qing-Lin; Li, Wei

2014-01-01

CD44 is a potentially interesting prognostic marker and therapeutic target in non-small cell lung cancer (NSCLC). Although the expression of CD44 has been reported to correlate with poor prognosis of NSCLC in most literatures, some controversies still exist. Since the limited patient numbers within independent studies, here we performed a meta-analysis to clarify the correlations between CD44 expression and prognosis and clinicopathological features in NSCLC. Relevant literatures were identified using PubMed, EMBASE and CNKI (China National Knowledge Infrastructure) databases (up to February 2014). Data from eligible studies were extracted and included into meta-analysis using a random effects model. Studies were pooled. Summary hazard ratios (HR) and clinical parameters were calculated. We performed a final analysis of 1772 patients from 23 evaluable studies for Prognostic Value and 2167 patients from 28 evaluable studies for clinicopathological features. Our study shows that the pooled hazard ratio (HR) of overexpression CD44-V6 for overall survival in NSCLC was 1.63 [95% confidence interval (CI): 1.20-2.21] by univariate analysis and 1.29 (95% CI: 0.71-2.37) by multivariate analysis.The pooled HR of overexprssion panCD44 for overall survival in NSCLC was 1.53 (95% CI: 0.58-4.04) by univariate analysis and 3.00 (95% CI: 1.53-5.87) by multivariate analysis. Overexpression of CD44-V6 is associated with tumor differentiation (poor differentiation, OR = 1.66, 95% CI: 1.12-2.45), tumor histological type [squamous cell carcinomas (SCC), OR = 2.6, 95% CI: 1.63-5.02], clinical TMN stage (TMN stage III, OR = 2.22, 95% CI: 1.44-3.43) and lymph node metastasis (N1-3, 3.52, 95% CI: 2.08-5.93) in patients with NSCLC. However, there was no significant association between CD44-V6 and tumor size [T category, OR = 1.42, 95% CI: 0.73-2.78]. Our meta-analysis showed that CD44-V6 is an efficient prognostic factor for NSCLC. Overexpression of CD44-V6 was significantly associated with tumor differentiation, tumor histological type, clinical TMN stage and lymph node metastasis. However, there was no significant association between CD44-V6 and tumor size. Large prospective studies are now needed to confirm the clinical utility of CD44 as an independent prognostic marker.

Cellular FLICE-inhibitory Protein (cFLIP) Isoforms Block CD95- and TRAIL Death Receptor-induced Gene Induction Irrespective of Processing of Caspase-8 or cFLIP in the Death-inducing Signaling Complex*

PubMed Central

Kavuri, Shyam M.; Geserick, Peter; Berg, Daniela; Dimitrova, Diana Panayotova; Feoktistova, Maria; Siegmund, Daniela; Gollnick, Harald; Neumann, Manfred; Wajant, Harald; Leverkus, Martin

2011-01-01

Death receptors (DRs) induce apoptosis but also stimulate proinflammatory “non-apoptotic” signaling (e.g. NF-κB and mitogen-activated protein kinase (MAPK) activation) and inhibit distinct steps of DR-activated maturation of procaspase-8. To examine whether isoforms of cellular FLIP (cFLIP) or its cleavage products differentially regulate DR signaling, we established HaCaT cells expressing cFLIPS, cFLIPL, or mutants of cFLIPL (cFLIPD376N and cFLIPp43). cFLIP variants blocked TRAIL- and CD95L-induced apoptosis, but the cleavage pattern of caspase-8 in the death inducing signaling complex was different: cFLIPL induced processing of caspase-8 to the p43/41 fragments irrespective of cFLIP cleavage. cFLIPS or cFLIPp43 blocked procaspase-8 cleavage. Analyzing non-apoptotic signaling pathways, we found that TRAIL and CD95L activate JNK and p38 within 15 min. cFLIP variants and different caspase inhibitors blocked late death ligand-induced JNK or p38 MAPK activation suggesting that these responses are secondary to cell death. cFLIP isoforms/mutants also blocked death ligand-mediated gene induction of CXCL-8 (IL-8). Knockdown of caspase-8 fully suppressed apoptotic and non-apoptotic signaling. Knockdown of cFLIP isoforms in primary human keratinocytes enhanced CD95L- and TRAIL-induced NF-κB activation, and JNK and p38 activation, underscoring the regulatory role of cFLIP for these DR-mediated signals. Whereas the presence of caspase-8 is critical for apoptotic and non-apoptotic signaling, cFLIP isoforms are potent inhibitors of TRAIL- and CD95L-induced apoptosis, NF-κB activation, and the late JNK and p38 MAPK activation. cFLIP-mediated inhibition of CD95 and TRAIL DR could be of crucial importance during keratinocyte skin carcinogenesis and for the activation of innate and/or adaptive immune responses triggered by DR activation in the skin. PMID:21454681

Clinically relevant morphological structures in breast cancer represent transcriptionally distinct tumor cell populations with varied degrees of epithelial-mesenchymal transition and CD44+CD24- stemness

PubMed Central

Denisov, Evgeny V.; Skryabin, Nikolay A.; Gerashchenko, Tatiana S.; Tashireva, Lubov A.; Wilhelm, Jochen; Buldakov, Mikhail A.; Sleptcov, Aleksei A.; Lebedev, Igor N.; Vtorushin, Sergey V.; Zavyalova, Marina V.; Cherdyntseva, Nadezhda V.; Perelmuter, Vladimir M.

2017-01-01

Intratumor morphological heterogeneity in breast cancer is represented by different morphological structures (tubular, alveolar, solid, trabecular, and discrete) and contributes to poor prognosis; however, the mechanisms involved remain unclear. In this study, we performed 3D imaging, laser microdissection-assisted array comparative genomic hybridization and gene expression microarray analysis of different morphological structures and examined their association with the standard immunohistochemistry scorings and CD44+CD24- cancer stem cells. We found that the intratumor morphological heterogeneity is not associated with chromosomal aberrations. By contrast, morphological structures were characterized by specific gene expression profiles and signaling pathways and significantly differed in progesterone receptor and Ki-67 expression. Most importantly, we observed significant differences between structures in the number of expressed genes of the epithelial and mesenchymal phenotypes and the association with cancer invasion pathways. Tubular (tube-shaped) and alveolar (spheroid-shaped) structures were transcriptionally similar and demonstrated co-expression of epithelial and mesenchymal markers. Solid (large shapeless) structures retained epithelial features but demonstrated an increase in mesenchymal traits and collective cell migration hallmarks. Mesenchymal genes and cancer invasion pathways, as well as Ki-67 expression, were enriched in trabecular (one/two rows of tumor cells) and discrete groups (single cells and/or arrangements of 2-5 cells). Surprisingly, the number of CD44+CD24- cells was found to be the lowest in discrete groups and the highest in alveolar and solid structures. Overall, our findings indicate the association of intratumor morphological heterogeneity in breast cancer with the epithelial-mesenchymal transition and CD44+CD24- stemness and the appeal of this heterogeneity as a model for the study of cancer invasion. PMID:28977854

PDGF Suppresses the Sulfation of CD44v and Potentiates CD44v-Mediated Binding of Colon Carcinoma Cells to Fibrin under Flow

PubMed Central

Alves, Christina S.; Konstantopoulos, Konstantinos

2012-01-01

Fibrin(ogen) mediates sustained tumor cell adhesion and survival in the pulmonary vasculature, thereby facilitating the metastatic dissemination of tumor cells. CD44 is the major functional fibrin receptor on colon carcinoma cells. Growth factors, such as platelet-derived growth factor (PDGF), induce post-translational protein modifications, which modulate ligand binding activity. In view of the roles of PDGF, fibrin(ogen) and CD44 in cancer metastasis, we aimed to delineate the effect of PDGF on CD44-fibrin recognition. By immunoprecipitating CD44 from PDGF-treated and untreated LS174T colon carcinoma cells, which express primarily CD44v, we demonstrate that PDGF enhances the adhesion of CD44v-coated beads to immobilized fibrin. Enzymatic inhibition studies coupled with flow-based adhesion assays and autoradiography reveal that PDGF augments the binding of CD44v to fibrin by significantly attenuating the extent of CD44 sulfation primarily on chondroitin and dermatan sulfate chains. Surface plasmon resonance assays confirm that PDGF enhances the affinity of CD44v-fibrin binding by markedly reducing its dissociation rate while modestly increasing the association rate. PDGF mildly reduces the affinity of CD44v-hyaluronan binding without affecting selectin-CD44v recognition. The latter is attributed to the fact that CD44v binds to selectins via sialofucosylated O-linked residues independent of heparan, dermatan and chondroitin sulfates. Interestingly, PDGF moderately reduces the sulfation of CD44s and CD44s-fibrin recognition. Collectively, these data offer a novel perspective into the mechanism by which PGDF regulates CD44-dependent binding of metastatic colon carcinoma cells to fibrin(ogen). PMID:23056168

Immunohistochemical Analysis of p53, Ki-67, CD44, HER-2/neu Expression Patterns in Gastric Cancer, and Their Association with One Year Survival in North-West of Iran

PubMed Central

Sanaat, Zohreh; Halimi, Monireh; Ghojezadeh, Morteza; Pirovi, Amir Hossein; Gharamaleki, Jalil Vaez; Ziae, Ali Esfahani Jamal Eivazi; Kermani, Iraj Aswadi

2013-01-01

Introduction Gastric cancer remains the second most common cause of cancer-related deaths worldwide. In many malignancies like, lung and breast, multiple prognostic factors are known, such as mutations in Ki-67, HER-2/neu, p53. In this study, we evaluated immunohistochemical protein expression patterns of cell-cycle-regulators p53, proliferation marker Ki-67, surface expression of CD44, HER-2/neu oncogene proposed as useful prognostic factors. Methods In this descriptive-analytic study, we evaluate 100 patients with gastric cancer who were referred to Shahid Ghazi Hospital or other oncology clinics of Tabriz University of Medical Sciences in 2005-2010. Patients with pathologic confirmation of gastric cancer were selected. Expression of p53, ki-67, CD-44, HER-2/neu were detected by immunohistochemical staining. Results In this study, 100 patients with gastric cancer participated. 76(76%) were men and 24(24%) were women with mean age of 64.02(8.05) years. Seventy two samples were intestinal type and 28 were diffuse type. CD44 was positive in 27(27%) patients. P53 was positive in 35(35%) patients. Ki-67 was positive in 53(53%) patients. HER-2/neu was positive in 51(51%) patients. Conclusion The frequency of positive p53, Ki-67, CD44 and HER-2/neu varied in different studies. Positive Ki-67 and HER-2/neu were not associated with changes in survival but positive p53 and CD44 were significantly associated with improved survival. PMID:24505530

Construction of Hyaluronic Tetrasaccharide Clusters Modified Polyamidoamine siRNA Delivery System.

PubMed

Ma, Yingcong; Sha, Meng; Cheng, Shixuan; Yao, Wang; Li, Zhongjun; Qi, Xian-Rong

2018-06-14

The CD44 protein, as a predominant receptor for hyaluronan (HA), is highly expressed on the surface of multiple tumor cells. HA, as a targeting molecule for a CD44-contained delivery system, increases intracellular drug concentration in tumor tissue. However, due to the weak binding ability of hyaluronan oligosaccharide to CD44, targeting for tumor drug delivery has been restricted. In this study, we first use a HA tetrasaccharide cluster as the target ligand to enhance the binding ability to CD44. A polyamidoamine (PAMAM) dendrimer was modified by a HA tetrasaccharide cluster as a nonviral vector for small interfering RNA (siRNA) delivery. The dendrimer/siRNA nanocomplexes increased the cellular uptake capacity of siRNA through the CD44 receptor-mediated endocytosis pathway, allowing the siRNA to successfully escape the endosome/lysosome. Compared with the control group, nanocomplexes effectively reduced the expression of GFP protein and mRNA in MDA-MB-231-GFP cells. This delivery system provides a foundation to increase the clinical applications of PAMAM nanomaterials.

CD44 Interacts with HIF-2α to Modulate the Hypoxic Phenotype of Perinecrotic and Perivascular Glioma Cells.

PubMed

Johansson, Elinn; Grassi, Elisa S; Pantazopoulou, Vasiliki; Tong, Bei; Lindgren, David; Berg, Tracy J; Pietras, Elin J; Axelson, Håkan; Pietras, Alexander

2017-08-15

Hypoxia-inducible factors enhance glioma stemness, and glioma stem cells have an amplified hypoxic response despite residing within a perivascular niche. Still, little is known about differential HIF regulation in stem versus bulk glioma cells. We show that the intracellular domain of stem cell marker CD44 (CD44ICD) is released at hypoxia, binds HIF-2α (but not HIF-1α), enhances HIF target gene activation, and is required for hypoxia-induced stemness in glioma. In a glioma mouse model, CD44 was restricted to hypoxic and perivascular tumor regions, and in human glioma, a hypoxia signature correlated with CD44. The CD44ICD was sufficient to induce hypoxic signaling at perivascular oxygen tensions, and blocking CD44 cleavage decreased HIF-2α stabilization in CD44-expressing cells. Our data indicate that the stem cell marker CD44 modulates the hypoxic response of glioma cells and that the pseudo-hypoxic phenotype of stem-like glioma cells is achieved by stabilization of HIF-2α through interaction with CD44, independently of oxygen. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Characterization of a CD44/CD122int memory CD8 T cell subset generated under sterile inflammatory conditions.

PubMed

Mbitikon-Kobo, Florentin-Martial; Vocanson, Marc; Michallet, Marie-Cécile; Tomkowiak, Martine; Cottalorda, Anne; Angelov, Georgi S; Coupet, Charles-Antoine; Djebali, Sophia; Marçais, Antoine; Dubois, Bertrand; Bonnefoy-Bérard, Nathalie; Nicolas, Jean-François; Arpin, Christophe; Marvel, Jacqueline

2009-03-15

Most memory CD8 T cell subsets that have been hitherto defined are generated in response to infectious pathogens. In this study, we have characterized the CD8 T cells that survive priming conditions, devoid of pathogen-derived danger signals. In both a TCR-transgenic model and a model of contact hypersensitivity, we show that the priming of naive CD8 T cells under sterile inflammatory conditions generates memory. The corresponding memory CD8 T cells can be identified by their intermediate expression levels of CD44 and CD122. We also show that CD44/122(int) memory CD8 T cells spontaneously develop in wild type mice and that they display intermediate levels of several other memory traits including functional (IFN-gamma secretion capacity, CCL5 messenger stores), phenotypic, and molecular (T-bet and eomesodermin expression levels) features. We finally show that they correspond to an early differentiation stage and can further differentiate in CD44/122(high) memory T cells. Altogether, our results identify a new memory CD8 T cell subset that is generated under sterile inflammatory conditions and involved in the recall contact hypersensitivity reactions that are responsible for allergic contact dermatitis.

Cooperativity of CD44 and CD49d in leukemia cell homing, migration, and survival offers a means for therapeutic attack.

PubMed

Singh, Vibuthi; Erb, Ulrike; Zöller, Margot

2013-11-15

A CD44 blockade drives leukemic cells into differentiation and apoptosis by dislodging from the osteogenic niche. Because anti-CD49d also supports hematopoietic stem cell mobilization, we sought to determine the therapeutic efficacy of a joint CD49d/CD44 blockade. To unravel the underlying mechanism, the CD49d(-) EL4 lymphoma was transfected with CD49d or point-mutated CD49d, prohibiting phosphorylation and FAK binding; additionally, a CD44(-) Jurkat subline was transfected with murine CD44, CD44 with a point mutation in the ezrin binding site, or with cytoplasmic tail-truncated CD44. Parental and transfected EL4 and Jurkat cells were evaluated for adhesion, migration, and apoptosis susceptibility in vitro and in vivo. Ligand-binding and Ab-blocking studies revealed CD44-CD49d cooperation in vitro and in vivo in adhesion, migration, and apoptosis resistance. The cooperation depends on ligand-induced proximity such that both CD44 and CD49d get access to src, FAK, and paxillin and via lck to the MAPK pathway, with the latter also supporting antiapoptotic molecule liberation. Accordingly, synergisms were only seen in leukemia cells expressing wild-type CD44 and CD49d. Anti-CD44 together with anti-CD49d efficiently dislodged EL4-CD49d/Jurkat-CD44 in bone marrow and spleen. Dislodging was accompanied by increased apoptosis susceptibility that strengthened low-dose chemotherapy, the combined treatment most strongly interfering with metastatic settlement and being partly curative. Ab treatment also promoted NK and Ab-dependent cellular cytotoxicity activation, which affected leukemia cells independent of CD44/CD49d tail mutations. Thus, mostly owing to a blockade of joint signaling, anti-CD44 and anti-CD49d hamper leukemic cell settlement and break apoptosis resistance, which strongly supports low-dose chemotherapy.

Assessing Specific Oligonucleotides and Small Molecule Antibiotics for the Ability to Inhibit the CRD-BP-CD44 RNA Interaction

PubMed Central

Thomsen, Dana; Lee, Chow H.

2014-01-01

Studies on Coding Region Determinant-Binding Protein (CRD-BP) and its orthologs have confirmed their functional role in mRNA stability and localization. CRD-BP is present in extremely low levels in normal adult tissues, but it is over-expressed in many types of aggressive human cancers and in neonatal tissues. Although the exact role of CRD-BP in tumour progression is unclear, cumulative evidence suggests that its ability to physically associate with target mRNAs is an important criterion for its oncogenic role. CRD-BP has high affinity for the 3′UTR of the oncogenic CD44 mRNA and depletion of CRD-BP in cells led to destabilization of CD44 mRNA, decreased CD44 expression, reduced adhesion and disruption of invadopodia formation. Here, we further characterize the CRD-BP-CD44 RNA interaction and assess specific antisense oligonucleotides and small molecule antibiotics for their ability to inhibit the CRD-BP-CD44 RNA interaction. CRD-BP has a high affinity for binding to CD44 RNA nts 2862–3055 with a Kd of 645 nM. Out of ten antisense oligonucleotides spanning nts 2862–3055, only three antisense oligonucleotides (DD4, DD7 and DD10) were effective in competing with CRD-BP for binding to 32P-labeled CD44 RNA. The potency of DD4, DD7 and DD10 in inhibiting the CRD-BP-CD44 RNA interaction in vitro correlated with their ability to specifically reduce the steady-state level of CD44 mRNA in cells. The aminoglycoside antibiotics neomycin, paramomycin, kanamycin and streptomycin effectively inhibited the CRD-BP-CD44 RNA interaction in vitro. Assessing the potential inhibitory effect of aminoglycoside antibiotics including neomycin on the CRD-BP-CD44 mRNA interaction in cells proved difficult, likely due to their propensity to non-specifically bind nucleic acids. Our results have important implications for future studies in finding small molecules and nucleic acid-based inhibitors that interfere with protein-RNA interactions. PMID:24622399

Assessing specific oligonucleotides and small molecule antibiotics for the ability to inhibit the CRD-BP-CD44 RNA interaction.

PubMed

King, Dustin T; Barnes, Mark; Thomsen, Dana; Lee, Chow H

2014-01-01

Studies on Coding Region Determinant-Binding Protein (CRD-BP) and its orthologs have confirmed their functional role in mRNA stability and localization. CRD-BP is present in extremely low levels in normal adult tissues, but it is over-expressed in many types of aggressive human cancers and in neonatal tissues. Although the exact role of CRD-BP in tumour progression is unclear, cumulative evidence suggests that its ability to physically associate with target mRNAs is an important criterion for its oncogenic role. CRD-BP has high affinity for the 3'UTR of the oncogenic CD44 mRNA and depletion of CRD-BP in cells led to destabilization of CD44 mRNA, decreased CD44 expression, reduced adhesion and disruption of invadopodia formation. Here, we further characterize the CRD-BP-CD44 RNA interaction and assess specific antisense oligonucleotides and small molecule antibiotics for their ability to inhibit the CRD-BP-CD44 RNA interaction. CRD-BP has a high affinity for binding to CD44 RNA nts 2862-3055 with a Kd of 645 nM. Out of ten antisense oligonucleotides spanning nts 2862-3055, only three antisense oligonucleotides (DD4, DD7 and DD10) were effective in competing with CRD-BP for binding to 32P-labeled CD44 RNA. The potency of DD4, DD7 and DD10 in inhibiting the CRD-BP-CD44 RNA interaction in vitro correlated with their ability to specifically reduce the steady-state level of CD44 mRNA in cells. The aminoglycoside antibiotics neomycin, paramomycin, kanamycin and streptomycin effectively inhibited the CRD-BP-CD44 RNA interaction in vitro. Assessing the potential inhibitory effect of aminoglycoside antibiotics including neomycin on the CRD-BP-CD44 mRNA interaction in cells proved difficult, likely due to their propensity to non-specifically bind nucleic acids. Our results have important implications for future studies in finding small molecules and nucleic acid-based inhibitors that interfere with protein-RNA interactions.

Viremic HIV Infected Individuals with High CD4 T Cells and Functional Envelope Proteins Show Anti-gp41 Antibodies with Unique Specificity and Function

PubMed Central

Curriu, Marta; Fausther-Bovendo, Hughes; Pernas, María; Massanella, Marta; Carrillo, Jorge; Cabrera, Cecilia; López-Galíndez, Cecilio; Clotet, Bonaventura; Debré, Patrice; Vieillard, Vincent; Blanco, Julià

2012-01-01

Background CD4 T-cell decay is variable among HIV-infected individuals. In exceptional cases, CD4 T-cell counts remain stable despite high plasma viremia. HIV envelope glycoprotein (Env) properties, namely tropism, fusion or the ability to induce the NK ligand NKp44L, or host factors that modulate Env cytopathic mechanisms may be modified in such situation. Methods We identified untreated HIV-infected individuals showing non-cytopathic replication (VL>10,000 copies/mL and CD4 T-cell decay<50 cells/µL/year, Viremic Non Progressors, VNP) or rapid progression (CD4 T-cells<350 cells/µL within three years post-infection, RP). We isolated full-length Env clones and analyzed their functions (tropism, fusion activity and capacity to induce NKp44L expression on CD4 cells). Anti-Env humoral responses were also analyzed. Results Env clones isolated from VNP or RP individuals showed no major phenotypic differences. The percentage of functional clones was similar in both groups. All clones tested were CCR5-tropic and showed comparable expression and fusogenic activity. Moreover, no differences were observed in their capacity to induce NKp44L expression on CD4 T cells from healthy donors through the 3S epitope of gp41. In contrast, anti- Env antibodies showed clear functional differences: plasma from VNPs had significantly higher capacity than RPs to block NKp44L induction by autologous viruses. Consistently, CD4 T-cells isolated from VNPs showed undetectable NKp44L expression and specific antibodies against a variable region flanking the highly conserved 3S epitope were identified in plasma samples from these patients. Conversely, despite continuous antigen stimulation, VNPs were unable to mount a broad neutralizing response against HIV. Conclusions Env functions (fusion and induction of NKp44L) were similar in viremic patients with slow or rapid progression to AIDS. However, differences in humoral responses against gp41 epitopes nearby 3S sequence may contribute to the lack of CD4 T cell decay in VNPs by blocking the induction of NKp44L by gp41. PMID:22312424

CD44 increases the efficiency of distant metastasis of breast cancer

PubMed Central

McFarlane, Suzanne; Coulter, Jonathan A.; Tibbits, Paul; O'Grady, Anthony; McFarlane, Cheryl; Montgomery, Nicola; Hill, Ashleigh; McCarthy, Helen O.; Young, Leonie S.; Kay, Elaine W.; Isacke, Clare M.; Waugh, David J.J.

2015-01-01

Metastasis is the predominant cause of death from cancer yet we have few biomarkers to predict patients at increased risk of metastasis and are unable to effectively treat disseminated disease. Analysis of 448 primary breast tumors determined that expression of the hylauronan receptor CD44 associated with high grade (p = 0.046), ER- (p = 0.001) and PR-negative tumors (p = 0.029), and correlated with increased distant recurrence and reduced disease-free survival in patients with lymph-node positive or large tumors. To determine its functional role in distant metastasis, CD44 was knocked-down in MDA-MB-231 cells using two independent shRNA sequences. Loss of CD44 attenuated tumor cell adhesion to endothelial cells and reduced cell invasion but did not affect proliferation in vitro. To verify the importance of CD44 to post-intravasation events, tumor formation was assessed by quantitative in vivo imaging and post-mortem tissue analysis following an intra-cardiac injection of transfected cells. CD44 knock-down increased survival and decreased overall tumor burden at multiple sites, including the skeleton in vivo. We conclude that elevated CD44 expression on tumour cells within the systemic circulation increases the efficiency of post-intravasation events and distant metastasis in vivo, consistent with its association with increased distant recurrence and reduced disease-free survival in patients. PMID:25888636

Is sphere assay useful for the identification of cancer initiating cells of the ovary?

PubMed

Martínez-Serrano, María José; Caballero-Baños, Miguel; Vilella, Ramon; Vidal, Laura; Pahisa, Jaume; Martínez-Roman, Sergio

2015-01-01

Current evidence suggests that the presence of tumor-initiating cells (TICs) in epithelial ovarian cancer (EOC) has a role in chemoresistance and relapse. Surface markers such as CD44(+)/CD24(-), CD117(+), and CD133(+) expression have been reported as potential markers for TICs related to ovarian cancer and tumorigenic cell lines. In this study, we have investigated if spheroid forms are TIC specific or whether they can also be produced by somatic stem cells from healthy tissue in vitro. In addition, we also investigated the specificity of surface markers to identify TICs from papillary serous EOC patients. Cells were obtained from fresh tumors from 10 chemotherapy-naive patients with EOC, and cells from ovarian and tubal epithelium were obtained from 5 healthy menopausal women undergoing surgery for benign pathology and cultured in standard and in selective medium. Cells forming nonadherent spheroids were considered TICs, and the adherent cells were considered as non-TIC-like. Percentages of CD24(+), CD44(+), CD117(+), CD133(+), and vascular endothelial growth factor receptor (VEGF-R)(+) cell surface markers were analyzed by flow cytometry. Four of 10 EOC cell tissues were excluded from the study. Tumor cells cultured in selective medium developed spheroid forms after 1 to 7 weeks in 5 of 6 EOC patients. No spheroid forms were observed in cultures of cells from healthy women. Unlike previously published data, low levels of CD24(+), CD44(+), CD117(+), and VEGF-R(+) expression were observed in spheroid cells, whereas expression of CD133(+) was moderate but higher in adherent cells from papillary serous EOC cells in comparison with adherent cells from controls. Papillary serous EOC contains TICs that form spheroids with low expression of CD44(+), CD24(+), CD117(+) and VEGF-R(+). Further research is required to find specific surface markers to identify papillary serous TICs.

Expression of CD44 and CD29 by PEComa cells suggests their possible origin of mesenchymal stem cells

PubMed Central

Liu, Ruixue; Jia, Wei; Zou, Hong; Wang, Xinhua; Ren, Yan; Zhao, Jin; Wang, Lianghai; Li, Man; Qi, Yan; Shen, Yaoyuan; Liang, Weihua; Jiang, Jinfang; Sun, Zhenzhu; Pang, Lijuan; Li, Feng

2015-01-01

Background: Perivascular epithelioid cell tumor (PEComa) is a rare mesenchymal tumor composed of histologically and immunohistochemically distinctive perivascular epithelioid cells. The perivascular epithelioid cell (PEC) co-expresses melanocytic and muscle markers. Since no normal counterpart to the PEC has ever been identified in any normal tissue, the cell origin of these tumors is still uncertain. Although, several hypotheses have recently been advanced to explain the histogenesis of PEComa, it remains unclear. Methods: The aim of this study was to discuss whether differential expression of stem cell-associated proteins could be used to aid in determining the histogenesis of PEComa. For this purpose, we detected the immunoexpression of 5 kinds of stem cell markers on PEComas, including CD29, CD44, CD133, ALDH1, and nestin. In addition to observed histopathologic morphology, we also performed PEComa relevant clinical diagnostic markers (HMB-45, SMA, melan-A, Desmin, Ki-67, S-100 and TFE3) to identify whether they belonged to PEComas. Results: Our study included 13 PEComa samples, and we obtained positive immunoexpression results as follows: CD29 (13/13), CD44 (8/13), ALDH1 (10/13), nestin (1/13), and CD133 (0/13). Conclusions: Since CD44 and CD29 are surface proteins associated with MSCs, these results suggest that PEComa might arise from MSCs. However, whether MSCs are the origin of PEComa needs to be further explored in the future. PMID:26722497

TGF-β reduces DNA ds-break repair mechanisms to heighten genetic diversity and adaptability of CD44+/CD24- cancer cells.

PubMed

Pal, Debjani; Pertot, Anja; Shirole, Nitin H; Yao, Zhan; Anaparthy, Naishitha; Garvin, Tyler; Cox, Hilary; Chang, Kenneth; Rollins, Fred; Kendall, Jude; Edwards, Leyla; Singh, Vijay A; Stone, Gary C; Schatz, Michael C; Hicks, James; Hannon, Gregory J; Sordella, Raffaella

2017-01-16

Many lines of evidence have indicated that both genetic and non-genetic determinants can contribute to intra-tumor heterogeneity and influence cancer outcomes. Among the best described sub-population of cancer cells generated by non-genetic mechanisms are cells characterized by a CD44+/CD24- cell surface marker profile. Here, we report that human CD44+/CD24- cancer cells are genetically highly unstable because of intrinsic defects in their DNA-repair capabilities. In fact, in CD44+/CD24- cells, constitutive activation of the TGF-beta axis was both necessary and sufficient to reduce the expression of genes that are crucial in coordinating DNA damage repair mechanisms. Consequently, we observed that cancer cells that reside in a CD44+/CD24- state are characterized by increased accumulation of DNA copy number alterations, greater genetic diversity and improved adaptability to drug treatment. Together, these data suggest that the transition into a CD44+/CD24- cell state can promote intra-tumor genetic heterogeneity, spur tumor evolution and increase tumor fitness.

Analysis of Cd44-Containing Lipid Rafts

PubMed Central

Oliferenko, Snezhana; Paiha, Karin; Harder, Thomas; Gerke, Volker; Schwärzler, Christoph; Schwarz, Heinz; Beug, Hartmut; Günthert, Ursula; Huber, Lukas A.

1999-01-01

CD44, the major cell surface receptor for hyaluronic acid (HA), was shown to localize to detergent-resistant cholesterol-rich microdomains, called lipid rafts, in fibroblasts and blood cells. Here, we have investigated the molecular environment of CD44 within the plane of the basolateral membrane of polarized mammary epithelial cells. We show that CD44 partitions into lipid rafts that contain annexin II at their cytoplasmic face. Both CD44 and annexin II were released from these lipid rafts by sequestration of plasma membrane cholesterol. Partition of annexin II and CD44 to the same type of lipid rafts was demonstrated by cross-linking experiments in living cells. First, when CD44 was clustered at the cell surface by anti-CD44 antibodies, annexin II was recruited into the cytoplasmic leaflet of CD44 clusters. Second, the formation of intracellular, submembranous annexin II–p11 aggregates caused by expression of a trans-dominant mutant of annexin II resulted in coclustering of CD44. Moreover, a frequent redirection of actin bundles to these clusters was observed. These basolateral CD44/annexin II–lipid raft complexes were stabilized by addition of GTPγS or phalloidin in a semipermeabilized and cholesterol-depleted cell system. The low lateral mobility of CD44 in the plasma membrane, as assessed with fluorescent recovery after photobleaching (FRAP), was dependent on the presence of plasma membrane cholesterol and an intact actin cytoskeleton. Disruption of the actin cytoskeleton dramatically increased the fraction of CD44 which could be recovered from the light detergent-insoluble membrane fraction. Taken together, our data indicate that in mammary epithelial cells the vast majority of CD44 interacts with annexin II in lipid rafts in a cholesterol-dependent manner. These CD44-containing lipid microdomains interact with the underlying actin cytoskeleton. PMID:10459018

β1,4-galactosyltransferase 1 is a novel receptor for IgA in human mesangial cells.

PubMed

Molyneux, Karen; Wimbury, David; Pawluczyk, Izabella; Muto, Masahiro; Bhachu, Jasraj; Mertens, Peter R; Feehally, John; Barratt, Jonathan

2017-12-01

IgA nephropathy is characterized by mesangial deposition of IgA, mesangial cell proliferation, and extracellular matrix production. Mesangial cells bind IgA, but the identity of all potential receptors involved remains incomplete. The transferrin receptor (CD71) acts as a mesangial cell IgA receptor and its expression is upregulated in many forms of glomerulonephritis, including IgA nephropathy. CD71 is not expressed in healthy glomeruli and blocking CD71 does not completely abrogate mesangial cell IgA binding. Previously we showed that mesangial cells express a receptor that binds the Fc portion of IgA and now report that this receptor is an isoform of β-1,4-galactosyltransferase. A human mesangial cell cDNA library was screened for IgA binding proteins and β-1,4-galactosyltransferase identified. Cell surface expression of the long isoform of β-1,4-galactosyltransferase was shown by flow cytometry and confocal microscopy and confirmed by immunoblotting. Glomerular β-1,4-galactosyltransferase expression was increased in IgA nephropathy. IgA binding and IgA-induced mesangial cell phosphorylation of spleen tyrosine kinase and IL-6 synthesis were inhibited by a panel of β-1,4-galactosyltransferase-specific antibodies, suggesting IgA binds to the catalytic domain of β-1,4-galactosyltransferase. Thus, β-1,4-galactosyltransferase is a constitutively expressed mesangial cell IgA receptor with an important role in both mesangial IgA clearance and the initial response to IgA deposition. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

FGF-2 signal promotes proliferation of cerebellar progenitor cells and their oligodendrocytic differentiation at early postnatal stage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Naruse, Masae; Shibasaki, Koji; Ishizaki, Yasuki, E-mail: yasukiishizaki@gunma-u.ac.jp

The origins and developmental regulation of cerebellar oligodendrocytes are largely unknown, although some hypotheses of embryonic origins have been suggested. Neural stem cells exist in the white matter of postnatal cerebellum, but it is unclear whether these neural stem cells generate oligodendrocytes at postnatal stages. We previously showed that cerebellar progenitor cells, including neural stem cells, widely express CD44 at around postnatal day 3. In the present study, we showed that CD44-positive cells prepared from the postnatal day 3 cerebellum gave rise to neurospheres, while CD44-negative cells prepared from the same cerebellum did not. These neurospheres differentiated mainly into oligodendrocytesmore » and astrocytes, suggesting that CD44-positive neural stem/progenitor cells might generate oligodendrocytes in postnatal cerebellum. We cultured CD44-positive cells from the postnatal day 3 cerebellum in the presence of signaling molecules known as mitogens or inductive differentiation factors for oligodendrocyte progenitor cells. Of these, only FGF-2 promoted survival and proliferation of CD44-positive cells, and these cells differentiated into O4+ oligodendrocytes. Furthermore, we examined the effect of FGF-2 on cerebellar oligodendrocyte development ex vivo. FGF-2 enhanced proliferation of oligodendrocyte progenitor cells and increased the number of O4+ and CC1+ oligodendrocytes in slice cultures. These results suggest that CD44-positive cells might be a source of cerebellar oligodendrocytes and that FGF-2 plays important roles in their development at an early postnatal stage. - Highlights: • CD44 is expressed in cerebellar neural stem/progenitor cells at postnatal day 3 (P3). • FGF-2 promoted proliferation of CD44-positive progenitor cells from P3 cerebellum. • FGF-2 promoted oligodendrocytic differentiation of CD44-positive progenitor cells. • FGF-2 increased the number of oligodendrocytes in P3 cerebellar slice culture.« less

CD44-engineered mesoporous silica nanoparticles for overcoming multidrug resistance in breast cancer

NASA Astrophysics Data System (ADS)

Wang, Xin; Liu, Ying; Wang, Shouju; Shi, Donghong; Zhou, Xianguang; Wang, Chunyan; Wu, Jiang; Zeng, Zhiyong; Li, Yanjun; Sun, Jing; Wang, Jiandong; Zhang, Longjiang; Teng, Zhaogang; Lu, Guangming

2015-03-01

Multidrug resistance is a major impediment for the successful chemotherapy in breast cancer. CD44 is over-expressed in multidrug resistant human breast cancer cells. CD44 monoclonal antibody exhibits anticancer potential by inhibiting proliferation and regulating P-glycoprotein-mediated drug efflux activity in multidrug resistant cells. Thereby, CD44 monoclonal antibody in combination with chemotherapeutic drug might be result in enhancing chemosensitivity and overcoming multidrug resistance. The purpose of this study is to investigate the effects of the CD44 monoclonal antibody functionalized mesoporous silica nanoparticles containing doxorubicin on human breast resistant cancer MCF-7 cells. The data showed that CD44-modified mesoporous silica nanoparticles increased cytotoxicity and enhanced the downregulation of P-glycoprotein in comparison to CD44 antibody. Moreover, CD44-engineered mesoporous silica nanoparticles provided active target, which promoted more cellular uptake of DOX in the resistant cells and more retention of DOX in tumor tissues than unengineered counterpart. Animal studies of the resistant breast cancer xenografts demonstrated that CD44-engineered drug delivery system remarkably induced apoptosis and inhibited the tumor growth. Our results indicated that the CD44-engineered mesoporous silica nanoparticle-based drug delivery system offers an effective approach to overcome multidrug resistance in human breast cancer.

Functional Elements on SIRPα IgV domain Mediate Cell Surface Binding to CD47

PubMed Central

Liu, Yuan; Tong, Qiao; Zhou, Yubin; Lee, Hsiau-Wei; Yang, Jenny J.; Bühring, Hans-Jörg; Chen, Yi-Tien; Ha, Binh; Chen, Celia X-J.; Zen, Ke

2007-01-01

Summary SIRPα and SIRPβ1, the two major isoforms of the signal regulatory protein (SIRP) family, are co-expressed in human leukocytes but mediate distinct extracellular binding interactions and divergent cell signaling responses. Previous studies have demonstrated that binding of SIRPα with CD47, another important cell surface molecule, through the extracellular IgV domain regulates important leukocyte functions including macrophage recognition, leukocyte adhesion and transmigration. Although SIRPβ1 shares highly homologous extracellular IgV structure with SIRPα, it does not bind to CD47. In this study, we defined key amino acid residues exclusively expressing in the IgV domain of SIRPα, but not SIRPβ1, which determine the extracellular binding interaction of SIRPα to CD47. These key residues include Gln67, a small hydrophobic amino acid (Ala or Val) at the 57th position and Met102. We found that Gln67 and Ala/Val57 are critical. Mutation of either of these residues abates SIRPα directly binding to CD47. Functional cell adhesion and leukocyte transmigration assays further demonstrated central roles of Gln67 and Ala/Val57 in SIRPα extracellular binding mediated cell interactions and cell migration. Another SIRPα-specific residue, Met102, appears to assist SIRPα IgV binding through Gln67 and Ala/Val57. An essential role of these amino acids in SIRPα binding to CD47 was further confirmed by introducing these residues into the SIRPβ1 IgV domain, which dramatically converts SIRPβ1 into a CD47-binding molecule. Our results thus revealed the molecular basis by which SIRPα selectively binds to CD47 and shed new light into the structural mechanisms of SIRP isoform mediated distinctive extracellular interactions and cellular responses. PMID:17070842

Functional elements on SIRPalpha IgV domain mediate cell surface binding to CD47.

PubMed

Liu, Yuan; Tong, Qiao; Zhou, Yubin; Lee, Hsiau-Wei; Yang, Jenny J; Bühring, Hans-Jörg; Chen, Yi-Tien; Ha, Binh; Chen, Celia X-J; Yang, Yang; Zen, Ke

2007-01-19

SIRPalpha and SIRPbeta1, the two major isoforms of the signal regulatory protein (SIRP) family, are co-expressed in human leukocytes but mediate distinct extracellular binding interactions and divergent cell signaling responses. Previous studies have demonstrated that binding of SIRPalpha with CD47, another important cell surface molecule, through the extracellular IgV domain regulates important leukocyte functions including macrophage recognition, leukocyte adhesion and transmigration. Although SIRPbeta1 shares highly homologous extracellular IgV structure with SIRPalpha, it does not bind to CD47. Here, we defined key amino acid residues exclusively expressing in the IgV domain of SIRPalpha, but not SIRPbeta1, which determine the extracellular binding interaction of SIRPalpha to CD47. These key residues include Gln67, a small hydrophobic amino acid (Ala or Val) at the 57th position and Met102. We found that Gln67 and Ala/Val57 are critical. Mutation of either of these residues abates SIRPalpha directly binding to CD47. Functional cell adhesion and leukocyte transmigration assays further demonstrated central roles of Gln67 and Ala/Val57 in SIRPalpha extracellular binding mediated cell interactions and cell migration. Another SIRPalpha-specific residue, Met102, appears to assist SIRPalpha IgV binding through Gln67 and Ala/Val57. An essential role of these amino acid residues in SIRPalpha binding to CD47 was further confirmed by introducing these residues into the SIRPbeta1 IgV domain, which dramatically converts SIRPbeta1 into a CD47-binding molecule. Our results thus revealed the molecular basis by which SIRPalpha binds to CD47 and shed new light into the structural mechanisms of SIRP isoform mediated distinctive extracellular interactions and cellular responses.

Multifunctionalized iron oxide nanoparticles for selective drug delivery to CD44-positive cancer cells

NASA Astrophysics Data System (ADS)

Aires, Antonio; Ocampo, Sandra M.; Simões, Bruno M.; Josefa Rodríguez, María; Cadenas, Jael F.; Couleaud, Pierre; Spence, Katherine; Latorre, Alfonso; Miranda, Rodolfo; Somoza, Álvaro; Clarke, Robert B.; Carrascosa, José L.; Cortajarena, Aitziber L.

2016-02-01

Nanomedicine nowadays offers novel solutions in cancer therapy and diagnosis by introducing multimodal treatments and imaging tools in one single formulation. Nanoparticles acting as nanocarriers change the solubility, biodistribution and efficiency of therapeutic molecules, reducing their side effects. In order to successfully apply these novel therapeutic approaches, efforts are focused on the biological functionalization of the nanoparticles to improve the selectivity towards cancer cells. In this work, we present the synthesis and characterization of novel multifunctionalized iron oxide magnetic nanoparticles (MNPs) with antiCD44 antibody and gemcitabine derivatives, and their application for the selective treatment of CD44-positive cancer cells. The lymphocyte homing receptor CD44 is overexpressed in a large variety of cancer cells, but also in cancer stem cells (CSCs) and circulating tumor cells (CTCs). Therefore, targeting CD44-overexpressing cells is a challenging and promising anticancer strategy. Firstly, we demonstrate the targeting of antiCD44 functionalized MNPs to different CD44-positive cancer cell lines using a CD44-negative non-tumorigenic cell line as a control, and verify the specificity by ultrastructural characterization and downregulation of CD44 expression. Finally, we show the selective drug delivery potential of the MNPs by the killing of CD44-positive cancer cells using a CD44-negative non-tumorigenic cell line as a control. In conclusion, the proposed multifunctionalized MNPs represent an excellent biocompatible nanoplatform for selective CD44-positive cancer therapy in vitro.

Apigenin inhibited hypoxia induced stem cell marker expression in a head and neck squamous cell carcinoma cell line.

PubMed

Ketkaew, Yuwaporn; Osathanon, Thanaphum; Pavasant, Prasit; Sooampon, Sireerat

2017-02-01

Cancer stem cells contribute to tumor recurrence, and a hypoxic environment is critical for maintaining cancer stem cells. Apigenin is a natural product with anticancer activity. However, the effect of apigenin on cancer stem cells remains unclear. Our aim was to investigate the effect of apigenin on cancer stem cell marker expression in head and neck squamous cell carcinoma cells under hypoxia. We used three head and neck squamous cell carcinoma cell lines; HN-8, HN-30, and HSC-3. The mRNA expression of cancer stem cell markers was determined by semiquantitative RT-PCR and Real-time PCR. The cytotoxic effect of apigenin was determined by MTT colorimetric assay. Flow cytometry was used to reveal the number of cells expressing cancer stem cell surface markers. HN-30 cells, a cancer cell line from the pharynx, showed the greatest response to hypoxia by increasing their expression of CD44, CD105, NANOG, OCT-4, REX-1, and VEGF. Apigenin significantly decreased HN-30 cell viability in dose- and time-dependent manners. In addition, 40μM apigenin significantly down-regulated the mRNA expression of CD44, NANOG, and CD105. Consistent with these results, the hypoxia-induced increase in CD44 + cells, CD105 + cells, and STRO-1 + cells was significantly abolished by apigenin. Apigenin suppresses cancer stem cell marker expression and the number of cells expressing cell surface markers under hypoxia. Copyright © 2016 Elsevier Ltd. All rights reserved.

Intracellular targeting of CD44+ cells with self-assembling, protein only nanoparticles.

PubMed

Pesarrodona, Mireia; Ferrer-Miralles, Neus; Unzueta, Ugutz; Gener, Petra; Tatkiewicz, Witold; Abasolo, Ibane; Ratera, Imma; Veciana, Jaume; Schwartz, Simó; Villaverde, Antonio; Vazquez, Esther

2014-10-01

CD44 is a multifunctional cell surface protein involved in proliferation and differentiation, angiogenesis and signaling. The expression of CD44 is up-regulated in several types of human tumors and particularly in cancer stem cells, representing an appealing target for drug delivery in the treatment of cancer. We have explored here several protein ligands of CD44 for the construction of self-assembling modular proteins designed to bind and internalize target cells. Among five tested ligands, two of them (A5G27 and FNI/II/V) drive the formation of protein-only, ring-shaped nanoparticles of about 14 nm that efficiently bind and penetrate CD44(+) cells by an endosomal route. The potential of these newly designed nanoparticles is evaluated regarding the need of biocompatible nanostructured materials for drug delivery in CD44-linked conditions. Copyright © 2014 Elsevier B.V. All rights reserved.

Association of Glioblastoma Multiforme Stem Cell Characteristics, Differentiation, and Microglia Marker Genes with Patient Survival

PubMed Central

Balz, Ellen; Herzog, Susann; Plantera, Laura; Vogelgesang, Silke; Seifert, Carolin; Bialke, Angela; Venugopal, Chitra; Singh, Sheila K.; Hoffmann, Wolfgang; Schroeder, Henry W. S.

2018-01-01

Patients with glioblastoma multiforme (GBM) are at high risk to develop a relapse despite multimodal therapy. Assumedly, glioma stem cells (GSCs) are responsible for treatment resistance of GBM. Identification of specific GSC markers may help to develop targeted therapies. Here, we performed expression analyses of stem cell (ABCG2, CD44, CD95, CD133, ELF4, Nanog, and Nestin) as well as differentiation and microglia markers (GFAP, Iba1, and Sparc) in GBM compared to nonmalignant brain. Furthermore, the role of these proteins for patient survival and their expression in LN18 stem-like neurospheres was analyzed. At mRNA level, ABCG2 and CD95 were reduced, GFAP was unchanged; all other investigated markers were increased in GBM. At protein level, CD44, ELF4, Nanog, Nestin, and Sparc were elevated in GBM, but only CD133 and Nestin were strongly associated with survival time. In addition, ABCG2 and GFAP expression was decreased in LN18 neurospheres whereas CD44, CD95, CD133, ELF4, Nanog, Nestin, and Sparc were upregulated. Altogether only CD133 and Nestin were associated with survival rates. This raises concerns regarding the suitability of the other target structures as prognostic markers, but makes both CD133 and Nestin candidates for GBM therapy. Nevertheless, a search for more specific marker proteins is urgently needed. PMID:29535786

Conserved regulation of mesenchymal gene expression by Fgf-8 in face and limb development.

PubMed

Tucker, A S; Al Khamis, A; Ferguson, C A; Bach, I; Rosenfeld, M G; Sharpe, P T

1999-01-01

Clim-2 (NLI, Lbd1) is one of two related mouse proteins that interact with Lim-domain homeoproteins. In the mouse, embryonic expression of Clim-2 is particularly pronounced in facial ectomesenchyme and limb bud mesenchyme in association with Lim genes, Lhx-6 and Lmx-1 respectively. We show that in common with both these Lim genes, Clim-2 expression is regulated by signals from overlying epithelium. In both the developing face and the limb buds we identify Fgf-8 as the likely candidate signalling molecule that regulates Clim-2 expression. We show that in the mandibular arch, as in the limb, Fgf-8 functions in combination with CD44, a cell surface binding protein, and that blocking CD44 binding results in inhibition of Fgf8-induced expression of Clim-2 and Lhx-6. Regulation of gene expression by Fgf8 in association with CD44 is thus conserved between limb and mandibular arch development.

Differential cross-reactivity of monoclonal antibody OPD4 (anti-CD45RO) in macaques.

PubMed

Wang, Xiaolei; Pahar, Bapi; Rasmussen, Terri; Alvarez, Xavier; Dufour, Jason; Rasmussen, Kelsi; Lackner, Andrew A; Veazey, Ronald S

2008-01-01

Immunologic research in nonhuman primates is occasionally limited by the availability of reagents that cross-react in nonhuman primates. One major limitation has been the lack of a monoclonal antibody to CD45RO. Although the monoclonal antibody UCHL-1 is used to detect CD45RO isoforms in humans, it does not react with nonhuman primates, mandating the use of alternative strategies to define "memory" T cell responses in nonhuman primates. The current study examined the reactivity and specificity of another antibody against CD45RO, clone OPD4, in macaques. Here we demonstrate that OPD4 specifically labels memory CD4+ T cells in approximately 44% of rhesus macaques (Macaca mulatta) of Indian but not Chinese origin. In contrast, tissues from pigtail macaques (Macaca nemestrina) react with this clone, indicating that OPD4 may be useful for examining memory CD4+ T cells in certain macaques, but its utility may be limited in other species or even among individual macaques.

Differential cross-reactivity of monoclonal antibody OPD4 (anti-CD45RO) in macaques

PubMed Central

Wang, Xiaolei; Pahar, Bapi; Rasmussen, Terri; Alvarez, Xavier; Dufour, Jason; Rasmussen, Kelsi; Lackner, Andrew A.; Veazey, Ronald S.

2008-01-01

Immunologic research in nonhuman primates is occasionally limited by the availability of reagents that cross react in nonhuman primates. One major limitation has been the lack of a monoclonal antibody to CD45RO. Although the monoclonal antibody UCHL-1 is used to detect CD45RO isoforms in humans, it does not react with nonhuman primates, mandating the use of alternative strategies to define “memory” T cell responses in nonhuman primates. The current study examined the reactivity and specificity of another antibody against CD45RO, clone OPD4, in macaques. Here we demonstrate that OPD4 specifically labels memory CD4+ T cells in ~44% of rhesus macaques (Macaca mulatta) of Indian, but not Chinese origin. In contrast, tissues from pigtail macaques (Macaca nemestrina) react with this clone, indicating that OPD4 may be useful for examining memory CD4+ T cells in certain macaques, but its utility may be limited in other species or even among individual macaques. PMID:18304631

TGF-β reduces DNA ds-break repair mechanisms to heighten genetic diversity and adaptability of CD44+/CD24− cancer cells

PubMed Central

Pal, Debjani; Pertot, Anja; Shirole, Nitin H; Yao, Zhan; Anaparthy, Naishitha; Garvin, Tyler; Cox, Hilary; Chang, Kenneth; Rollins, Fred; Kendall, Jude; Edwards, Leyla; Singh, Vijay A; Stone, Gary C; Schatz, Michael C; Hicks, James; Hannon, Gregory J; Sordella, Raffaella

2017-01-01

Many lines of evidence have indicated that both genetic and non-genetic determinants can contribute to intra-tumor heterogeneity and influence cancer outcomes. Among the best described sub-population of cancer cells generated by non-genetic mechanisms are cells characterized by a CD44+/CD24− cell surface marker profile. Here, we report that human CD44+/CD24− cancer cells are genetically highly unstable because of intrinsic defects in their DNA-repair capabilities. In fact, in CD44+/CD24− cells, constitutive activation of the TGF-beta axis was both necessary and sufficient to reduce the expression of genes that are crucial in coordinating DNA damage repair mechanisms. Consequently, we observed that cancer cells that reside in a CD44+/CD24− state are characterized by increased accumulation of DNA copy number alterations, greater genetic diversity and improved adaptability to drug treatment. Together, these data suggest that the transition into a CD44+/CD24− cell state can promote intra-tumor genetic heterogeneity, spur tumor evolution and increase tumor fitness. DOI: http://dx.doi.org/10.7554/eLife.21615.001 PMID:28092266

CD33: increased inclusion of exon 2 implicates the Ig V-set domain in Alzheimer's disease susceptibility

PubMed Central

Raj, Towfique; Ryan, Katie J.; Replogle, Joseph M.; Chibnik, Lori B.; Rosenkrantz, Laura; Tang, Anna; Rothamel, Katie; Stranger, Barbara E.; Bennett, David A.; Evans, Denis A.; De Jager, Philip L.; Bradshaw, Elizabeth M.

2014-01-01

We previously demonstrated that the Alzheimer's disease (AD) associated risk allele, rs3865444C, results in a higher surface density of CD33 on monocytes. Here, we find alternative splicing of exon 2 to be the primary mechanism of the genetically driven differential expression of CD33 protein. We report that the risk allele, rs3865444C, is associated with greater cell surface expression of CD33 in both subjects of European and African–American ancestry and that there is a single haplotype influencing CD33 surface expression. A meta-analysis of the two populations narrowed the number of significant SNPs in high linkage disequilibrium (LD) (r2 > 0.8) with rs3865444 to just five putative causal variants associated with increased protein expression. Using gene expression data from flow-sorted CD14+CD16− monocytes from 398 healthy subjects of three populations, we show that the rs3865444C risk allele is strongly associated with greater expression of CD33 exon 2 (pMETA = 2.36 × 10−60). Western blotting confirms increased protein expression of the full-length CD33 isoform containing exon 2 relative to the rs3865444C allele (P < 0.0001). Of the variants in strong LD with rs3865444, rs12459419, which is located in a putative SRSF2 splice site of exon 2, is the most likely candidate to mediate the altered alternative splicing of CD33's Immunoglobulin V-set domain 2 and ultimately influence AD susceptibility. PMID:24381305

Cancer stem cell markers in patterning differentiation and in prognosis of oral squamous cell carcinoma.

PubMed

Mohanta, Simple; Siddappa, Gangotri; Valiyaveedan, Sindhu Govindan; Dodda Thimmasandra Ramanjanappa, Ravindra; Das, Debashish; Pandian, Ramanan; Khora, Samanta Sekhar; Kuriakose, Moni Abraham; Suresh, Amritha

2017-06-01

Differentiation is a major histological parameter determining tumor aggressiveness and prognosis of the patient; cancer stem cells with their slow dividing and undifferentiated nature might be one of the factors determining the same. This study aims to correlate cancer stem cell markers (CD44 and CD147) with tumor differentiation and evaluate their subsequent effect on prognosis. Immunohistochemical analysis in treatment naïve oral cancer patients (n = 53) indicated that the expression of CD147 was associated with poorly differentiated squamous cell carcinoma and moderately differentiated squamous cell carcinoma (p < 0.01). Furthermore, co-expression analysis showed that 45% each of moderately differentiated squamous cell carcinoma and poorly differentiated squamous cell carcinoma patients were CD44 high /CD147 high as compared to only 10% of patients with well-differentiated squamous cell carcinoma. A three-way analysis indicated that differentiation correlated with recurrence and survival (p < 0.05) in only the patients with CD44 high /CD147 high cohort. Subsequently, relevance of these cancer stem cell markers in patterning the differentiation characteristics was evaluated in oral squamous cell carcinoma cell lines originating from different grades of oral cancer. Flowcytometry-based analysis indicated an increase in CD44 + /CD147 + cells in cell lines of poorly differentiated squamous cell carcinoma (94.35 ± 1.14%, p < 0.001) and moderately differentiated squamous cell carcinoma origin (93.49 ± 0.47%, p < 0.001) as compared to cell line of well-differentiated squamous cell carcinoma origin (23.12% ± 0.49%). Expression profiling indicated higher expression of cancer stem cell and epithelial-mesenchymal transition markers in SCC029B (poorly differentiated squamous cell carcinoma originated; p ≤ 0.001), which was further translated into increased spheroid formation, migration, and invasion (p < 0.001) as compared to cell line of well-differentiated squamous cell carcinoma origin. This study suggests that CD44 and CD147 together improve the prognostic efficacy of tumor differentiation; in vitro results further point out that these markers might be determinant of differentiation characteristics, imparting properties of increased self-renewal, migration, and invasion.

Association of CD147 and Calcium Exporter PMCA4 Uncouples IL-2 Expression from Early TCR Signaling.

PubMed

Supper, Verena; Schiller, Herbert B; Paster, Wolfgang; Forster, Florian; Boulègue, Cyril; Mitulovic, Goran; Leksa, Vladimir; Ohradanova-Repic, Anna; Machacek, Christian; Schatzlmaier, Philipp; Zlabinger, Gerhard J; Stockinger, Hannes

2016-02-01

The Ig superfamily member CD147 is upregulated following T cell activation and was shown to serve as a negative regulator of T cell proliferation. Thus, Abs targeting CD147 are being tested as new treatment strategies for cancer and autoimmune diseases. How CD147 mediates immunosuppression and whether association with other coreceptor complexes is needed have remained unknown. In the current study, we show that silencing of CD147 in human T cells increases IL-2 production without affecting the TCR proximal signaling components. We mapped the immunosuppressive moieties of CD147 to its transmembrane domain and Ig-like domain II. Using affinity purification combined with mass spectrometry, we determined the domain specificity of CD147 interaction partners and identified the calcium exporter plasma membrane calcium ATPase isoform 4 (PMCA4) as the interaction partner of the immunosuppressive moieties of CD147. CD147 does not control the proper membrane localization of PMCA4, but PMCA4 is essential for the CD147-dependent inhibition of IL-2 expression via a calcium-independent mechanism. In summary, our data show that CD147 interacts via its immunomodulatory domains with PMCA4 to bypass TCR proximal signaling and inhibit IL-2 expression. Copyright © 2016 by The American Association of Immunologists, Inc.

Glycogen Synthase Kinase 3 Inactivation Drives T-bet-Mediated Downregulation of Co-receptor PD-1 to Enhance CD8+ Cytolytic T Cell Responses

PubMed Central

Taylor, Alison; Harker, James A.; Chanthong, Kittiphat; Stevenson, Philip G.; Zuniga, Elina I.; Rudd, Christopher E.

2016-01-01

Summary Despite the importance of the co-receptor PD-1 in T cell immunity, the upstream signaling pathway that regulates PD-1 expression has not been defined. Glycogen synthase kinase 3 (GSK-3, isoforms α and β) is a serine-threonine kinase implicated in cellular processes. Here, we identified GSK-3 as a key upstream kinase that regulated PD-1 expression in CD8+ T cells. GSK-3 siRNA downregulation, or inhibition by small molecules, blocked PD-1 expression, resulting in increased CD8+ cytotoxic T lymphocyte (CTL) function. Mechanistically, GSK-3 inactivation increased Tbx21 transcription, promoting enhanced T-bet expression and subsequent suppression of Pdcd1 (encodes PD-1) transcription in CD8+ CTLs. Injection of GSK-3 inhibitors in mice increased in vivo CD8+ OT-I CTL function and the clearance of murine gamma-herpesvirus 68 and lymphocytic choriomeningitis clone 13 and reversed T cell exhaustion. Our findings identify GSK-3 as a regulator of PD-1 expression and demonstrate the applicability of GSK-3 inhibitors in the modulation of PD-1 in immunotherapy. PMID:26885856

The Prognostic Role of Cancer Stem Cell Markers for Long-term Outcome After Resection of Colonic Liver Metastases.

PubMed

Spelt, Lidewij; Sasor, Agata; Ansari, Daniel; Hilmersson, Katarzyna Said; Andersson, Roland

2018-01-01

To assess the expression of cancer stem cell (CSC) markers CD44, CD133 and CD24 in colon cancer liver metastases and analyse their predictive value for overall survival (OS) and disease-free survival (DFS) after liver resection. Patients operated on for colon cancer liver metastases were included. CSC marker expression was determined through immunohistochemistry analysis. OS and DFS were compared between marker-positive and marker-negative patients. Multivariate analysis was performed to select predictive variables for OS and DFS. CD133-positive patients had a worse DFS than CD133-negative patients, with a median DFS of 12 and 25 months (p=0.051). Multivariate analysis selected CD133 expression as a significant predictor for DFS. CD44 and CD24 were not found to predict OS or DFS. CD133 expression in colonic liver metastases is a negative prognostic factor for DFS after liver resection. In the future, CD133 could be used as a biomarker for risk stratification, and possibly for developing novel targeted therapy. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

[Effect of Biejiajian Pills on Wnt signal pathway molecules β-catenin and GSK-3β and the target genes CD44v6 and VEGF in hepatocellular carcinoma cells].

PubMed

Sun, Haitao; He, Songqi; Wen, Bin; Jia, Wenyan; Fan, Eryan; Zheng, Yan

2014-10-01

To investigate the effect of Biejiajian Pills on the expressions of the signal molecules and target genes of Wnt signal pathway in HepG2 cells and explore the mechanisms by which Biejiajian pills suppress the invasiveness of hepatocellular carcinoma. HepG2 cells were cultured for 48 h in the presence of serum collected from rats fed with Biejiajian Pills. The expressions of β-catenin, GSK-3β and P-GSK-3β in the cultured cells were assessed by Western blotting and the expressions of CD44v6 and VEGF were detected using immunohistochemistry. HepG2 cells cultured with the serum of rats fed with Biejiajian Pills showed lowered expressions of β-catenin protein both in the cytoplasm and the nuclei with also inhibition of phosphorylation of GSK-3β and reduced expression of CD44v6 and VEGF. Biejiajian Pills can significantly reduce the expression of β-catenin by decreasing the phosphorylation of GSK-3β and blocking the Wnt/β-catenin signaling pathway to cause down-regulation of the target genes CD44v6 and VEGF, which may be one of the molecular mechanisms by which Biejiajian Pills suppress the proliferation and invasiveness of hepatocellular carcinoma.

Glomerular parietal epithelial cells contribute to adult podocyte regeneration in experimental focal segmental glomerulosclerosis

PubMed Central

Eng, Diana G.; Sunseri, Maria W.; Kaverina, Natalya; Roeder, Sebastian S.; Pippin, Jeffrey W.; Shankland, Stuart J.

2015-01-01

Since adult podocytes cannot adequately proliferate following depletion in disease states there has been interest in the potential role of progenitors in podocyte repair and regeneration. To determine if parietal epithelial cells (PECs) can serve as adult podocyte progenitors following disease-induced podocyte depletion, PECs were permanently labeled in adult PECrtTA/LC1/R26 reporter mice. In normal mice, labeled PECs were confined to Bowman's capsule, while in disease (cytotoxic sheep anti-podocyte antibody), labeled PECs were found in the glomerular tuft in progressively higher numbers by days 7, 14 and 28. Early in disease, the majority of PECs in the tuft co-expressed CD44. By day 28, when podocyte numbers were significantly higher and disease severity was significantly lower, the majority of labeled PECs co-expressed podocyte proteins but not CD44. Neither labeled PECs on the tuft, nor podocytes stained for the proliferation marker BrdU. The de novo expression of phospho-ERK colocalized to CD44 expressing PECs, but not to PECs expressing podocyte markers. Thus, in a mouse model of focal segmental glomerulosclerosis typified by abrupt podocyte depletion followed by regeneration, PECs undergo two phenotypic changes once they migrate to the glomerular tuft. Initially these cells are predominantly activated CD44 expressing cells coinciding with glomerulosclerosis, and later they predominantly exhibit a podocyte phenotype which is likely reparative. PMID:25993321

Angiotensin II AT1 receptor alters ACE2 activity, eNOS expression and CD44-hyaluronan interaction in rats with hypertension and myocardial fibrosis.

PubMed

Bai, Feng; Pang, Xue-Fen; Zhang, Li-Hui; Wang, Ning-Ping; McKallip, Robert J; Garner, Ronald E; Zhao, Zhi-Qing

2016-05-15

This study tested the hypothesis that angiotensin II (Ang II) AT1 receptor is involved in development of hypertension and cardiac fibrosis via modifying ACE2 activity, eNOS expression and CD44-hyaluronan interaction. Male Sprague-Dawley rats were subjected to Ang II infusion (500ng/kg/min) using osmotic minipumps up to 4weeks and the AT1 receptor blocker, telmisartan was administered by gastric gavage (10mg/kg/day) during Ang II infusion. Our results indicated that Ang II enhances AT1 receptor, downregulates AT2 receptor, ACE2 activity and eNOS expression, and increases CD44 expression and hyaluronidase activity, an enzyme for hyaluronan degradation. Further analyses revealed that Ang II increases blood pressure and augments vascular/interstitial fibrosis. Comparison of the Ang II group, treatment with telmisartan significantly increased ACE2 activity and eNOS expression in the intracardiac vessels and intermyocardium. These changes occurred in coincidence with decreased blood pressure. Furthermore, the locally-expressed AT1 receptor was downregulated, as evidenced by an increased ratio of the AT2 over AT1 receptor (1.4±0.4% vs. 0.4±0.1% in Ang II group, P<0.05). Along with these modulations, telmisartan inhibited membrane CD44 expression and hyaluronidase activity, decreased populations of macrophages and myofibroblasts, and reduced expression of TGFβ1 and Smads. Collagen I synthesis and tissue fibrosis were attenuated as demonstrated by the less extensive collagen-rich area. These results suggest that the AT1 receptor is involved in development of hypertension and cardiac fibrosis. Selective activating ACE2/eNOS and inhibiting CD44/HA interaction might be considered as the therapeutic targets for attenuating Ang II induced deleterious cardiovascular effects. Copyright © 2016 Elsevier Inc. All rights reserved.

Interplay between estrogen receptor and AKT in Estradiol-induced alternative splicing

PubMed Central

2013-01-01

Background Alternative splicing is critical for generating complex proteomes in response to extracellular signals. Nuclear receptors including estrogen receptor alpha (ERα) and their ligands promote alternative splicing. The endogenous targets of ERα:estradiol (E2)-mediated alternative splicing and the influence of extracellular kinases that phosphorylate ERα on E2-induced splicing are unknown. Methods MCF-7 and its anti-estrogen derivatives were used for the majority of the assays. CD44 mini gene was used to measure the effect of E2 and AKT on alternative splicing. ExonHit array analysis was performed to identify E2 and AKT-regulated endogenous alternatively spliced apoptosis-related genes. Quantitative reverse transcription polymerase chain reaction was performed to verify alternative splicing. ERα binding to alternatively spliced genes was verified by chromatin immunoprecipitation assay. Bromodeoxyuridine incorporation-ELISA and Annexin V labeling assays were done to measure cell proliferation and apoptosis, respectively. Results We identified the targets of E2-induced alternative splicing and deconstructed some of the mechanisms surrounding E2-induced splicing by combining splice array with ERα cistrome and gene expression array. E2-induced alternatively spliced genes fall into at least two subgroups: coupled to E2-regulated transcription and ERα binding to the gene without an effect on rate of transcription. Further, AKT, which phosphorylates both ERα and splicing factors, influenced ERα:E2 dependent splicing in a gene-specific manner. Genes that are alternatively spliced include FAS/CD95, FGFR2, and AXIN-1. E2 increased the expression of FGFR2 C1 isoform but reduced C3 isoform at mRNA level. E2-induced alternative splicing of FAS and FGFR2 in MCF-7 cells correlated with resistance to FAS activation-induced apoptosis and response to keratinocyte growth factor (KGF), respectively. Resistance of MCF-7 breast cancer cells to the anti-estrogen tamoxifen was associated with ERα-dependent overexpression of FGFR2, whereas resistance to fulvestrant was associated with ERα-dependent isoform switching, which correlated with altered response to KGF. Conclusion E2 may partly alter cellular proteome through alternative splicing uncoupled to its effects on transcription initiation and aberration in E2-induced alternative splicing events may influence response to anti-estrogens. PMID:23758675

Dual-targeting hybrid nanoparticles for the delivery of SN38 to Her2 and CD44 overexpressed human gastric cancer

NASA Astrophysics Data System (ADS)

Yang, Zhe; Luo, Huiyan; Cao, Zhong; Chen, Ya; Gao, Jinbiao; Li, Yingqin; Jiang, Qing; Xu, Ruihua; Liu, Jie

2016-06-01

Gastric cancer (GC), particularly of the type with high expression of both human epidermal growth factor receptor 2 (Her2) and cluster determinant 44 (CD44), is one of the most malignant human tumors which causes a high mortality rate due to rapid tumor growth and metastasis. To develop effective therapeutic treatments, a dual-targeting hybrid nanoparticle (NP) system was designed and constructed to deliver the SN38 agent specifically to human solid gastric tumors bearing excessive Her2 and CD44. The hybrid NPs consist of a particle core made of the biodegradable polymer PLGA and a lipoid shell prepared by conjugating the AHNP peptides and n-hexadecylamine (HDA) to the carboxyl groups of hyaluronic acid (HA). Upon encapsulation of the SN38 agent in the NPs, the AHNP peptides and HA on the NP surface allow preferential delivery of the drug to gastric cancer cells (e.g., HGC27 cells) by targeting Her2 and CD44. Cellular uptake and in vivo biodistribution experiments verified the active targeting and prolonged in vivo circulation properties of the dual-targeting hybrid NPs, leading to enhanced accumulation of the drug in tumors. Furthermore, the anti-proliferation mechanism studies revealed that the inhibition of the growth and invasive activity of HGC27 cells was not only attributed to the enhanced cellular uptake of dual-targeting NPs, but also benefited from the suppression of CD44 and Her2 expression by HA and AHNP moieties. Finally, intravenous administration of the SN38-loaded dual-targeting hybrid NPs induced significant growth inhibition of HGC27 tumor xenografted in nude mice compared with a clinical antitumor agent, Irinotecan (CPT-11), and the other NP formulations. These results demonstrate that the designed dual-targeting hybrid NPs are promising for targeted anti-cancer drug delivery to treat human gastric tumors over-expressing Her2 and CD44.Gastric cancer (GC), particularly of the type with high expression of both human epidermal growth factor receptor 2 (Her2) and cluster determinant 44 (CD44), is one of the most malignant human tumors which causes a high mortality rate due to rapid tumor growth and metastasis. To develop effective therapeutic treatments, a dual-targeting hybrid nanoparticle (NP) system was designed and constructed to deliver the SN38 agent specifically to human solid gastric tumors bearing excessive Her2 and CD44. The hybrid NPs consist of a particle core made of the biodegradable polymer PLGA and a lipoid shell prepared by conjugating the AHNP peptides and n-hexadecylamine (HDA) to the carboxyl groups of hyaluronic acid (HA). Upon encapsulation of the SN38 agent in the NPs, the AHNP peptides and HA on the NP surface allow preferential delivery of the drug to gastric cancer cells (e.g., HGC27 cells) by targeting Her2 and CD44. Cellular uptake and in vivo biodistribution experiments verified the active targeting and prolonged in vivo circulation properties of the dual-targeting hybrid NPs, leading to enhanced accumulation of the drug in tumors. Furthermore, the anti-proliferation mechanism studies revealed that the inhibition of the growth and invasive activity of HGC27 cells was not only attributed to the enhanced cellular uptake of dual-targeting NPs, but also benefited from the suppression of CD44 and Her2 expression by HA and AHNP moieties. Finally, intravenous administration of the SN38-loaded dual-targeting hybrid NPs induced significant growth inhibition of HGC27 tumor xenografted in nude mice compared with a clinical antitumor agent, Irinotecan (CPT-11), and the other NP formulations. These results demonstrate that the designed dual-targeting hybrid NPs are promising for targeted anti-cancer drug delivery to treat human gastric tumors over-expressing Her2 and CD44. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr01749e

Cell Homogeneity Indispensable for Regenerative Medicine by Cultured Human Corneal Endothelial Cells.

PubMed

Hamuro, Junji; Toda, Munetoyo; Asada, Kazuko; Hiraga, Asako; Schlötzer-Schrehardt, Ursula; Montoya, Monty; Sotozono, Chie; Ueno, Morio; Kinoshita, Shigeru

2016-09-01

To identify the subpopulation (SP) among heterogeneous cultured human corneal endothelial cells (cHCECs) devoid of cell-state transition applicable for cell-based therapy. Subpopulation presence in cHCECs was confirmed via surface CD-marker expression level by flow cytometry. CD markers effective for distinguishing distinct SPs were selected by analyzing those on established cHCECs with a small cell area and high cell density. Contrasting features among three typical cHCEC SPs was confirmed by PCR array for extracellular matrix (ECM). Combined analysis of CD markers was performed to identify the SP (effector cells) applicable for therapy. ZO-1 and Na+/K+ ATPase, CD200, and HLA expression were compared among heterogeneous SPs. Flow cytometry analysis identified the effector cell expressing CD166+CD105-CD44-∼+/-CD26-CD24-, but CD200-, and the presence of other SPs with CD166+ CD105-CD44+++ (CD26 and CD24, either + or -) was confirmed. PCR array revealed three distinct ECM expression profiles. Some SPs expressed ZO-1 and Na+/K+ ATPase at comparable levels with effector cells, while only one SP expressed CD200, but not on effector cells. Human leukocyte antigen expression was most reduced in the effector SP. The proportion of effector cells (E-ratio) inversely paralleled donor age and decreased during prolonged culture passages. The presence of Rho-associated protein kinase (ROCK) inhibitor increased the E-ratio in cHCECs. The average area of effector cells was approximately 200∼220 μm2, and the density of cHCECs exceeded 2500 cells/mm2. A specified cultured effector cell population sharing the surface phenotypes with mature HCECs in corneal tissues may serve as an alternative to donor corneas for the treatment of corneal endothelial dysfunction.

[Expression of molecular markers detected by immunohistochemistry and risk of lymph node metastasis in stage T1 and T2 colorecrectal cancers].

PubMed

Wang, Fu-long; Wan, De-sen; Lu, Zhen-hai; Fang, Yu-jing; Li, Li-ren; Chen, Gong; Wu, Xiao-jun; Ding, Pei-rong; Kong, Ling-heng; Lin, Jun-zhong; Pan, Zhi-zhong

2013-04-01

To study the molecular risk factors of lymph node metastasis in stage T1 and T2 colorectal cancers by tissue microarray and immunohistochemistry techniques. Two hundred and three patients with stage T1 and T2 colorectal carcinoma who underwent radical surgery from 1999 to 2010 in our department were included in this study. Their clinicopathological data were retrospectively analyzed. Expression of the following 14 molecular markers were selected and assayed by tissue microarray and immunohistochemistry: VEGFR-3, HER2, CD44v6, CXCR4, TIMP-1, EGFR, IGF-1R, IGF-2, IGFBP-1, ECAD, MMP-9, RKIP, CD133, MSI. Chi-squared test and logistic regression were used to evaluate the variables as potential risk factors for lymph node metastasis. The positive expression rates of biomarkers were as following: VEGFR-3 (44.3%), EGFR (30.5%), HER-2 (28.1%), IGF-1R (63.5%), IGF-2 (44.8%), IGFBP-1 (70.9%), ECAD (45.8%), CD44v6 (51.2%), MMP-9 (44.3%), TIMP-1 (41.4%), RKIP (45.3%), CXCR4 (40.9%), and CD133 (49.8%). The positive rate of MSI expression was 22.2%. Both univariate and multivariate analyses showed that VEGFR-3, HER-2, and TIMP-1 were significant predictors of lymph node metastasis. Univariate analysis showed that CD44v6 and CXCR4 were significant significant predictors of lymph node metastasis. VEGFR-3, HER2 and TIMP-1 are independent factors for lymph node metastasis in stage T1 and T2 colorectal cancers.

Characterization and Classification of Mesenchymal Stem Cells in Several Species Using Surface Markers for Cell Therapy Purposes.

PubMed

Ghaneialvar, Hori; Soltani, Leila; Rahmani, Hamid Reza; Lotfi, Abbas Sahebghadam; Soleimani, Masoud

2018-01-01

Mesenchymal stem cells are multipotent cells capable of replicating as undifferentiated cells, and have the potential of differentiating into mesenchymal tissue lineages such as osteocytes, adipocytes and chondrocytes. Such lineages can then be used in cell therapy. The aim of present study was to characterize bone marrow derived mesenchymal stem cells in four different species, including: sheep, goat, human and mouse. Human bone-marrow mesenchymal stem cells were purchased, those of sheep and goat were isolated from fetal bone marrow, and those of mouse were collected by washing bone cavity of femur and tibia with DMEM/F12. Using flow-cytometry, they were characterized by CD surface antigens. Furthermore, cells of third passage were examined for their osteogenic and adipogenic differentiation potential by oil red and alizarin red staining respectively. According to the results, CD markers studied in the four groups of mesenchymal stem cells showed a different expression. Goat and sheep expressed CD44 and CD166, and weakly expressed CD34, CD45, CD105 and CD90. Similarly, human and mouse mesenchymal cells expressed CD44, CD166, CD105 and CD90 whereas the expression of CD34 and CD45 was negative. In conclusion, although all mesenchymal stem cells display plastic adherence and tri-lineage differentiation, not all express the same panel of surface antigens described for human mesenchymal stem cells. Additional panel of CD markers are necessary to characterize regenerative potential and possible application of these stem cells in regenerative medicine and implantology.

Prognostic value of E-cadherin, beta-catenin, CD44v6, and HER2/neu in metastatic cutaneous adenocarcinoma.

PubMed

Pozdnyakova, Olga; Hoang, Mai M P; Dresser, Karen A; Mahalingam, Meera

2009-08-01

Our recent experience with a patient developing cutaneous metastases within 3 months of diagnosis of esophageal adenocarcinoma suggests that altered expression of the cellular adhesion molecules, E-cadherin and CD44v6, may have had a role to play in the rapid onset of metastases. To corroborate these findings, we designed a cross-sectional study to investigate the expression of select molecules involved in the metastatic cascade. E-cadherin, beta-catenin, CD44v6, and HER2/neu immunohistochemical stains were performed on archival materials of metastatic adenocarcinoma to the skin from 27 patients and the available corresponding primary tumors in 10 patients. The primary sites included breast (n = 10; 37%), gastrointestinal tract (n = 10; 37%), ovary (n = 1; 4%), thyroid (n = 2; 7%), lung (n = 1; 4%), and unknown primary (n = 3; 11%). Expression of all markers was noted with the most significant increases observed in beta-catenin (26 of 27 cases; 96%), followed by CD44v6 (24 of 27 cases; 89%), E-cadherin (22 of 27 cases; 82%), and HER2/neu (11 of 27 cases; 41%). Contrasting expression of these molecules in the primary versus the metastatic tumors, enhanced expression of CD44v6 was observed in the cutaneous metastases relative to the primary in 6 of 10 (60%) cases. Of interest, 2 of these 6 cases (33%) also showed reduction in E-cadherin--a member of the cadherin family functioning as an invasion suppressor molecule. These findings reinforce the complexities of the metastatic cascade and imply that the variation in adhesive properties of tumor cells is, perhaps, a consequence of the difference in density of the molecules mediating this process.

CD1d expression by hepatocytes is a main restriction element for intrahepatic T-cell recognition.

PubMed

Agrati, C; Martini, F; Nisii, C; Oliva, A; D'Offizi, G; Narciso, P; Nardacci, R; Piacentini, M; Dieli, F; Pucillo, L P; Poccia, F

2005-01-01

The liver has specific mechanisms to protect itself from infectious agents and to avoid autoimmunity, indicating an important role of the hepatic tissues in antigen presentation and tolerance induction. Since intrahepatic lymphocytes may contribute to the innate immunity and to the liver pathology, it is of interest to analyze the expression of antigen presenting molecules and of the related T cell recognition in liver, and how these change in relation to different diseases. We analyzed the expression of MHC class I, and of CD1-a, -b, -c, and -d proteins on liver tissues from patients with different hepatic diseases. Moreover, in the same patients we studied the intrahepatic and peripheral NKT cell recognition of alpha-galactosyl ceramide antigen in the context of CD1d. Unlike in other tissues, classical MHC class I molecules were poorly expressed in the hepatic compartment, suggesting that inflamed hepatocytes may trigger weak MHC-restricted T cell responses. Nevertheless, we observed a prevalent expression of HLA class I-like CD1d isoform on the hepatocyte surface, indicating that CD1d is the main restriction element in the liver. In patients with viral hepatitis, the intrahepatic CD1d expression parallels the recruitment of CD56+Valpha24Vbeta11+ NKT cells in the liver which recognize CD1d presenting glycolipids such as alpha-galactosyl ceramide, suggesting that the intrahepatic T cell immunity may focus on glycolipid antigens.

Functional and molecular characterization of multiple K-Cl cotransporter isoforms in corneal epithelial cells

PubMed Central

Capó-Aponte, José E.; Wang, Zheng; Bildin, Victor N.; Iserovich, Pavel; Pan, Zan; Zhang, Fan; Pokorny, Kathryn S.; Reinach, Peter S.

2009-01-01

The dependence of regulatory volume decrease (RVD) activity on potassium–chloride cotransporter (KCC) isoform expression was characterized in corneal epithelial cells (CEC). During exposure to a 50% hypotonic challenge, the RVD response was larger in SV40-immortalized human CEC (HCEC) than in SV40-immortalized rabbit CEC (RCEC). A KCC inhibitor—[(dihydroindenyl)oxy] alkanoic acid (DIOA)—blocked RVD more in HCEC than RCEC. Under isotonic conditions, N-ethylmaleimide (NEM) produced KCC activation and transient cell shrinkage. Both of these changes were greater in HCEC than in RCEC. Immunoblot analysis of HCEC, RCEC, primary human CEC (pHCEC), and primary bovine CEC (BCEC) plasma membrane enriched fractions revealed KCC1, KCC3, and KCC4 isoform expression, whereas KCC2 was undetectable. During a hypotonic challenge, KCC1 membrane content increased more rapidly in HCEC than in RCEC. Such a challenge induced a larger increase and more transient p44/42MAPK activation in HCEC than RCEC. On the other hand, HCEC and RCEC p38MAPK phosphorylation reached peak activations at 2.5 and 15 min, respectively. Only in HCEC, pharmacological manipulation of KCC activity modified the hypotonicity-induced activation of p44/42MAPK, whereas p38MAPK phosphorylation was insensitive to such procedures in both cell lines. Larger increases in HCEC KCC1 membrane protein content correlate with their ability to undergo faster and more complete RVD. Furthermore, pharmacological activation of KCC increased p44/42MAPK phosphorylation in HCEC but not in RCEC, presumably a reflection of low KCC membrane expression in RCEC. These findings suggest that KCC1 plays a role in (i) maintaining isotonic steady-state cell volume homeostasis, (ii) recovery of isotonic cell volume after a hypotonic challenge through RVD, and (iii) regulating hypotonicity-induced activation of the p44/42MAPK signaling pathway required for cell proliferation. PMID:17418819

Survival in acute myeloid leukemia is associated with NKp44 splice variants

PubMed Central

Hadad, Uzi; Teltsh, Omri; Edri, Avishay; Rubin, Eitan; Campbell, Kerry S.; Rosental, Benyamin; Porgador, Angel

2016-01-01

NKp44 is a receptor encoded by the NCR2 gene, which is expressed by cytokine-activated natural killer (NK) cells that are involved in anti-AML immunity. NKp44 has three splice variants corresponding to NKp44ITIM+ (NKp44-1) and NKp44ITIM− (NKp44-2, and NKp44-3) isoforms. RNAseq data of AML patients revealed similar survival of NKp46+NKp44+ and NKp46+NKp44− patients. However, if grouped according to the NKp44 splice variant profile, NKp44-1 expression was significantly associated with poor survival of AML patients. Moreover, activation of PBMC from healthy controls showed co-dominant expression of NKp44-1 and NKp44-3, while primary NK clones show more diverse NKp44 splice variant profiles. Cultured primary NK cells resulted in NKp44-1 dominance and impaired function associated with PCNA over-expression by target cells. This impaired functional phenotype could be rescued by blocking of NKp44 receptor. Human NK cell lines revealed co-dominant expression of NKp44-1 and NKp44-3 and showed a functional phenotype that was not inhibited by PCNA over-expression. Furthermore, transfection-based overexpression of NKp44-1, but not NKp44-2/NKp44-3, reversed the endogenous resistance of NK-92 cells to PCNA-mediated inhibition, and resulted in poor formation of stable lytic immune synapses. This research contributes to the understanding of AML prognosis by shedding new light on the functional implications of differential splicing of NKp44. PMID:27102296

Heat shock protein-90 beta is expressed at the surface of multipotential mesenchymal precursor cells: generation of a novel monoclonal antibody, STRO-4, with specificity for mesenchymal precursor cells from human and ovine tissues.

PubMed

Gronthos, Stan; McCarty, Rosa; Mrozik, Krzysztof; Fitter, Stephen; Paton, Sharon; Menicanin, Danijela; Itescu, Silviu; Bartold, P Mark; Xian, Cory; Zannettino, Andrew C W

2009-11-01

Mesenchymal stromal cells (MSCs) and their precursor cells (MPCs) can proliferate and differentiate into multiple mesodermal and some ectodermal and endodermal tissues. Culture-expanded MSCs are currently being evaluated as a possible cell therapy to replace/repair injured or diseased tissues. While a number of mAb reagents with specificity to human MSCs, including STRO-1, STRO-3 (BLK ALP), CD71 (SH2, SH3), CD106 (VCAM-1), CD166, and CD271, have facilitated the isolation of purified populations of human MSCs from primary tissues, few if any mAb reagents have been described that can be used to isolate equivalent cells from other species. This is of particular relevance when assessing the tissue regenerative efficacy of MSCs in large immunocompetent, preclinical animal models of disease. In light of this, we sought to generate novel monoclonal antibodies (mAb) with specific reactivity against a cell surface molecule that is expressed at high levels by MSCs from different species. Using CD106 (VCAM-1)-selected ovine MSCs as an immunogen, mAb-producing hybridomas were selected for their reactivity to both human and ovine MSCs. One such hybridoma, termed STRO-4, produced an IgG mAb that reacted with <5% of human and ovine bone marrow (BM) mononuclear cells. As a single selection reagent, STRO-4 mAb was able to enrich colony-forming fibroblasts (CFU-F) in both human and ovine BM by 16- and 8-folds, respectively. Cells isolated with STRO-4 exhibited reactivity with markers commonly associated with MSCs isolated by plastic adherence including CD29, CD44, and CD166. Moreover, when placed in inductive culture conditions in vitro, STRO-4(+) MSCs exhibited multilineage differentiation potential and were capable of forming a mineralized matrix, lipid-filled adipocytes, and chondrocytes capable of forming a glycosaminoglycan-rich matrix. Biochemical analysis revealed that STRO-4 identified the beta isoform of heat shock protein-90 (Hsp90beta). In addition to identifying an antibody reagent that identifies a highly conserved epitope expressed by MSCs from different species, our study also points to a potential role for Hsp90beta in MSC biology.

[Epstein-Barr virus associated gastric carcinoma: the genetic alteration and the expression of CD44 variant].

PubMed

Chong, J M; Fukayama, M

1997-02-01

Epstein-Barr virus (EBV), a ubiquitous human herpes virus, was recently identified in 2-16% of gastric carcinomas. EBV-encoded small RNA was found in nearly all of the carcinoma cells even at the intramucosal stage. EBV in EBV associated gastric carcinoma (EBVaGC) is monoclonal based on Southern blot hybridization using probes adjacent to the unique terminal repeat of EBV-DNA. Furthermore, the genetic pathway of this carcinogenesis is different of EBVaGC: deletion of 5q and/or 17p and microsatellite instability are extremely rare in EBVaGC, in contrast to their high frequency in EBV-negative carcinomas. We also examined the relationship between the expression of CD44 variants and EBVaGC, and found the expression of CD44 variants was significantly correlated with EBV-etiology.

Prognostic value of CD8CD45RO tumor infiltrating lymphocytes in patients with extrahepatic cholangiocarcinoma

PubMed Central

Kim, Richard; Coppola, Domenico; Wang, Emilie; Chang, Young Doo; Kim, Yuhree; Anaya, Daniel; Kim, Dae Won

2018-01-01

Cholangiocarcinoma is a malignancy arising from the biliary tract epithelial cells with poor prognosis. Tumor infiltrating lymphocytes (TIL)s and programmed cell death receptor ligand 1 (PD-L1) have a prognostic impact in various solid tumors. We aimed to investigate TILs and PD-L1 expression and their clinical relevance in cholangiocarcinoma. Tumor samples from 44 patients with resected and histologically verified extrahepatic cholangiocarcinoma were evaluated for CD8, CD45RO and PD-L1 expression, and their correlations with clinicopathological data and survival data were analyzed. Total 44 extrahepatic cholangiocarcinoma tissues were evaluated. CD8+ tumor infiltrating lymphocytes (TIL)s were observed in 30 (68%) tumors. Among them, 14 had CD8+CD45RO+ TILs. PD-L1 was expressed on cancer cells in 10 (22.7%) tumors in 34 evaluable extrahepatic cholangiocarciniomas. The presence of CD8+ TILs or CD8+CD45RO+ TILs was not associated with clinical staging or tumor differentiation. Extrahepatic cholangiocarcinoma with CD8+CD45RO+ TILs had longer overall survival (OS) on univariate (P = 0.013) and multivariate (P = 0.012) analysis. Neither CD8+TIL nor PD-L1 expression on cancer cells correlated significantly with OS. These results add to the understanding of the clinical features associated with CD8 TILs and PD-L1 expression in extrahepatic cholangiocarcinoma, and they support the potential rationale of using PD-1 blockade immunotherapy in cholangiocarcinoma.

Hyaluronan-CD44v3 interaction with Oct4-Sox2-Nanog promotes miR-302 expression leading to self-renewal, clonal formation, and cisplatin resistance in cancer stem cells from head and neck squamous cell carcinoma.

PubMed

Bourguignon, Lilly Y W; Wong, Gabriel; Earle, Christine; Chen, Liqun

2012-09-21

Human head and neck squamous cell carcinoma (HNSCC) is a highly malignant cancer associated with major morbidity and mortality. In this study, we determined that human HNSCC-derived HSC-3 cells contain a subpopulation of cancer stem cells (CSCs) characterized by high levels of CD44v3 and aldehyde dehydrogenase-1 (ALDH1) expression. These tumor cells also express several stem cell markers (the transcription factors Oct4, Sox2, and Nanog) and display the hallmark CSC properties of self-renewal/clonal formation and the ability to generate heterogeneous cell populations. Importantly, hyaluronan (HA) stimulates the CD44v3 (an HA receptor) interaction with Oct4-Sox2-Nanog leading to both a complex formation and the nuclear translocation of three CSC transcription factors. Further analysis reveals that microRNA-302 (miR-302) is controlled by an upstream promoter containing Oct4-Sox2-Nanog-binding sites, whereas chromatin immunoprecipitation (ChIP) assays demonstrate that stimulation of miR-302 expression by HA-CD44 is Oct4-Sox2-Nanog-dependent in HNSCC-specific CSCs. This process results in suppression of several epigenetic regulators (AOF1/AOF2 and DNMT1) and the up-regulation of several survival proteins (cIAP-1, cIAP-2, and XIAP) leading to self-renewal, clonal formation, and cisplatin resistance. These CSCs were transfected with a specific anti-miR-302 inhibitor to silence miR-302 expression and block its target functions. Our results demonstrate that the anti-miR-302 inhibitor not only enhances the expression of AOF1/AOF2 and DNMT1 but also abrogates the production of cIAP-1, cIAP-2, and XIAP and HA-CD44v3-mediated cancer stem cell functions. Taken together, these findings strongly support the contention that the HA-induced CD44v3 interaction with Oct4-Sox2-Nanog signaling plays a pivotal role in miR-302 production leading to AOF1/AOF2/DNMT1 down-regulation and survival of protein activation. All of these events are critically important for the acquisition of cancer stem cell properties, including self-renewal, clonal formation, and chemotherapy resistance in HA-CD44v3-activated head and neck cancer.

Hyaluronan-CD44v3 Interaction with Oct4-Sox2-Nanog Promotes miR-302 Expression Leading to Self-renewal, Clonal Formation, and Cisplatin Resistance in Cancer Stem Cells from Head and Neck Squamous Cell Carcinoma*

PubMed Central

Bourguignon, Lilly Y. W.; Wong, Gabriel; Earle, Christine; Chen, Liqun

2012-01-01

Human head and neck squamous cell carcinoma (HNSCC) is a highly malignant cancer associated with major morbidity and mortality. In this study, we determined that human HNSCC-derived HSC-3 cells contain a subpopulation of cancer stem cells (CSCs) characterized by high levels of CD44v3 and aldehyde dehydrogenase-1 (ALDH1) expression. These tumor cells also express several stem cell markers (the transcription factors Oct4, Sox2, and Nanog) and display the hallmark CSC properties of self-renewal/clonal formation and the ability to generate heterogeneous cell populations. Importantly, hyaluronan (HA) stimulates the CD44v3 (an HA receptor) interaction with Oct4-Sox2-Nanog leading to both a complex formation and the nuclear translocation of three CSC transcription factors. Further analysis reveals that microRNA-302 (miR-302) is controlled by an upstream promoter containing Oct4-Sox2-Nanog-binding sites, whereas chromatin immunoprecipitation (ChIP) assays demonstrate that stimulation of miR-302 expression by HA-CD44 is Oct4-Sox2-Nanog-dependent in HNSCC-specific CSCs. This process results in suppression of several epigenetic regulators (AOF1/AOF2 and DNMT1) and the up-regulation of several survival proteins (cIAP-1, cIAP-2, and XIAP) leading to self-renewal, clonal formation, and cisplatin resistance. These CSCs were transfected with a specific anti-miR-302 inhibitor to silence miR-302 expression and block its target functions. Our results demonstrate that the anti-miR-302 inhibitor not only enhances the expression of AOF1/AOF2 and DNMT1 but also abrogates the production of cIAP-1, cIAP-2, and XIAP and HA-CD44v3-mediated cancer stem cell functions. Taken together, these findings strongly support the contention that the HA-induced CD44v3 interaction with Oct4-Sox2-Nanog signaling plays a pivotal role in miR-302 production leading to AOF1/AOF2/DNMT1 down-regulation and survival of protein activation. All of these events are critically important for the acquisition of cancer stem cell properties, including self-renewal, clonal formation, and chemotherapy resistance in HA-CD44v3-activated head and neck cancer. PMID:22847005

Novel Adult Stem Cells for Peripheral Nerve Regeneration

DTIC Science & Technology

2012-09-01

were also positive for MSC surface marker CD29 and CD44 (Fig. 1F-G). However, CD29 and CD44 are also expressed in SMCs, so we will not use these non...tubulin. In addition, MVSCs were negative for perivascular MSC marker CD146 (Fig. 1H) and SMC progenitor marker Sca-1 (Fig. 1I). MVSCs were also...University of California, Berkeley, California 94720, USA. 2 UC Berkeley-UCSF Graduate Program in Bioengineering, Berkeley, California 94720, USA. 3

Triple negative breast tumors in African-American and Hispanic/Latina women are high in CD44+, low in CD24+, and have loss of PTEN.

PubMed

Wu, Yanyuan; Sarkissyan, Marianna; Elshimali, Yahya; Vadgama, Jaydutt V

2013-01-01

African-American women have higher mortality from breast cancer than other ethnic groups. The association between poor survival and differences with tumor phenotypes is not well understood. The purpose of this study is to assess the clinical significance of (1) Stem cell-like markers CD44 and CD24; (2) PI3K/Akt pathway associated targets PTEN, activation of Akt, and FOXO1; and (3) the Insulin-like growth factor-1 (IGF-I) and IGF binding protein-3 (IGFBP3) in different breast cancer subtypes, and compare the differences between African-American and Hispanic/Latina women who have similar social-economic-status. A total of N=318 African-American and Hispanic/Latina women, with clinically-annotated information within the inclusion criteria were included. Formalin fixed paraffin embedded tissues from these patients were tested for the different markers using immunohistochemistry techniques. Kaplan-Meier survival-curves and Cox-regression analyses were used to assess Relative Risk and Disease-Free-Survival (DFS). The triple-negative-breast-cancer (TNBC) receptor-subtype was more prevalent among premenopausal women, and the Hormonal Receptor (HR) positive subtype was most common overall. TNBC tumors were more likely to have loss of PTEN, express high Ki67, and have increased CD44+/CD24- expression. TNBC was also associated with higher plasma-IGF-I levels. HR-/HER2+ tumors showed high pAkt, decreased FOXO1, and high CD24+ expression. The loss of PTEN impacted DFS significantly in African Americans, but not in Hispanics/Latinas after adjusted for treatment and other tumor pathological factors. The CD44+/CD24- and CD24+/CD44- phenotypes decreased DFS, but were not independent predictors for DFS. HER2-positive and TNBC type of cancers continued to exhibit significant decrease in DFS after adjusting for the selected biomarkers and treatment. TNBC incidence is high among African-American and Hispanic/Latino women residing in South Los Angeles. Our study also shows for the first time that TNBC was significantly associated with PTEN loss, high Ki67 and the CD44+/CD24- phenotype. The loss of PTEN impacts DFS significantly in African Americans.

Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

PubMed Central

Shafaghat, Farzaneh; Abbasi-Kenarsari, Hajar; Majidi, Jafar; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

2015-01-01

Purpose: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. Methods: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. Conclusion: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems. PMID:25789221

Elevated Expression of Osteopontin May Be Related to Adipose Tissue Macrophage Accumulation and Liver Steatosis in Morbid Obesity

PubMed Central

Bertola, Adeline; Deveaux, Vanessa; Bonnafous, Stéphanie; Rousseau, Déborah; Anty, Rodolphe; Wakkach, Abdelilah; Dahman, Moncef; Tordjman, Joan; Clément, Karine; McQuaid, Siobhán E.; Frayn, Keith N.; Huet, Pierre-Michel; Gugenheim, Jean; Lotersztajn, Sophie; Le Marchand-Brustel, Yannick; Tran, Albert; Gual, Philippe

2009-01-01

OBJECTIVE—Osteopontin (OPN) plays an important role in the development of insulin resistance and liver complications in dietary murine models. We aimed to determine the expression pattern of OPN and its receptor CD44 in obese patients and mice according to insulin resistance and liver steatosis. RESEARCH DESIGN AND METHODS—OPN and CD44 expressions were studied in 52 morbidly obese patients and in mice. Cellular studies were performed in HepG2 cells. RESULTS—Hepatic OPN and CD44 expressions were strongly correlated with liver steatosis and insulin resistance in obese patients and mice. This increased OPN expression could be due to the accumulation of triglycerides, since fat loading in HepG2 promotes OPN expression. In contrast, OPN expression in adipose tissue (AT) was enhanced independently of insulin resistance and hepatic steatosis in obese patients. The elevated OPN expression in AT was paralleled with the AT macrophage infiltration, and both phenomena were reversed after weight loss. The circulating OPN level was slightly elevated in obese patients and was not related to liver steatosis. Further, AT did not appear to secrete OPN. In contrast, bariatric surgery–induced weight loss induced a strong increase in circulating OPN. CONCLUSIONS—The modestly elevated circulating OPN levels in morbidly obese patients were not related to liver steatosis and did not appear to result from adipose tissue secretion. In subcutaneous AT, expression of OPN was directly related to macrophage accumulation independently from liver complications. In contrast, hepatic OPN and CD44 expressions were related to insulin resistance and steatosis, suggesting their local implication in the progression of liver injury. PMID:18952835

Melanoma upregulates ICAM-1 expression on endothelial cells through engagement of tumor CD44 with endothelial E-selectin and activation of a PKCα–p38–SP-1 pathway

PubMed Central

Zhang, Pu; Goodrich, Chris; Fu, Changliang; Dong, Cheng

2014-01-01

Cancer metastasis involves multistep adhesive interactions between tumor cells (TCs) and endothelial cells (ECs), but the molecular mechanisms of intercellular communication in the tumor microenvironment remain elusive. Using static and flow coculture systems in conjunction with flow cytometry, we discovered that certain receptors on the ECs are upregulated on melanoma cell adhesion. Direct contact but not separate coculture between human umbilical endothelial cells (HUVECs) and a human melanoma cell line (Lu1205) increased intercellular adhesion molecule 1 (ICAM-1) and E-selectin expression on HUVECs by 3- and 1.5-fold, respectively, compared with HUVECs alone. The nonmetastatic cell line WM35 failed to promote ICAM-1 expression changes in HUVECs on contact. Enzyme-linked immunosorbent assay (ELISA) revealed that EC–TC contact has a synergistic effect on the expression of the cytokines interleukin (IL)-8, IL-6, and growth-related oncogene α (Gro-α). By using E-selectin cross-linking and beads coated with CD44 immunopurified from Lu1205 cells, we showed that CD44/selectin ligation was responsible for the ICAM-1 up-regulation on HUVECs. Protein kinase Cα (PKC-α) activation was found to be the downstream target of the CD44/selectin-initiated signaling, as ICAM-1 elevation was inhibited by siRNA targeting PKCα or a dominant negative form of PKCα (PKCα DN). Western blot analysis and electrophoretic mobility shift assays (EMSAs) showed that TC–EC contact mediated p38 phosphorylation and binding of the transcription factor SP-1 to its regulation site. In conclusion, CD44/selectin binding signals ICAM-1 up-regulation on the EC surface through a PKCα–p38–SP-1 pathway, which further enhances melanoma cell adhesion to ECs during metastasis.—Zhang, P., Goodrich, C., Fu, C., Dong, C. Melanoma upregulates ICAM-1 expression on ECs through engagement of tumor CD44 with endothelial E-selectin and activation of a PKCα–p38–SP-1 pathway. PMID:25138157

Tcf7 Is an Important Regulator of the Switch of Self-Renewal and Differentiation in a Multipotential Hematopoietic Cell Line

PubMed Central

Schulz, Vincent P.; Hariharan, Manoj; Tuck, David; Lian, Jin; Du, Jiang; Shi, Minyi; Ye, Zhijia; Gerstein, Mark; Snyder, Michael P.; Weissman, Sherman

2012-01-01

A critical problem in biology is understanding how cells choose between self-renewal and differentiation. To generate a comprehensive view of the mechanisms controlling early hematopoietic precursor self-renewal and differentiation, we used systems-based approaches and murine EML multipotential hematopoietic precursor cells as a primary model. EML cells give rise to a mixture of self-renewing Lin-SCA+CD34+ cells and partially differentiated non-renewing Lin-SCA-CD34− cells in a cell autonomous fashion. We identified and validated the HMG box protein TCF7 as a regulator in this self-renewal/differentiation switch that operates in the absence of autocrine Wnt signaling. We found that Tcf7 is the most down-regulated transcription factor when CD34+ cells switch into CD34− cells, using RNA–Seq. We subsequently identified the target genes bound by TCF7, using ChIP–Seq. We show that TCF7 and RUNX1 (AML1) bind to each other's promoter regions and that TCF7 is necessary for the production of the short isoforms, but not the long isoforms of RUNX1, suggesting that TCF7 and the short isoforms of RUNX1 function coordinately in regulation. Tcf7 knock-down experiments and Gene Set Enrichment Analyses suggest that TCF7 plays a dual role in promoting the expression of genes characteristic of self-renewing CD34+ cells while repressing genes activated in partially differentiated CD34− state. Finally a network of up-regulated transcription factors of CD34+ cells was constructed. Factors that control hematopoietic stem cell (HSC) establishment and development, cell growth, and multipotency were identified. These studies in EML cells demonstrate fundamental cell-intrinsic properties of the switch between self-renewal and differentiation, and yield valuable insights for manipulating HSCs and other differentiating systems. PMID:22412390

Characterization of 14-3-3 isoforms expressed in the Echinococcus granulosus pathogenic larval stage.

PubMed

Teichmann, Aline; Vargas, Daiani M; Monteiro, Karina M; Meneghetti, Bruna V; Dutra, Cristine S; Paredes, Rodolfo; Galanti, Norbel; Zaha, Arnaldo; Ferreira, Henrique B

2015-04-03

The 14-3-3 protein family of eukaryotic regulators was studied in Echinococcus granulosus, the causative agent of cystic hydatid disease. These proteins mediate important cellular processes in eukaryotes and are expected to play important roles in parasite biology. Six isoforms of E. granulosus 14-3-3 genes and proteins (Eg14-3-3.1-6) were analyzed, and their phylogenetic relationships were established with bona fide 14-3-3 orthologous proteins from eukaryotic species. Eg14-3-3 isoforms with previous evidence of expression (Eg14-3-3.1-4) in E. granulosus pathogenic larval stage (metacestode) were cloned, and recombinant proteins were used for functional studies. These protein isoforms were detected in different components of E. granulosus metacestode, including interface components with the host. The roles that are played by Eg14-3-3 proteins in parasite biology were inferred from the repertoires of interacting proteins with each isoform, as assessed by gel overlay, cross-linking, and affinity chromatography assays. A total of 95 Eg14-3-3 protein ligands were identified by mass spectrometry. Eg14-3-3 isoforms have shared partners (44 proteins), indicating some overlapping functions; however, they also bind exclusive partners (51 proteins), suggesting Eg14-3-3 functional specialization. These ligand repertoires indicate the involvement of Eg14-3-3 proteins in multiple biochemical pathways in the E. granulosus metacestode and note some degree of isoform specialization.

A novel mechanism of regulating breast cancer cell migration via palmitoylation-dependent alterations in the lipid raft affiliation of CD44.

PubMed

Babina, Irina S; McSherry, Elaine A; Donatello, Simona; Hill, Arnold D K; Hopkins, Ann M

2014-02-10

Most breast cancer-related deaths result from metastasis, a process involving dynamic regulation of tumour cell adhesion and migration. The adhesion protein CD44, a key regulator of cell migration, is enriched in cholesterol-enriched membrane microdomains termed lipid rafts. We recently reported that raft affiliation of CD44 negatively regulates interactions with its migratory binding partner ezrin. Since raft affiliation is regulated by post-translational modifications including palmitoylation, we sought to establish the contribution of CD44 palmitoylation and lipid raft affiliation to cell migration. Recovery of CD44 and its binding partners from raft versus non-raft membrane microdomains was profiled in non-migrating and migrating breast cancer cell lines. Site-directed mutagenesis was used to introduce single or double point mutations into both CD44 palmitoylation sites (Cys286 and Cys295), whereupon the implications for lipid raft recovery, phenotype, ezrin co-precipitation and migratory behaviour was assessed. Finally CD44 palmitoylation status and lipid raft affiliation was assessed in primary cultures from a small panel of breast cancer patients. CD44 raft affiliation was increased during migration of non-invasive breast cell lines, but decreased during migration of highly-invasive breast cells. The latter was paralleled by increased CD44 recovery in non-raft fractions, and exclusive non-raft recovery of its binding partners. Point mutation of CD44 palmitoylation sites reduced CD44 raft affiliation in invasive MDA-MB-231 cells, increased CD44-ezrin co-precipitation and accordingly enhanced cell migration. Expression of palmitoylation-impaired (raft-excluded) CD44 mutants in non-invasive MCF-10a cells was sufficient to reversibly induce the phenotypic appearance of epithelial-to-mesenchymal transition and to increase cell motility. Interestingly, cell migration was associated with temporal reductions in CD44 palmitoylation in wild-type breast cells. Finally, the relevance of these findings is underscored by the fact that levels of palmitoylated CD44 were lower in primary cultures from invasive ductal carcinomas relative to non-tumour tissue, while CD44 co-localisation with a lipid raft marker was less in invasive ductal carcinoma relative to ductal carcinoma in situ cultures. Our results support a novel mechanism whereby CD44 palmitoylation and consequent lipid raft affiliation inversely regulate breast cancer cell migration, and may act as a new therapeutic target in breast cancer metastasis.

Immunohistochemistry Analysis of CD44, EGFR, and p16 in Oral Cavity and Oropharyngeal Squamous Cell Carcinoma.

PubMed

Cohen, Erin R; Reis, Isildinha M; Gomez, Carmen; Pereira, Lutecia; Freiser, Monika E; Hoosien, Gia; Franzmann, Elizabeth J

2017-08-01

Objectives We analyze the relationship between CD44, epidermal growth factor receptor (EGFR), and p16 expression in oral cavity and oropharyngeal cancers in a diverse population. We also describe whether particular patterns of staining are associated with progression-free survival and overall survival. Study Design Prospective study, single-blind to pathologist and laboratory technologist. Setting Hospital based. Subjects and Methods Immunohistochemistry, comprising gross staining and cellular expression, was performed and interpreted in a blinded fashion on 24 lip/oral cavity and 40 oropharyngeal cancer specimens collected between 2007 and 2012 from participants of a larger study. Information on overall survival and progression-free survival was obtained from medical records. Results Nineteen cases were clinically p16 positive, 16 of which were oropharyngeal. Oral cavity lesions were more likely to exhibit strong CD44 membrane staining ( P = .0002). Strong CD44 membrane and strong EGFR membrane and/or cytoplasmic staining were more common in p16-negative cancers ( P = .006). Peripheral/mixed gross p16 staining pattern was associated with worse survival than the universal staining on univariate and multivariate analyses ( P = .006, P = .030). This held true when combining gross and cellular localization for p16. For CD44, universal gross staining demonstrated poorer overall survival compared with the peripheral/mixed group ( P = .039). CD44 peripheral/mixed group alone and when combined with universal p16 demonstrated the best survival on multivariate analysis ( P = .010). Conclusion In a diverse population, systematic analysis applying p16, CD44, and EGFR gross staining and cellular localization on immunohistochemistry demonstrates distinct patterns that may have prognostic potential exceeding current methods. Larger studies are warranted to investigate these findings further.

Stage 3 immature human natural killer cells found in secondary lymphoid tissue constitutively and selectively express the TH17 cytokine interleukin-22

PubMed Central

Hughes, Tiffany; Becknell, Brian; McClory, Susan; Briercheck, Edward; Freud, Aharon G.; Zhang, Xiaoli; Mao, Hsiaoyin; Nuovo, Gerard; Yu, Jianhua

2009-01-01

Considerable functional heterogeneity within human natural killer (NK) cells has been revealed through the characterization of distinct NK-cell subsets. Accordingly, a small subset of CD56+NKp44+NK cells, termed NK-22 cells, was recently described within secondary lymphoid tissue (SLT) as IL-22− when resting, with a minor fraction of this population becoming IL-22+ when activated. Here we discover that the vast majority of stage 3 immature NK (iNK) cells in SLT constitutively and selectively express IL-22, a TH17 cytokine important for mucosal immunity, whereas earlier and later stages of NK developmental intermediates do not express IL-22. These iNK cells have a surface phenotype of CD34−CD117+CD161+CD94−, largely lack expression of NKp44 and CD56, and do not produce IFN-γ or possess cytolytic activity. In summary, stage 3 iNK cells are highly enriched for IL-22 and IL-26 messenger RNA, and IL-22 protein production, but do not express IL-17A or IL-17F. PMID:19244159

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barbieri, Federica; Wurth, Roberto; Ratto, Alessandra

Current carcinogenesis theory states that only a small subset of tumor cells, the cancer stem cells or tumor initiating cells (TICs), are responsible for tumor formation and progression. Human breast cancer-initiating cells have been identified as CD44-expressing cells, which retain tumorigenic activity and display stem cell-like properties. Spontaneous feline mammary carcinoma (FMC) is an aggressive cancer, which shows biological similarities to the human tumor counterpart. We report the isolation and phenotypic characterization of FMC-derived stem/progenitor cells, showing in vitro self-renewal, long-lasting proliferation and in vivo tumorigenicity. Twenty-one FMC samples were collected, histologically classified and characterized for the expression of Ki67,more » EGFR, ER-{alpha} and CD44, by immunohistochemistry. By culture in stem cell permissive conditions, we isolated, from 13 FMCs, a CD44-positive subpopulation able to survive and proliferate in vitro as mammospheres of different sizes and morphologies. When injected in NOD/SCID mice, FMC stem-like cells initiate tumors, generating cell heterogeneity and recapitulating the original histotype. In serum-containing medium, spheroid cells showed differentiation properties as shown by morphological changes, the loss of CD44 expression and tumorigenic potential. These data show that stem-defined culture of FMC enriches for TICs and validate the use of these cells as a suitable model for comparative oncology studies of mammary biology and testing therapeutic strategies aimed at eradicating TICs. -- Highlights: Black-Right-Pointing-Pointer Feline mammary carcinoma contain a sub-population of stem-like cells expressing CD44 Black-Right-Pointing-Pointer These grow as spheres in serum-free medium and self-renew Black-Right-Pointing-Pointer Isolated stem-like cancer cells initiate tumor in immunodeficient mice Black-Right-Pointing-Pointer Xenografted tumors are phenotypically similar to the original tumor Black-Right-Pointing-Pointer Upon differentiation, cells grow as monolayers, loosing the tumorigenic potential.« less

Extracellular matrix components expression in human pluripotent stem cell-derived retinal organoids recapitulates retinogenesis in vivo and reveals an important role for IMPG1 and CD44 in the development of photoreceptors and interphotoreceptor matrix.

PubMed

Felemban, Majed; Dorgau, Birthe; Hunt, Nicola Claire; Hallam, Dean; Zerti, Darin; Bauer, Roman; Ding, Yuchun; Collin, Joseph; Steel, David; Krasnogor, Natalio; Al-Aama, Jumana; Lindsay, Susan; Mellough, Carla; Lako, Majlinda

2018-05-17

The extracellular matrix (ECM) plays an important role in numerous processes including cellular proliferation, differentiation, migration, maturation, adhesion guidance and axonal growth. To date, there has been no detailed analysis of the ECM distribution during retinal ontogenesis in humans and the functional importance of many ECM components is poorly understood. In this study, the expression of key ECM components in adult mouse and monkey retina, developing and adult human retina and retinal organoids derived from human pluripotent stem cells was studied. Our data indicate that basement membrane ECMs (Fibronectin and Collagen IV) were expressed in Bruch's membrane and the inner limiting membrane of the developing human retina, whilst the hyalectins (Versican and Brevican), cluster of differentiation 44 (CD44), photoreceptor-specific ECMs Interphotoreceptor Matrix Proteoglycan 1 (IMPG1) and Interphotoreceptor Matrix Proteoglycan 2 (IMPG2) were detected in the developing interphotoreceptor matrix (IPM). The expression of IMPG1, Versican and Brevican in the developing IPM was conserved between human developing retina and human pluripotent stem cell-derived retinal organoids. Blocking the action of CD44 and IMPG1 in pluripotent stem cell derived retinal organoids affected the development of photoreceptors, their inner/outer segments and connecting cilia and disrupted IPM formation, with IMPG1 having an earlier and more significant impact. Together, our data suggest an important role for IMPG1 and CD44 in the development of photoreceptors and IPM formation during human retinogenesis. The expression and the role of many extracellular matrix (ECM) components during human retinal development is not fully understood. In this study, expression of key ECM components (Collagen IV, Fibronectin, Brevican, Versican, IMPG1 and IMPG2) was investigated during human retinal ontogenesis. Collagen IV and Fibronectin were expressed in Bruch's membrane; whereas Brevican, Versican, IMPG1 & IMPG2 in the developing interphotoreceptor matrix (IPM). Retinal organoids were successfully generated from pluripotent stem cells. The expression of ECM components was examined in the retinal organoids and found to recapitulate human retinal development in vivo. Using functional blocking experiments, we were able to highlight an important role for IMPG1 and CD44 in the development of photoreceptors and IPM formation. Copyright © 2018 Acta Materialia Inc. All rights reserved.

Premature aging induced by radiation exhibits pro-atherosclerotic effects mediated by epigenetic activation of CD44 expression

PubMed Central

Lowe, Donna; Raj, Kenneth

2014-01-01

Age is undoubtedly a major risk factor for heart disease. However, the reason for this is not entirely clear. In the course of our investigation into the mechanism of radiation-induced cardiovascular disease, we made several unexpected findings that inform us on this question. We observed that human coronary endothelial cells, while being able to initiate repair of radiation-induced DNA damage, often fail to complete the repair and become senescent. Such radiation-induced cellular aging occurs through a mutation-independent route. Endothelial cells that aged naturally through replication or as a result of radiation exhibited indistinguishable characteristics. The promoter regions of the CD44 gene in aging endothelial cells become demethylated, and the proteins are highly expressed on the cell surface, making the cells adhesive for monocytes. Adhesion is a cardinal feature that recruits monocytes to the endothelium, allowing them to infiltrate the vessel wall and initiate atherosclerosis. The epigenetic activation of CD44 expression is particularly significant as it causes persistent elevated CD44 protein expression, making senescent endothelial cells chronically adhesive. In addition to understanding why cardiovascular disease increases with age, these observations provide insights into the puzzling association between radiation and cardiovascular disease and highlight the need to consider premature aging as an additional risk of radiation to human health. PMID:25059316

Isolation, identification and multipotential differentiation of mouse adipose tissue-derived stem cells.

PubMed

Taha, Masoumeh Fakhr; Hedayati, Vahideh

2010-08-01

Bone marrow and adipose tissue have provided two suitable sources of mesenchymal stem cells. Although previous studies have confirmed close similarities between bone marrow-derived stem cells (BM-MSCs) and adipose tissue-derived stem cells (ADSCs), the molecular phenotype of ADSCs is still poorly identified. In the present study, mouse ADSCs were isolated from the inguinal fat pad of 12-14 weeks old mice. Freshly isolated and three passaged ADSCs were analyzed for the expression of OCT4, Sca-1, c-kit and CD34 by RT-PCR. Three passaged ADSCs were analyzed by flow cytometry for the presence of CD11b, CD45, CD31, CD29 and CD44. Moreover, cardiogenic, adipogenic and neurogenic differentiation of ADSCs were induced in vitro. Freshly isolated ADSCs showed the expression of OCT4, Sca-1, c-kit and CD34, and two days cultured ADSCs were positively immunostained with anti-OCT4 monoclonal antibody. After three passages, the expression of OCT4, c-kit and CD34 eliminated, while the expression of Sca-1 showed a striking enhancement. These cells were identified positive for CD29 and CD44 markers, and they showed the lack of CD45 and CD31 expression. Three passaged ADSCs were differentiated to adipocyte-, cardiomyocyte- and neuron-like cells that were identified based on the positive staining with Sudan black, anti-cardiac troponin I antibody and anti-map-2 antibody, respectively. In conclusion, adipose tissue contains a stem cell population that seems to be a good multipotential cell candidate for the future cell replacement therapy. Copyright 2010 Elsevier Ltd. All rights reserved.

Pursuing Intracellular Pathogens with Hyaluronan. From a 'Pro-Infection' Polymer to a Biomaterial for 'Trojan Horse' Systems.

PubMed

Montanari, Elita; Di Meo, Chiara; Oates, Angela; Coviello, Tommasina; Matricardi, Pietro

2018-04-18

Hyaluronan (HA) is among the most important bioactive polymers in mammals, playing a key role in a number of biological functions. In the last decades, it has been increasingly studied as a biomaterial for drug delivery systems, thanks to its physico-chemical features and ability to target and enter certain cells. The most important receptor of HA is ‘Cluster of Differentiation 44’ (CD44), a cell surface glycoprotein over-expressed by a number of cancers and heavily involved in HA endocytosis. Moreover, CD44 is highly expressed by keratinocytes, activated macrophages and fibroblasts, all of which can act as ‘reservoirs’ for intracellular pathogens. Interestingly, both CD44 and HA appear to play a key role for the invasion and persistence of such microorganisms within the cells. As such, HA is increasingly recognised as a potential target for nano-carriers development, to pursuit and target intracellular pathogens, acting as a ‘Trojan Horse’. This review describes the biological relationship between HA, CD44 and the entry and survival of a number of pathogens within the cells and the subsequent development of HA-based nano-carriers for enhancing the intracellular activity of antimicrobials.

Identification, expansion and characterization of cancer cells with stem cell properties from head and neck squamous cell carcinomas

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaseb, Hatem O.; Department of Clinical Pathology, National Cancer Institute; Fohrer-Ting, Helene

Head and neck squamous cell carcinoma (HNSCC) is a major public health concern. Recent data indicate the presence of cancer stem cells (CSC) in many solid tumors, including HNSCC. Here, we assessed the stem cell (SC) characteristics, including cell surface markers, radioresistance, chromosomal instability, and in vivo tumorigenic capacity of CSC isolated from HNSCC patient specimens. We show that spheroid enrichment of CSC from early and short-term HNSCC cell cultures was associated with increased expression of CD44, CD133, SOX2 and BMI1 compared with normal oral epithelial cells. On immunophenotyping, five of 12 SC/CSC markers were homogenously expressed in all tumormore » cultures, while one of 12 was negative, four of 12 showed variable expression, and two of the 12 were expressed heterogeneously. We showed that irradiated CSCs survived and retained their self-renewal capacity across different ionizing radiation (IR) regimens. Fluorescence in situ hybridization (FISH) analyses of parental and clonally-derived tumor cells revealed different chromosome copy numbers from cell to cell, suggesting the presence of chromosomal instability in HNSCC CSC. Further, our in vitro and in vivo mouse engraftment studies suggest that CD44+/CD66− is a promising, consistent biomarker combination for HNSCC CSC. Overall, our findings add further evidence to the proposed role of HNSCC CSCs in therapeutic resistance. - Highlights: • Spheroid enrichment selects cancer stem cells (CSC) from head & neck tumors (HNSCC). • Compared to normal epithelial cells, isolated CSC express increased SC/CSC markers. • Isolated CSC display enhanced radioresistance, clonogenicity and tumorigenicity. • HNSCC CSC express chromosomal instability. • CD44+/CD66− is a promising, consistent biomarker for HNSCC CSC.« less

A novel mechanism of regulating breast cancer cell migration via palmitoylation-dependent alterations in the lipid raft affiliation of CD44

PubMed Central

2014-01-01

Introduction Most breast cancer-related deaths result from metastasis, a process involving dynamic regulation of tumour cell adhesion and migration. The adhesion protein CD44, a key regulator of cell migration, is enriched in cholesterol-enriched membrane microdomains termed lipid rafts. We recently reported that raft affiliation of CD44 negatively regulates interactions with its migratory binding partner ezrin. Since raft affiliation is regulated by post-translational modifications including palmitoylation, we sought to establish the contribution of CD44 palmitoylation and lipid raft affiliation to cell migration. Methods Recovery of CD44 and its binding partners from raft versus non-raft membrane microdomains was profiled in non-migrating and migrating breast cancer cell lines. Site-directed mutagenesis was used to introduce single or double point mutations into both CD44 palmitoylation sites (Cys286 and Cys295), whereupon the implications for lipid raft recovery, phenotype, ezrin co-precipitation and migratory behaviour was assessed. Finally CD44 palmitoylation status and lipid raft affiliation was assessed in primary cultures from a small panel of breast cancer patients. Results CD44 raft affiliation was increased during migration of non-invasive breast cell lines, but decreased during migration of highly-invasive breast cells. The latter was paralleled by increased CD44 recovery in non-raft fractions, and exclusive non-raft recovery of its binding partners. Point mutation of CD44 palmitoylation sites reduced CD44 raft affiliation in invasive MDA-MB-231 cells, increased CD44-ezrin co-precipitation and accordingly enhanced cell migration. Expression of palmitoylation-impaired (raft-excluded) CD44 mutants in non-invasive MCF-10a cells was sufficient to reversibly induce the phenotypic appearance of epithelial-to-mesenchymal transition and to increase cell motility. Interestingly, cell migration was associated with temporal reductions in CD44 palmitoylation in wild-type breast cells. Finally, the relevance of these findings is underscored by the fact that levels of palmitoylated CD44 were lower in primary cultures from invasive ductal carcinomas relative to non-tumour tissue, while CD44 co-localisation with a lipid raft marker was less in invasive ductal carcinoma relative to ductal carcinoma in situ cultures. Conclusion Our results support a novel mechanism whereby CD44 palmitoylation and consequent lipid raft affiliation inversely regulate breast cancer cell migration, and may act as a new therapeutic target in breast cancer metastasis. PMID:24512624

Expression and distribution of hyaluronic acid and CD44 in unphonated human vocal fold mucosa.

PubMed

Sato, Kiminori; Umeno, Hirohito; Nakashima, Tadashi; Nonaka, Satoshi; Harabuchi, Yasuaki

2009-11-01

The tension caused by phonation (vocal fold vibration) is hypothesized to stimulate vocal fold stellate cells (VFSCs) in the maculae flavae (MFe) to accelerate production of extracellular matrices. The distribution of hyaluronic acid (HA) and expression of CD44 (a cell surface receptor for HA) were examined in human vocal fold mucosae (VFMe) that had remained unphonated since birth. Five specimens of VFMe (3 adults, 2 children) that had remained unphonated since birth were investigated with Alcian blue staining, hyaluronidase digestion, and immunohistochemistry for CD44. The VFMe containing MFe were hypoplastic and rudimentary. The VFMe did not have a vocal ligament, Reinke's space, or a layered structure, and the lamina propria appeared as a uniform structure. In the children, HA was distributed in the VFMe containing MFe. In the adults, HA had decreased in the VFMe containing MFe. In both groups, the VFSCs in the MFe and the fibroblasts in the lamina propria expressed little CD44. This study supports the hypothesis that the tensions caused by vocal fold vibration stimulate the VFSCs in the MFe to accelerate production of extracellular matrices and form the layered structure. Phonation after birth is one of the important factors in the growth and development of the human VFMe.

Integrin alpha 10, CD44, PTEN, cadherin-11 and lactoferrin expressions are potential biomarkers for selecting patients in need of central nervous system prophylaxis in diffuse large B-cell lymphoma

PubMed Central

Lemma, Siria A; Kuusisto, Milla; Haapasaari, Kirsi-Maria; Sormunen, Raija; Lehtinen, Tuula; Klaavuniemi, Tuula; Eray, Mine; Jantunen, Esa; Soini, Ylermi; Vasala, Kaija; Böhm, Jan; Salokorpi, Niina; Koivunen, Petri; Karihtala, Peeter; Vuoristo, Jussi; Turpeenniemi-Hujanen, Taina; Kuittinen, Outi

2017-01-01

Abstract Central nervous system (CNS) relapse is a devastating complication that occurs in about 5% of diffuse large B-cell lymphoma (DLBCL) patients. Currently, there are no predictive biological markers. We wanted to study potential biomarkers of CNS tropism that play a role in adhesion, migration and/or in the regulation of inflammatory responses. The expression levels of ITGA10, CD44, PTEN, cadherin-11, CDH12, N-cadherin, P-cadherin, lactoferrin and E-cadherin were studied with IHC and IEM. GEP was performed to see whether found expressional changes are regulated at DNA/RNA level. IHC included 96 samples of primary CNS lymphoma (PCNSL), secondary CNS lymphoma (sCNSL) and systemic DLBCL (sDLBCL). IEM included two PCNSL, one sCNSL, one sDLBCL and one reactive lymph node samples. GEP was performed on two DLBCL samples, one with and one without CNS relapse. CNS disease was associated with enhanced expression of cytoplasmic and membranous ITGA10 and nuclear PTEN (P < 0.0005, P = 0.002, P = 0.024, respectively). sCNSL presented decreased membranous CD44 and nuclear and cytoplasmic cadherin-11 expressions (P = 0.001, P = 0.006, P = 0.048, respectively). In PCNSL lactoferrin expression was upregulated (P < 0.0005). IEM results were mainly supportive of the IHC results. In GEP CD44, cadherin-11, lactoferrin and E-cadherin were under-expressed in CNS disease. Our results are in line with previous studies, where gene expressions in extracellular matrix and adhesion-related pathways are altered in CNS lymphoma. This study gives new information on the DLBCL CNS tropism. If further verified, these markers might become useful in predicting CNS relapses. PMID:28854563

Induction of resistance to diabetes in non-obese diabetic mice by targeting CD44 with a specific monoclonal antibody

PubMed Central

Weiss, Lola; Slavin, Shimon; Reich, Shoshana; Cohen, Patrizia; Shuster, Svetlana; Stern, Robert; Kaganovsky, Ella; Okon, Elimelech; Rubinstein, Ariel M.; Naor, David

2000-01-01

Inflammatory destruction of insulin-producing β cells in the pancreatic islets is the hallmark of insulin-dependent diabetes mellitus, a spontaneous autoimmune disease of non-obese diabetic mice resembling human juvenile (type I) diabetes. Histochemical analysis of diabetic pancreata revealed that mononuclear cells infiltrating the islets and causing autoimmune insulitis, as well as local islet cells, express the CD44 receptor; hyaluronic acid, the principal ligand of CD44, is detected in the islet periphery and islet endothelium. Injection of anti-CD44 mAb 1 hr before cell transfer of diabetogenic splenocytes and subsequently on alternate days for 4 weeks induced considerable resistance to diabetes in recipient mice, reflected by reduced insulitis. Contact sensitivity to oxazolone was not influenced by this treatment. A similar antidiabetic effect was observed even when the anti-CD44 mAb administration was initiated at the time of disease onset: i.e., 4–7 weeks after cell transfer. Administration of the enzyme hyaluronidase also induced appreciable resistance to insulin-dependent diabetes mellitus, suggesting that the CD44–hyaluronic acid interaction is involved in the development of the disease. These findings demonstrate that CD44-positive inflammatory cells may be a potential therapeutic target in insulin-dependent diabetes. PMID:10618410

Expression of CD44 3'-untranslated region regulates endogenous microRNA functions in tumorigenesis and angiogenesis.

PubMed

Jeyapalan, Zina; Deng, Zhaoqun; Shatseva, Tatiana; Fang, Ling; He, Chengyan; Yang, Burton B

2011-04-01

The non-coding 3'-untranslated region (UTR) plays an important role in the regulation of microRNA (miRNA) functions, since it can bind and inactivate multiple miRNAs. Here, we show the 3'-UTR of CD44 is able to antagonize cytoplasmic miRNAs, and result in the increased translation of CD44 and downstream target mRNA, CDC42. A series of cell function assays in the human breast cancer cell line, MT-1, have shown that the CD44 3'-UTR inhibits proliferation, colony formation and tumor growth. Furthermore, it modulated endothelial cell activities, favored angiogenesis, induced tumor cell apoptosis and increased sensitivity to Docetaxel. These results are due to the interaction of the CD44 3'-UTR with multiple miRNAs. Computational algorithms have predicted three miRNAs, miR-216a, miR-330 and miR-608, can bind to both the CD44 and CDC42 3'-UTRs. This was confirmed with luciferase assays, western blotting and immunohistochemical staining and correlated with a series of siRNA assays. Thus, the non-coding CD44 3'-UTR serves as a competitor for miRNA binding and subsequently inactivates miRNA functions, by freeing the target mRNAs from being repressed.

Expression of CD44 3′-untranslated region regulates endogenous microRNA functions in tumorigenesis and angiogenesis

PubMed Central

Jeyapalan, Zina; Deng, Zhaoqun; Shatseva, Tatiana; Fang, Ling; He, Chengyan; Yang, Burton B.

2011-01-01

The non-coding 3′-untranslated region (UTR) plays an important role in the regulation of microRNA (miRNA) functions, since it can bind and inactivate multiple miRNAs. Here, we show the 3′-UTR of CD44 is able to antagonize cytoplasmic miRNAs, and result in the increased translation of CD44 and downstream target mRNA, CDC42. A series of cell function assays in the human breast cancer cell line, MT-1, have shown that the CD44 3′-UTR inhibits proliferation, colony formation and tumor growth. Furthermore, it modulated endothelial cell activities, favored angiogenesis, induced tumor cell apoptosis and increased sensitivity to Docetaxel. These results are due to the interaction of the CD44 3′-UTR with multiple miRNAs. Computational algorithms have predicted three miRNAs, miR-216a, miR-330 and miR-608, can bind to both the CD44 and CDC42 3′-UTRs. This was confirmed with luciferase assays, western blotting and immunohistochemical staining and correlated with a series of siRNA assays. Thus, the non-coding CD44 3′-UTR serves as a competitor for miRNA binding and subsequently inactivates miRNA functions, by freeing the target mRNAs from being repressed. PMID:21149267

Effect of dacarbazine on CD44 in live melanoma cells as measured by atomic force microscopy-based nanoscopy.

PubMed

Huang, Xun; He, Jiexiang; Zhang, Huan-Tian; Sun, Kai; Yang, Jie; Wang, Huajun; Zhang, Hongxin; Guo, Zhenzhao; Zha, Zhen-Gang; Zhou, Changren

2017-01-01

CD44 ligand-receptor interactions are known to be involved in regulating cell migration and tumor cell metastasis. High expression levels of CD44 correlate with a poor prognosis of melanoma patients. In order to understand not only the mechanistic basis for dacarbazine (DTIC)-based melanoma treatment but also the reason for the poor prognosis of melanoma patients treated with DTIC, dynamic force spectroscopy was used to structurally map single native CD44-coupled receptors on the surface of melanoma cells. The effect of DTIC treatment was quantified by the dynamic binding strength and the ligand-binding free-energy landscape. The results demonstrated no obvious effect of DTIC on the unbinding force between CD44 ligand and its receptor, even when the CD44 nanodomains were reduced significantly. However, DTIC did perturb the kinetic and thermodynamic interactions of the CD44 ligand-receptor, with a resultant greater dissociation rate, lower affinity, lower binding free energy, and a narrower energy valley for the free-energy landscape. For cells treated with 25 and 75 μg/mL DTIC for 24 hours, the dissociation constant for CD44 increased 9- and 70-fold, respectively. The CD44 ligand binding free energy decreased from 9.94 for untreated cells to 8.65 and 7.39 kcal/mol for DTIC-treated cells, which indicated that the CD44 ligand-receptor complexes on DTIC-treated melanoma cells were less stable than on untreated cells. However, affinity remained in the micromolar range, rather than the millimolar range associated with nonaffinity ligands. Hence, the CD44 receptor could still be activated, resulting in intracellular signaling that could trigger a cellular response. These results demonstrate DTIC perturbs, but not completely inhibits, the binding of CD44 ligand to membrane receptors, suggesting a basis for the poor prognosis associated with DTIC treatment of melanoma. Overall, atomic force microscopy-based nanoscopic methods offer thermodynamic and kinetic insight into the effect of DTIC on the CD44 ligand-binding process.

Effect of dacarbazine on CD44 in live melanoma cells as measured by atomic force microscopy-based nanoscopy

PubMed Central

Huang, Xun; He, Jiexiang; Zhang, Huan-tian; Sun, Kai; Yang, Jie; Wang, Huajun; Zhang, Hongxin; Guo, Zhenzhao; Zha, Zhen-gang; Zhou, Changren

2017-01-01

CD44 ligand–receptor interactions are known to be involved in regulating cell migration and tumor cell metastasis. High expression levels of CD44 correlate with a poor prognosis of melanoma patients. In order to understand not only the mechanistic basis for dacarbazine (DTIC)-based melanoma treatment but also the reason for the poor prognosis of melanoma patients treated with DTIC, dynamic force spectroscopy was used to structurally map single native CD44-coupled receptors on the surface of melanoma cells. The effect of DTIC treatment was quantified by the dynamic binding strength and the ligand-binding free-energy landscape. The results demonstrated no obvious effect of DTIC on the unbinding force between CD44 ligand and its receptor, even when the CD44 nanodomains were reduced significantly. However, DTIC did perturb the kinetic and thermodynamic interactions of the CD44 ligand–receptor, with a resultant greater dissociation rate, lower affinity, lower binding free energy, and a narrower energy valley for the free-energy landscape. For cells treated with 25 and 75 μg/mL DTIC for 24 hours, the dissociation constant for CD44 increased 9- and 70-fold, respectively. The CD44 ligand binding free energy decreased from 9.94 for untreated cells to 8.65 and 7.39 kcal/mol for DTIC-treated cells, which indicated that the CD44 ligand–receptor complexes on DTIC-treated melanoma cells were less stable than on untreated cells. However, affinity remained in the micromolar range, rather than the millimolar range associated with nonaffinity ligands. Hence, the CD44 receptor could still be activated, resulting in intracellular signaling that could trigger a cellular response. These results demonstrate DTIC perturbs, but not completely inhibits, the binding of CD44 ligand to membrane receptors, suggesting a basis for the poor prognosis associated with DTIC treatment of melanoma. Overall, atomic force microscopy-based nanoscopic methods offer thermodynamic and kinetic insight into the effect of DTIC on the CD44 ligand-binding process. PMID:29296081

Macrophage Migration Inhibitory Factor Mediates Proliferative GN via CD74

PubMed Central

Djudjaj, Sonja; Lue, Hongqi; Rong, Song; Papasotiriou, Marios; Klinkhammer, Barbara M.; Zok, Stephanie; Klaener, Ole; Braun, Gerald S.; Lindenmeyer, Maja T.; Cohen, Clemens D.; Bucala, Richard; Tittel, Andre P.; Kurts, Christian; Moeller, Marcus J.; Floege, Juergen; Ostendorf, Tammo

2016-01-01

Pathologic proliferation of mesangial and parietal epithelial cells (PECs) is a hallmark of various glomerulonephritides. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that mediates inflammation by engagement of a receptor complex involving the components CD74, CD44, CXCR2, and CXCR4. The proliferative effects of MIF may involve CD74 together with the coreceptor and PEC activation marker CD44. Herein, we analyzed the effects of local glomerular MIF/CD74/CD44 signaling in proliferative glomerulonephritides. MIF, CD74, and CD44 were upregulated in the glomeruli of patients and mice with proliferative glomerulonephritides. During disease, CD74 and CD44 were expressed de novo in PECs and colocalized in both PECs and mesangial cells. Stress stimuli induced MIF secretion from glomerular cells in vitro and in vivo, in particular from podocytes, and MIF stimulation induced proliferation of PECs and mesangial cells via CD74. In murine crescentic GN, Mif-deficient mice were almost completely protected from glomerular injury, the development of cellular crescents, and the activation and proliferation of PECs and mesangial cells, whereas wild-type mice were not. Bone marrow reconstitution studies showed that deficiency of both nonmyeloid and bone marrow–derived Mif reduced glomerular cell proliferation and injury. In contrast to wild-type mice, Cd74-deficient mice also were protected from glomerular injury and ensuing activation and proliferation of PECs and mesangial cells. Our data suggest a novel molecular mechanism and glomerular cell crosstalk by which local upregulation of MIF and its receptor complex CD74/CD44 mediate glomerular injury and pathologic proliferation in GN. PMID:26453615

Duodenal intraepithelial T lymphocytes in patients with functional dyspepsia

PubMed Central

Gargala, Gilles; Lecleire, Stéphane; François, Arnaud; Jacquot, Serge; Déchelotte, Pierre; Ballet, Jean Jacques; Favennec, Loic; Ducrotté, Philippe

2007-01-01

AIM: To quantify the intraepithelial lymphocytes (IELs) and to document the membrane expression of CD4, CD8, TCRγδ and adhesion and/or activation-associated molecules (CD103, CD28, CD44, CD69, HLA-DR, CD95/Fas) in the duodenal mucosa of patients with functional dyspepsia (FD) in order to provide arguments for an immunological process in FD. METHODS: Twenty-six FD patients according to Rome II criteria (20 were H pylori negative) were studied and compared to 12 healthy adults. IELs were isolated from five duodenal biopsy samples, then quantified by microscopy and flow cytometry while the membrane phenotypes were determined by cytofluorometry. RESULTS: Duodenal histological examination was normal. In H pylori negative patients, the number of IELs was not different from that in healthy controls. Median percentage expression of CD4, CD8, or TCRγδ and CD103, CD44, CD28, CD69 on CD3+ IELs, among the adhesion/activation associated molecules tested, was not different from that in healthy controls. In contrast, the median percentage expression of CD95/Fas [22 (9-65) vs 45 (19-88), P = 0.03] and HLA-DR expressing CD3+ IELs [4 (0-30) vs 13 (4-42), P = 0.04] was significantly lower in the H pylori negative FD group than in healthy controls, respectively. The number of IELs was significantly greater in H pylori positive FD patients than in healthy controls [median ratiofor 100 enterocytes 27.5 (6.7-62.5) vs 10.8 (3-33.3), P = 0.02] due to a higher number of CD8+ CD3+ IELs. CONCLUSION: In H pylori negative FD patients, the phenotypic characterization of IELs suggests that we cannot exclude a role of IELs in FD. PMID:17511033

HOXA5 determines cell fate transition and impedes tumor initiation and progression in breast cancer through regulation of E-cadherin and CD24

PubMed Central

Teo, Wei Wen; Merino, Vanessa F.; Cho, Soonweng; Korangath, Preethi; Liang, Xiaohui; Wu, Ren-chin; Neumann, Neil M.; Ewald, Andrew J.; Sukumar, Saraswati

2016-01-01

Loss of HOXA5 expression occurs frequently in breast cancer and correlates with higher pathological grade and poorer disease outcome. However, how HOX proteins drive differentiation in mammalian cells is poorly understood. In this paper, we investigated cellular and molecular consequences of loss of HOXA5 in breast cancer, and the role played by retinoic acid in HOXA5 function. Analysis of global gene expression data from HOXA5-depleted MCF10A breast epithelial cells, followed by validation, pointed to a role for HOXA5 in maintaining several molecular traits typical of the epithelial lineage such as cell-cell adhesion, tight junctions and markers of differentiation. Depleting HOXA5 in immortalized MCF10A or transformed MCF10A-Kras cells reduced their CD24+/CD44lo population, enhanced self-renewal capacity, and reduced expression of E-cadherin (CDH1) and CD24. In the case of MCF10A-Kras, HOXA5 loss increased branching and protrusive morphology in Matrigel, all features suggestive of epithelial to basal transition. Further, orthotopically implanted xenografts of MCF10A-Kras-scr grew as well-differentiated pseudo-luminal carcinomas, while MCF10A-Kras-shHOXA5 cells formed aggressive, poorly differentiated carcinomas. Conversely, ectopic expression of HOXA5 in aggressive SUM149 or SUM159 breast cancer cells reversed the cellular and molecular alterations observed in the HOXA5-depleted cells. Retinoic acid is a known upstream regulator of HOXA5 expression. HOXA5 depletion in MCF10A cells engineered to express doxycycline-induced shHOXA5 slowed transition of cells from a less differentiated CD24−/CD44+ to the more differentiated CD24+/CD44+ state. This transition was promoted by retinal treatment which upregulated endogenous HOXA5 expression, and caused re-expression of, Occludin, and claudin-7 (CLDN7). Expression of CDH1 and CD24 was transcriptionally upregulated by direct binding of HOXA5 to their promoter sequences as demonstrated by luciferase and ChIP analyses. Thus, loss of HOXA5 in mammary cells leads to loss of epithelial traits, an increase in stemness and cell plasticity, and the acquisition of more aggressive phenotypes. PMID:27157614

Mycobacterium tuberculosis infection modulates adipose tissue biology

PubMed Central

Kühl, Anja A.; Kupz, Andreas; Vogelzang, Alexis; Mollenkopf, Hans-Joachim; Löwe, Delia; Bandermann, Silke; Dorhoi, Anca; Brinkmann, Volker

2017-01-01

Mycobacterium tuberculosis (Mtb) primarily resides in the lung but can also persist in extrapulmonary sites. Macrophages are considered the prime cellular habitat in all tissues. Here we demonstrate that Mtb resides inside adipocytes of fat tissue where it expresses stress-related genes. Moreover, perigonadal fat of Mtb-infected mice disseminated the infection when transferred to uninfected animals. Adipose tissue harbors leukocytes in addition to adipocytes and other cell types and we observed that Mtb infection induces changes in adipose tissue biology depending on stage of infection. Mice infected via aerosol showed infiltration of inducible nitric oxide synthase (iNOS) or arginase 1 (Arg1)-negative F4/80+ cells, despite recruitment of CD3+, CD4+ and CD8+ T cells. Gene expression analysis of adipose tissue of aerosol Mtb-infected mice provided evidence for upregulated expression of genes associated with T cells and NK cells at 28 days post-infection. Strikingly, IFN-γ-producing NK cells and Mtb-specific CD8+ T cells were identified in perigonadal fat, specifically CD8+CD44-CD69+ and CD8+CD44-CD103+ subpopulations. Gene expression analysis of these cells revealed that they expressed IFN-γ and the lectin-like receptor Klrg1 and down-regulated CD27 and CD62L, consistent with an effector phenotype of Mtb-specific CD8+ T cells. Sorted NK cells expressed higher abundance of Klrg1 upon infection, as well. Our results reveal the ability of Mtb to persist in adipose tissue in a stressed state, and that NK cells and Mtb-specific CD8+ T cells infiltrate infected adipose tissue where they produce IFN-γ and assume an effector phenotype. We conclude that adipose tissue is a potential niche for Mtb and that due to infection CD8+ T cells and NK cells are attracted to this tissue. PMID:29040326

Differential Expression and Enzymatic Activity of DPPIV/CD26 Affects Migration Ability of Cervical Carcinoma Cells

PubMed Central

Beckenkamp, Aline; Willig, Júlia Biz; Santana, Danielle Bertodo; Nascimento, Jéssica; Paccez, Juliano Domiraci; Zerbini, Luiz Fernando; Bruno, Alessandra Nejar; Pilger, Diogo André; Wink, Márcia Rosângela; Buffon, Andréia

2015-01-01

Dipeptidyl peptidase IV (DPPIV/CD26) is a transmembrane glycoprotein that inactivates or degrades some bioactive peptides and chemokines. For this reason, it regulates cell proliferation, migration and adhesion, showing its role in cancer processes. This enzyme is found mainly anchored onto the cell membrane, although it also has a soluble form, an enzymatically active isoform. In the present study, we investigated DPPIV/CD26 activity and expression in cervical cancer cell lines (SiHa, HeLa and C33A) and non-tumorigenic HaCaT cells. The effect of the DPPIV/CD26 inhibitor (sitagliptin phosphate) on cell migration and adhesion was also evaluated. Cervical cancer cells and keratinocytes exhibited DPPIV/CD26 enzymatic activity both membrane-bound and in soluble form. DPPIV/CD26 expression was observed in HaCaT, SiHa and C33A, while in HeLa cells it was almost undetectable. We observed higher migratory capacity of HeLa, when compared to SiHa. But in the presence of sitagliptin SiHa showed an increase in migration, indicating that, at least in part, cell migration is regulated by DPPIV/CD26 activity. Furthermore, in the presence of sitagliptin phosphate, SiHa and HeLa cells exhibited a significant reduction in adhesion. However this mechanism seems to be mediated independent of DPPIV/CD26. This study demonstrates, for the first time, the activity and expression of DPPIV/CD26 in cervical cancer cells and the effect of sitagliptin phosphate on cell migration and adhesion. PMID:26222679

Differential Expression and Enzymatic Activity of DPPIV/CD26 Affects Migration Ability of Cervical Carcinoma Cells.

PubMed

Beckenkamp, Aline; Willig, Júlia Biz; Santana, Danielle Bertodo; Nascimento, Jéssica; Paccez, Juliano Domiraci; Zerbini, Luiz Fernando; Bruno, Alessandra Nejar; Pilger, Diogo André; Wink, Márcia Rosângela; Buffon, Andréia

2015-01-01

Dipeptidyl peptidase IV (DPPIV/CD26) is a transmembrane glycoprotein that inactivates or degrades some bioactive peptides and chemokines. For this reason, it regulates cell proliferation, migration and adhesion, showing its role in cancer processes. This enzyme is found mainly anchored onto the cell membrane, although it also has a soluble form, an enzymatically active isoform. In the present study, we investigated DPPIV/CD26 activity and expression in cervical cancer cell lines (SiHa, HeLa and C33A) and non-tumorigenic HaCaT cells. The effect of the DPPIV/CD26 inhibitor (sitagliptin phosphate) on cell migration and adhesion was also evaluated. Cervical cancer cells and keratinocytes exhibited DPPIV/CD26 enzymatic activity both membrane-bound and in soluble form. DPPIV/CD26 expression was observed in HaCaT, SiHa and C33A, while in HeLa cells it was almost undetectable. We observed higher migratory capacity of HeLa, when compared to SiHa. But in the presence of sitagliptin SiHa showed an increase in migration, indicating that, at least in part, cell migration is regulated by DPPIV/CD26 activity. Furthermore, in the presence of sitagliptin phosphate, SiHa and HeLa cells exhibited a significant reduction in adhesion. However this mechanism seems to be mediated independent of DPPIV/CD26. This study demonstrates, for the first time, the activity and expression of DPPIV/CD26 in cervical cancer cells and the effect of sitagliptin phosphate on cell migration and adhesion.

In vivo characterization of the novel CD44v6-targeting Fab fragment AbD15179 for molecular imaging of squamous cell carcinoma: a dual-isotope study

PubMed Central

2014-01-01

Background Patients with squamous cell carcinoma in the head and neck region (HNSCC) offer a diagnostic challenge due to difficulties to detect small tumours and metastases. Imaging methods available are not sufficient, and radio-immunodiagnostics could increase specificity and sensitivity of diagnostics. The objective of this study was to evaluate, for the first time, the in vivo properties of the radiolabelled CD44v6-targeting fragment AbD15179 and to assess its utility as a targeting agent for radio-immunodiagnostics of CD44v6-expressing tumours. Methods The fully human CD44v6-targeting Fab fragment AbD15179 was labelled with 111In or 125I, as models for radionuclides suitable for imaging with SPECT or PET. Species specificity, antigen specificity and internalization properties were first assessed in vitro. In vivo specificity and biodistribution were then evaluated in tumour-bearing mice using a dual-tumour and dual-isotope setup. Results Both species-specific and antigen-specific binding of the conjugates were demonstrated in vitro, with no detectable internalization. The in vivo studies demonstrated specific tumour binding and favourable tumour targeting properties for both conjugates, albeit with higher tumour uptake, slower tumour dissociation, higher tumour-to-blood ratio and higher CD44v6 sensitivity for the 111In-labelled fragment. In contrast, the 125I-Fab demonstrated more favourable tumour-to-organ ratios for liver, spleen and kidneys. Conclusions We conclude that AbD15179 efficiently targets CD44v6-expressing squamous cell carcinoma xenografts, and particularly, the 111In-Fab displayed high and specific tumour uptake. CD44v6 emerges as a suitable target for radio-immunodiagnostics, and a fully human antibody fragment such as AbD15179 can enable further clinical imaging studies. PMID:24598405

Dual pharmacological inhibition of glutathione and thioredoxin systems synergizes to kill colorectal carcinoma stem cells.

PubMed

Tanaka, Genki; Inoue, Ken-Ichi; Shimizu, Takayuki; Akimoto, Kazumi; Kubota, Keiichi

2016-09-01

NRF2 stabilizes redox potential through genes for glutathione and thioredoxin antioxidant systems. Whether blockade of glutathione and thioredoxin is useful in eliminating cancer stem cells remain unknown. We used xenografts derived from colorectal carcinoma patients to investigate the pharmacological inhibition of glutathione and thioredoxin systems. Higher expression of five glutathione S-transferase isoforms (GSTA1, A2, M4, O2, and P1) was observed in xenograft-derived spheroids than in fibroblasts. Piperlongumine (2.5-10 μmol/L) and auranofin (0.25-4 μmol/L) were used to inhibit glutathione S-transferase π and thioredoxin reductase, respectively. Piperlongumine or auranofin alone up-regulated the expression of NRF2 target genes, but not TP53 targets. While piperlongumine showed modest cancer-specific cell killing (IC50 difference between cancer spheroids and fibroblasts: P = 0.052), auranofin appeared more toxic to fibroblasts (IC50 difference between cancer spheroids and fibroblasts: P = 0.002). The synergism of dual inhibition was evaluated by determining the Combination Index, based on the number of surviving cells with combination treatments. Molar ratios indicated synergism in cancer spheroids, but not in fibroblasts: (auranofin:piperlongumine) = 2:5, 1:5, 1:10, and 1:20. Cancer-specific cell killing was achieved at the following drug concentrations (auranofin:piperlongumine): 0.25:2.5 μmol/L, 0.5:2.5 μmol/L, or 0.25:5 μmol/L. The dual inhibition successfully decreased CD44v9 surface presentation and delayed tumor emergence in nude mouse. However, a small subpopulation persistently survived and accumulated phosphorylated histone H2A. Such "persisters" still retained lesser but significant tumorigenicity. Thus, dual inhibition of glutathione S-transferase π and thioredoxin reductase could be a feasible option for decreasing the tumor mass and CD44v9-positive fraction by disrupting redox regulation. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Comparative Gene Expression Profiling of Primary and Metastatic Renal Cell Carcinoma Stem Cell-Like Cancer Cells

PubMed Central

Czarnecka, Anna M.; Lewicki, Sławomir; Helbrecht, Igor; Brodaczewska, Klaudia; Koch, Irena; Zdanowski, Robert; Król, Magdalena; Szczylik, Cezary

2016-01-01

Background Recent advancement in cancer research has shown that tumors are highly heterogeneous, and multiple phenotypically different cell populations are found in a single tumor. Cancer development and tumor growth are driven by specific types of cells—stem cell-like cancer cells (SCLCCs)—which are also responsible for metastatic spread and drug resistance. This research was designed to verify the presence of SCLCCs in renal cell cancer cell lines. Subsequently, we aimed to characterize phenotype and cell biology of CD105+ cells, defined previously as renal cell carcinoma tumor-initiating cells. The main goal of the project was to describe the gene-expression profile of stem cell-like cancer cells of primary tumor and metastatic origin. Materials and Methods Real-time PCR analysis of stemness genes (Oct-4, Nanog and Ncam) and soft agar colony formation assay were conducted to check the stemness properties of renal cell carcinoma (RCC) cell lines. FACS analysis of CD105+ and CD133+ cells was performed on RCC cells. Isolated CD105+ cells were verified for expression of mesenchymal markers—CD24, CD146, CD90, CD73, CD44, CD11b, CD19, CD34, CD45, HLA-DR and alkaline phosphatase. Hanging drop assay was used to investigate CD105+ cell-cell cohesion. Analysis of free-floating 3D spheres formed by isolated CD105+ was verified, as spheres have been hypothesized to contain undifferentiated multipotent progenitor cells. Finally, CD105+ cells were sorted from primary (Caki-2) and metastatic (ACHN) renal cell cancer cell lines. Gene-expression profiling of sorted CD105+ cells was performed with Agilent’s human GE 4x44K v2 microarrays. Differentially expressed genes were further categorized into canonical pathways. Network analysis and downstream analysis were performed with Ingenuity Pathway Analysis. Results Metastatic RCC cell lines (ACHN and Caki-1) demonstrated higher colony-forming ability in comparison to primary RCC cell lines. Metastatic RCC cell lines harbor numerous CD105+ cell subpopulations and have higher expression of stemness genes (Oct-4 and Nanog). CD105+ cells adopt 3D grape-like floating structures under handing drop conditions. Sorted CD105+ cells are positive for human mesenchymal stem cell (MSC) markers CD90, CD73, CD44, CD146, and alkaline phosphatase activity, but not for CD24 and hematopoietic lineage markers CD34, CD11b, CD19, CD45, and HLA-DR. 1411 genes are commonly differentially expressed in CD105+ cells (both from primary [Caki-2] and metastatic RCC [ACHN] cells) in comparison to a healthy kidney epithelial cell line (ASE-5063). TGF-β, Wnt/β-catenine, epithelial-mesenchymal transition (EMT), Rap1 signaling, PI3K-Akt signaling, and Hippo signaling pathway are deregulated in CD105+ cells. TGFB1, ERBB2, and TNF are the most significant transcriptional regulators activated in these cells. Conclusions All together, RCC-CD105+ cells present stemlike properties. These stem cell-like cancer cells may represent a novel target for therapy. A unique gene-expression profile of CD105+ cells could be used as initial data for subsequent functional studies and drug design. PMID:27812180

In vitro characterization of CD133lo cancer stem cells in Retinoblastoma Y79 cell line.

PubMed

Nair, Rohini M; Balla, Murali Ms; Khan, Imran; Kalathur, Ravi Kiran Reddy; Kondaiah, Paturu; Vemuganti, Geeta K

2017-11-21

Retinoblastoma (Rb), the most common childhood intraocular malignant tumor, is reported to have cancer stem cells (CSCs) similar to other tumors. Our previous investigation in primary tumors identified the small sized cells with low CD133 (Prominin-1) and high CD44 (Hyaluronic acid receptor) expression to be putative Rb CSCs using flow cytometry (FSC lo /SSC lo /CD133 lo /CD44 hi ). With this preliminary data, we have now utilized a comprehensive approach of in vitro characterization of Y79 Rb cell line following CSC enrichment using CD133 surface marker and subsequent validation to confirm the functional properties of CSCs. The cultured Rb Y79 cells were evaluated for surface markers by flow cytometry and CD133 sorted cells (CD133 lo /CD133 hi ) were compared for CSC characteristics by size/percentage, cell cycle assay, colony formation assay, differentiation, Matrigel transwell invasion assay, cytotoxicity assay, gene expression using microarray and validation by semi-quantitative PCR. Rb Y79 cell line shared the profile (CD133, CD90, CXCR4 and ABCB1) of primary tumors except for CD44 expression. The CD133 lo cells (16.1 ± 0.2%) were FSC lo /SSC lo , predominantly within the G0/G1 phase, formed larger and higher number of colonies with ability to differentiate to CD133 hi cells, exhibited increased invasive potential in a matrigel transwell assay (p < 0.05) and were resistant to Carboplatin treatment (p < 0.001) as compared to CD133 hi cells. The CD133 lo cells showed higher expression of several embryonic stem cell genes (HOXB2, HOXA9, SALL1, NANOG, OCT4, LEFTY), stem cells/progenitor genes (MSI2, BMI1, PROX1, ABCB1, ABCB5, ABCG2), and metastasis related gene- MACC1, when compared to the CD133 hi cells. This study validates the observation from our earlier primary tumor study that CSC properties in Rb Y79 cell line are endowed within the CD133 lo population, evident by their characteristics- i.e. small sized, dormant in nature, increased colony forming ability, differentiation to CD133 hi cells, higher invasiveness potential, drug resistance and primitive gene expression pattern. These findings provide a proof of concept for methodological characterization of the retinoblastoma CSCs with future implications for improved diagnostic and treatment strategies.

Caveolin-mediated endocytosis of the Chlamydia M278 outer membrane peptide encapsulated in poly(lactic acid)-Poly(ethylene glycol) nanoparticles by mouse primary dendritic cells enhances specific immune effectors mediated by MHC class II and CD4+ T cells.

PubMed

Dixit, Saurabh; Sahu, Rajnish; Verma, Richa; Duncan, Skyla; Giambartolomei, Guillermo H; Singh, Shree R; Dennis, Vida A

2018-03-01

We previously developed a Chlamydia trachomatis nanovaccine (PPM) by encapsulating a chlamydial M278 peptide within poly(lactic acid)-poly(ethylene glycol) biodegradable nanoparticles that immunopotentiated Chlamydia-specific immune effector responses in mice. Herein, we investigated the mechanistic interactions of PPM with mouse bone marrow-derived dendritic cells (DCs) for its uptake, trafficking, and T cell activation. Our results reveal that PPM triggered enhanced expression of effector cytokines and chemokines, surface activation markers (Cd1d2, Fcgr1), pathogen-sensing receptors (TLR2, Nod1), co-stimulatory (CD40, CD80, CD86) and MHC class I and II molecules. Co-culturing of PPM-primed DCs with T cells from C. muridarum vaccinated mice yielded an increase in Chlamydia-specific immune effector responses including CD3 + lymphoproliferation, CD3 + CD4 + IFN-γ-secreting cells along with CD3 + CD4 + memory (CD44 high and CD62L high ) and effector (CD44 high and CD62L low ) phenotypes. Intracellular trafficking analyses revealed an intense expression and colocalization of PPM predominantly in endosomes. PPM also upregulated the transcriptional and protein expression of the endocytic mediator, caveolin-1 in DCs. More importantly, the specific inhibition of caveolin-1 led to decreased expression of PPM-induced cytokines and co-stimulatory molecules. Our investigation shows that PPM provided enhancement of uptake, probably by exploiting the caveolin-mediated endocytosis pathway, endosomal processing, and MHC II presentation to immunopotentiate Chlamydia-specific immune effector responses mediated by CD4 + T cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comparison of EpCAMhighCD44+ cancer stem cells with EpCAMhighCD44- tumor cells in colon cancer by single-cell sequencing.

PubMed

Liu, Mingshan; Di, Jiabo; Liu, Yang; Su, Zhe; Jiang, Beihai; Wang, Zaozao; Su, Xiangqian

2018-03-26

Cancer stem cells (CSCs) are considered to be responsible for tumorigenesis and cancer relapse. EpCAM high CD44 + tumor cells are putative colorectal CSCs that express high levels of stem cell genes, while the EpCAM high CD44 - population mostly contains differentiated tumor cells (DTCs). This study aims to determine whether single CSC (EpCAM high CD44 + ) and DTC (EpCAM high CD44 - ) can be distinguished in terms of somatic copy number alterations (SCNAs). We applied fluorescence-activated cell sorting to isolate the CD45 - EpCAM high CD44 + and CD45 - EpCAM high CD44 - populations from two primary colon tumors, on which low-coverage single-cell whole-genome sequencing (WGS) was then performed ∼0.1x depth. We compared the SCNAs of the CSCs and DTCs at single-cell resolution. In total, 47 qualified single cells of the two populations underwent WGS. The single-cell SCNA profiles showed that there were obvious SCNAs in both the CSCs and DTCs of each patient, and each patient had a specific copy number alteration pattern. Hierarchical clustering and correlation analysis both showed that the SCNA profiles of CSCs and DTCs from the same patient had similar SCNA pattern, while there were regional differences in the CSCs and DTCs in certain patient. SCNAs of CSCs in the same patient were highly reproducible. Our data suggest that major SCNAs occurred at an early stage and were inherited steadily. The similarity of ubiquitous SCNAs between the CSCs and DTCs might have arisen from lineage differentiation. CSCs from the same patient had reproducible SCNA profiles, indicating that gain or loss in certain chromosome is required for colon cancer development.

Self-Assembled Polymeric Micelles Based on Hyaluronic Acid-g-Poly(d,l-lactide-co-glycolide) Copolymer for Tumor Targeting

PubMed Central

Son, Gyung Mo; Kim, Hyun Yul; Ryu, Je Ho; Chu, Chong Woo; Kang, Dae Hwan; Park, Su Bum; Jeong, Young-IL

2014-01-01

Graft copolymer composed hyaluronic acid (HA) and poly(d,l-lactide-co-glycolide) (PLGA) (HAgLG) was synthesized for antitumor targeting via CD44 receptor of tumor cells. The carboxylic end of PLGA was conjugated with hexamethylenediamine (HMDA) to have amine end group in the end of chain (PLGA-amine). PLGA-amine was coupled with carboxylic acid of HA. Self-assembled polymeric micelles of HAgLG have spherical morphologies and their sizes were around 50–200 nm. Doxorubicin (DOX)-incorporated polymeric micelles were prepared by dialysis procedure. DOX was released over 4 days and its release rate was accelerated by the tumoric enzyme hyaluronidase. To assess targetability of polymeric micelles, CD44-positive HepG2 cells were employed treated with fluorescein isothiocyanate (FITC)-labeled polymeric micelles. HepG2 cells strongly expressed green fluorescence at the cell membrane and cytosol. However, internalization of polymeric micelles were significantly decreased when free HA was pretreated to block the CD44 receptor. Furthermore, the CD44-specific anticancer activity of HAgLG polymeric micelles was confirmed using CD44-negative CT26 cells and CD44-positive HepG2 cells. These results indicated that polymeric micelles of HaLG polymeric micelles have targetability against CD44 receptor of tumor cells. We suggest HAgLG polymeric micelles as a promising candidate for specific drug targeting. PMID:25216338

A phase IIa study of HA-irinotecan, formulation of hyaluronic acid and irinotecan targeting CD44 in extensive-stage small cell lung cancer.

PubMed

Alamgeer, Muhammad; Neil Watkins, D; Banakh, Ilia; Kumar, Beena; Gough, Daniel J; Markman, Ben; Ganju, Vinod

2018-04-01

Preclinical studies in small cell lung cancer (SCLC) have shown that hyaluronic acid (HA) can be effectively used to deliver chemotherapy and selectively decrease CD44 expressing (stem cell-like) tumour cells. The current study aimed to replicate these findings and obtain data on safety and activity of HA-irinotecan (HA-IR). Eligible patients with extensive stage SCLC were consented. A safety cohort (n = 5) was treated with HA-IR and Carboplatin (C). Subsequently, the patients were randomised 1:1 to receive experimental (HA-IR + C) or standard (IR + C) treatment, to a maximum of 6 cycles. The second line patients were added to the study and treated with open label HA-IR + C. Tumour response was measured after every 2 cycles. Baseline tumour specimens were stained for CD44s and CD44v6 expression. Circulating tumour cells (CTCs) were enumerated before each treatment cycle. Out of 39 patients screened, 34 were evaluable for the study. The median age was 66 (range 39-83). The overall response rates were 69% and 75% for experimental and standard arms respectively. Median progression free survival was 42 and 28 weeks, respectively (p = 0.892). The treatments were well tolerated. The incidence of grade III/IV diarrhea was more common in the standard arm, while anaemia was more common in the experimental arm. IHC analysis suggested that the patients with CD44s positive tumours may gain survival benefit from HA-IR. HA-IR is well tolerated and active in ES-SCLC. The effect of HA-IR on CD44s + cancer stem-like cells provide an early hint towards a potential novel target.

Metastatic Potential of Cancer Stem Cells in Head and Neck Squamous Cell Carcinoma

PubMed Central

Davis, Samantha J.; Divi, Vasu; Owen, John H.; Papagerakis, Silvana; Bradford, Carol R.; Carey, Thomas E.; Prince, Mark E. P.

2012-01-01

Objective Subpopulations of highly tumorigenic cells, which have the unique capacity to self-renew and produce differentiated progeny, have been identified in multiple malignancies. In head and neck squamous cell carcinoma (HNSCC), this subpopulation of cells, termed cancer stem cells (CSCs) are contained within the population with high CD44 expression. It has been postulated that CSCs play a role in invasion and metastasis; however, there is little evidence to support this theory. We designed in vitro and in vivo models of metastasis to study the behavior of CSCs in HNSCC. Design Cells were sorted for CD44 expression using flow cytometry. Sorted cells were used in an in vitro invasion assay. For in vivo studies, CSCs and non-CSCs were injected into the tail veins of mice, and lungs were either harvested or imaged to evaluate for metastases. Results In vitro, CD44high cells were more motile but less invasive than CD44low cells. In vivo, 4/5 mice injected with CD44high cells and 0/5 mice injected with CD44low cells formed lung metastases. Two of the metastases arose from CSCs from a primary tumor and three from CSCs from HNSCC cell lines. Conclusions In vitro, CSCs do not have an increased ability to invade through basement membrane, but they do migrate more efficiently through a porous barrier. In contrast, CSCs formed metastases quite efficiently in vivo, whereas non-CSCs did not form metastases at all. This phenomenon could be due to enhanced migratory capacity of CSCs, which may be more important than basement membrane degradation in vivo. PMID:21173377

Hyaluronic Acid Surface Modified Liposomes Prepared via Orthogonal Aminoxy Coupling: Synthesis of Nontoxic Aminoxylipids Based on Symmetrically α-Branched Fatty Acids, Preparation of Liposomes by Microfluidic Mixing, and Targeting to Cancer Cells Expressing CD44.

PubMed

Bartheldyová, Eliška; Effenberg, Roman; Mašek, Josef; Procházka, Lubomír; Knötigová, Pavlína Turánek; Kulich, Pavel; Hubatka, František; Velínská, Kamila; Zelníčková, Jaroslava; Zouharová, Darina; Fojtíková, Martina; Hrebík, Dominik; Plevka, Pavel; Mikulík, Robert; Miller, Andrew D; Macaulay, Stuart; Zyka, Daniel; Drož, Ladislav; Raška, Milan; Ledvina, Miroslav; Turánek, Jaroslav

2018-06-25

New synthetic aminoxy lipids are designed and synthesized as building blocks for the formulation of functionalized nanoliposomes by microfluidization using a NanoAssemblr. Orthogonal binding of hyaluronic acid onto the outer surface of functionalized nanoliposomes via aminoxy coupling ( N-oxy ligation) is achieved at hemiacetal function of hyaluronic acid and the structure of hyaluronic acid-liposomes is visualized by transmission electron microscopy and cryotransmission electron microscopy. Observed structures are in a good correlation with data obtained by dynamic light scattering (size and ζ-potential). In vitro experiments on cell lines expressing CD44 receptors demonstrate selective internalization of fluorochrome-labeled hyaluronic acid-liposomes, while cells with down regulated CD44 receptor levels exhibit very low internalization of hyaluronic acid-liposomes. A method based on microfluidization mixing was developed for preparation of monodispersive unilamellar liposomes containing aminoxy lipids and orthogonal binding of hyaluronic acid onto the liposomal surface was demonstrated. These hyaluronic acid-liposomes represent a potentially new drug delivery platform for CD44-targeted anticancer drugs as well as for immunotherapeutics and vaccines.

Aggressive Phenotype of Cells Disseminated via Hematogenous and Lymphatic Route in Breast Cancer Patients.

PubMed

Markiewicz, Aleksandra; Nagel, Anna; Szade, Jolanta; Majewska, Hanna; Skokowski, Jaroslaw; Seroczynska, Barbara; Stokowy, Tomasz; Welnicka-Jaskiewicz, Marzena; Zaczek, Anna J

2018-06-01

Intratumoral heterogeneity of breast cancer remains a major challenge in successful treatment. Failure of cancer therapies can also be accredited to inability to systemically eradicate cancer stem cells (CSCs). Recent evidence points to the role of epithelial-mesenchymal transition (EMT) in expanding the pool of tumor cells with CSCs features. Thus, we assessed expression level as well as heterogeneity of CSCs markers in primary tumors (PT), lymph node metastasis (LNM), and circulating tumor cells (CTCs)-enriched blood fractions in order to correlate them with signs of EMT activation as well as clinicopathological data of breast cancer patients. Level of CSCs markers (ALDH1, CD44, CD133, OCT-4, NANOG) and EMT markers was quantified in PT (N=107), LNM (N=56), and CTCs-enriched blood fractions (N=85). Heterogeneity of CSCs markers expression within each PT and LNM was assessed by calculating Gini Index. Percentage of ALDH1-positive cells was elevated in PT in comparison to LNM (P = .005). However, heterogeneity of the four CSCs markers: ALDH1 (P = .019), CD133 (P = .009), OCT-4 (P = .027), and CD44 (P < .001) was decreased in LNM. Samples classified as mesenchymal (post-EMT) showed elevated expression of CSCs markers (OCT-4 and CD44 in PT; OCT-4 in LNM; ALDH1, OCT-4, NANOG, CD44 in CTCs). Patients with mesenchymal-like CTCs had worse prognosis than patients with epithelial-like or no CTCs (P = .0025). CSCs markers are enriched in PT, LNM, and CTCs with mesenchymal features, but their heterogeneity is decreased in metastatic lymph nodes. Mesenchymal CTCs phenotype correlates with poor prognosis of the patients. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

[Expressions of angiogenesis-related factors: CD105, EphA2 and EphrinA1 in laryngeal squamous cell carcinoma and clinical implication].

PubMed

Su, J; Ji, X B; Xie, J H; Li, W

2016-12-07

Objective: To investigate the expressions of endoglin (CD105), erythropoietin-producing hepatocyte receptor A2 (EphA2) and its ligand ephrinA1 proteins in laryngeal squamous cell carcinoma (LSCC) and the relationship between their expressions and the clinicopathological factors of LSCC. Methods: The expressions of CD105, EphA2 and EphrinA1 proteins were detected with immunohistochemical staining in LSCC in 76 cases and adjacent normal laryngeal tissues (ANLT) (S-P) in 25 cases.SPSS 17.0 software was used to analyze the data. Results: The mean microvessel density (MVD) value marked by CD105 staining in LSCC was 10.33±2.29, which was significantly higher than that in ANLT(1.20±1.04, t =18.732, P <0.05). The CD105-MVD was correlated with T stage, histological grading, clinical stage, lymph node metastasis, recurrence and prognosis in LSCC (F value was 5.34, 4.79, 5.36, t value was -2.70, 2.56, all P <0.05). The positive expression rates of EphA2 and EphrinA1 in LSCC were 78.95% (60/76), and 81.85% (62/76), which were respectively significantly higher than 40% (10/25) for EphA2 expression and 44% (11/25) for EphrinA1, expression in ANLT (χ 2 value was 13.41, 13.26, both P <0.05). EphA2 expression was correlated with histological grading, T stage, clinical stage, lymph node metastasis, recurrence and prognosis in LSCC (χ 2 value was 6.25, 14.60, 15.11, 8.52, 5.54, all P <0.05). EphrinA1 expression was correlated with T stage, clinical stage, lymph node metastasis, recurrence and prognosis in LSCC (χ 2 value was 6.44, 12.28, 16.78, 6.44, all P <0.05). The expressions of CD105, EphA2 and EphrinA1 were positively correlated with each other r value was 0.72, 0.74, 0.64, all P <0.05. Survival analysis indicated that the expressions of CD105 and EphA2, histological grading, lymph node metastasis, clinical stage and recurrence were independent factors for tumor prognosis in LSCC ( P <0.05). Conclusions: The expressions of CD105, EphA2 and EphrinA1 protein were positively correlated with each other in LSCC. They may play important roles in the tumorigenesis, malignant progression and poor prognosis of LSCC.

Galunisertib suppresses the staminal phenotype in hepatocellular carcinoma by modulating CD44 expression.

PubMed

Rani, Bhavna; Malfettone, Andrea; Dituri, Francesco; Soukupova, Jitka; Lupo, Luigi; Mancarella, Serena; Fabregat, Isabel; Giannelli, Gianluigi

2018-03-07

Cancer stem cells (CSCs) niche in the tumor microenvironment is responsible for cancer recurrence and therapy failure. To better understand its molecular and biological involvement in hepatocellular carcinoma (HCC) progression, one can design more effective therapies and tailored then to individual patients. While sorafenib is currently the only approved drug for first-line treatment of advanced stage HCC, its role in modulating the CSC niche is estimated to be small. By contrast, transforming growth factor (TGF)-β pathway seems to influence the CSC and thus may impact hallmarks of HCC, such as liver fibrosis, cirrhosis, and tumor progression. Therefore, blocking this pathway may offer an appealing and druggable target. In our study, we have used galunisertib (LY2157299), a selective ATP-mimetic inhibitor of TGF-β receptor I (TGFβI/ALK5) activation, currently under clinical investigation in HCC patients. Because the drug resistance is mainly mediated by CSCs, we tested the effects of galunisertib on stemness phenotype in HCC cells to determine whether TGF-β signaling modulates CSC niche and drug resistance. Galunisertib modulated the expression of stemness-related genes only in the invasive (HLE and HLF) HCC cells inducing a decreased expression of CD44 and THY1. Furthermore, galunisertib also reduced the stemness-related functions of invasive HCC cells decreasing the formation of colonies, liver spheroids and invasive growth ability. Interestingly, CD44 loss of function mimicked the galunisertib effects on HCC stemness-related functions. Galunisertib treatment also reduced the expression of stemness-related genes in ex vivo human HCC specimens. Our observations are the first evidence that galunisertib effectiveness overcomes stemness-derived aggressiveness via decreased expression CD44 and THY1.

Spatiotemporal expression dynamics of selectins govern the sequential extravasation of neutrophils and monocytes in the acute inflammatory response.

PubMed

Zuchtriegel, Gabriele; Uhl, Bernd; Hessenauer, Maximilian E T; Kurz, Angela R M; Rehberg, Markus; Lauber, Kirsten; Krombach, Fritz; Reichel, Christoph A

2015-04-01

Leukocyte recruitment to the site of inflammation is a key event in a variety of cardiovascular pathologies. Infiltrating neutrophils constitute the first line of defense that precedes a second wave of emigrating monocytes reinforcing the inflammatory reaction. The mechanisms initiating this sequential process remained largely obscure. Using advanced in vivo microscopy and in vitro/ex vivo techniques, we identified individual spatiotemporal expression patterns of selectins and their principal interaction partners on neutrophils, resident/inflammatory monocytes, and endothelial cells. Coordinating the intraluminal trafficking of neutrophils and inflammatory monocytes to common sites of extravasation, selectins assign different sites to these immune cells for their initial interactions with the microvascular endothelium. Whereas constitutively expressed leukocyte L-selectin/CD62L and endothelial P-selectin/CD62P together with CD44 and P-selectin glycoprotein ligand-1/CD162 initiate the emigration of neutrophils, de novo synthesis of endothelial E-selectin/CD62E launches the delayed secondary recruitment of inflammatory monocytes. In this context, P-selectin/CD62P and L-selectin/CD62L together with P-selectin glycoprotein ligand-1/CD162 and CD44 were found to regulate the flux of rolling neutrophils and inflammatory monocytes, whereas E-selectin/CD62E selectively adjusts the rolling velocity of inflammatory monocytes. Moreover, selectins and their interaction partners P-selectin glycoprotein ligand-1/CD162 and CD44 differentially control the intraluminal crawling behavior of neutrophils and inflammatory monocytes collectively enabling the sequential extravasation of these immune cells to inflamed tissue. Our findings provide novel insights into the mechanisms initiating the sequential infiltration of the perivascular tissue by neutrophils and monocytes in the acute inflammatory response and might thereby contribute to the development of targeted therapeutic strategies for prevention and treatment of cardiovascular diseases. © 2015 American Heart Association, Inc.

BRCA1-IRIS overexpression promotes and maintains the tumor initiating phenotype: implications for triple negative breast cancer early lesions

PubMed Central

Sullivan, Lisa M.; Sims, Hillary; Bastawisy, Ahmed El; Yousef, Hend F.; Zekri, Abdel-Rahman N.; Bahnassy, Abeer A.; ElShamy, Wael M.

2017-01-01

Tumor-initiating cells (TICs) are cancer cells endowed with self-renewal, multi-lineage differentiation, increased chemo-resistance, and in breast cancers the CD44+/CD24-/ALDH1+ phenotype. Triple negative breast cancers show lack of BRCA1 expression in addition to enhanced basal, epithelial-to-mesenchymal transition (EMT), and TIC phenotypes. BRCA1-IRIS (hereafter IRIS) is an oncogene produced by the alternative usage of the BRCA1 locus. IRIS is involved in induction of replication, transcription of selected oncogenes, and promoting breast cancer cells aggressiveness. Here, we demonstrate that IRIS overexpression (IRISOE) promotes TNBCs through suppressing BRCA1 expression, enhancing basal-biomarkers, EMT-inducers, and stemness-enforcers expression. IRISOE also activates the TIC phenotype in TNBC cells through elevating CD44 and ALDH1 expression/activity and preventing CD24 surface presentation by activating the internalization pathway EGFR→c-Src→cortactin. We show that the intrinsic sensitivity to an anti-CD24 cross-linking antibody-induced cell death in membranous CD24 expressing/luminal A cells could be acquired in cytoplasmic CD24 expressing IRISOE TNBC/TIC cells through IRIS silencing or inactivation. We show that fewer IRISOE TNBC/TICs cells form large tumors composed of TICs, resembling TNBCs early lesions in patients that contain metastatic precursors capable of disseminating and metastasizing at an early stage of the disease. IRIS-inhibitory peptide killed these IRISOE TNBC/TICs, in vivo and prevented their dissemination and metastasis. We propose IRIS inactivation could be pursued to prevent dissemination and metastasis from early TNBC tumor lesions in patients. PMID:28052035

The metastasis suppressor, NDRG1, inhibits “stemness” of colorectal cancer via down-regulation of nuclear β-catenin and CD44

PubMed Central

Wangpu, Xiongzhi; Yang, Xiao; Zhao, Jingkun; Lu, Jiaoyang; Guan, Shaopei; Lu, Jun; Kovacevic, Zaklina; Liu, Wensheng; Mi, Lan; Jin, Runsen; Sun, Jing; Yue, Fei; Ma, Junjun; Lu, Aiguo; Richardson, Des R.; Wang, Lishun; Zheng, Minhua

2015-01-01

N-myc downstream-regulated gene 1 (NDRG1), has been identified as an important metastasis suppressor for colorectal cancer (CRC). In this study, we investigated: (1) the effects of NDRG1 on CRC stemness and tumorigenesis; (2) the molecular mechanisms involved; and (3) the relationship between NDRG1 expression and colorectal cancer prognosis. Our investigation demonstrated that CRC cells with silenced NDRG1 showed more tumorigenic ability and stem cell-like properties, such as: colony and sphere formation, chemoresistance, cell invasion, high expression of CD44, and tumorigenicity in vivo. Moreover, NDRG1 silencing reduced β-catenin expression on the cell membrane, while increasing its nuclear expression. The anti-tumor activity of NDRG1 was demonstrated to be mediated by preventing β-catenin nuclear translocation, as silencing of this latter molecule could reverse the effects of silencing NDRG1 expression. NDRG1 expression was also demonstrated to be negatively correlated to CRC prognosis. In addition, there was a negative correlation between NDRG1 and nuclear β-catenin and also NDRG1 and CD44 expression in clinical CRC specimens. Taken together, our investigation demonstrates that the anti-metastatic activity of NDRG1 in CRC occurs through the down-regulation of nuclear β-catenin and suggests that NDRG1 is a significant therapeutic target. PMID:26418878

Effects of CD44 and E-cadherin overexpression on the proliferation, adhesion and invasion of ovarian cancer cells.

PubMed

Mao, Meiya; Zheng, Xiaojiao; Jin, Bohong; Zhang, Fubin; Zhu, Linyan; Cui, Lining

2017-12-01

CD44 is a prognostic indicator of shorter survival time in ovarian cancer. E-cadherin fragmentation promotes the progression of ovarian cancer. However, the effects of CD44 and E-cadherin overexpression on ovarian cancer cells have remained elusive. The present study aimed to investigate the effects of overexpression of CD44 and E-cadherin on cell proliferation, adhesion and invasion of SKOV-3 and OVCAR-3 ovarian cancer cells. Overexpression of CD44 and E-cadherin was achieved by transfecting SKOV-3 and OVCAR-3 cells with viruses carrying the CD44 or E-cadherin gene, respectively. Expression of CD44 and E-cadherin was detected by western blot analysis. The proliferation of SKOV-3 and OVCAR-3 cells was measured by a Cell Counting Kit-8 at 0, 24 and 48 h after viral transfection. The adhesion ability of SKOV-3 and OVCAR-3 cells to the endothelial layer was detected. A Transwell invasion assay was utilized to assess the invasion ability of the cells. Overexpression of CD44 and E-cadherin in SKOV-3 and OVCAR-3 cells was confirmed by western blot. Compared with the blank or negative control groups, the CD44 overexpression groups of SKOV-3 and OVCAR-3 cells exhibited an increased cell proliferation rate at 24 and 48 h, whereas overexpression of E-cadherin did not alter the proliferation of these cells. Furthermore, compared with the blank and negative control groups, the cell adhesion and invasion ability in the CD44 overexpression groups of SKOV-3 and OVCAR-3 cells was markedly higher. There were no significant differences in adhesion ability between the E-cadherin overexpression group and the blank/negative control group. Of note, overexpression of E-cadherin decreased the invasive ability of SKOV-3 and OVCAR-3 cells. In conclusion, Overexpression of CD44 increased the proliferation, adhesion and invasion of ovarian cancer cells, while overexpression of E-cadherin decreased the invasion of ovarian cancer cells.

Proinsulin Expression Shapes the TCR Repertoire but Fails to Control the Development of Low-Avidity Insulin-Reactive CD8+ T Cells

PubMed Central

Pearson, James A.; Thayer, Terri C.; McLaren, James E.; Ladell, Kristin; De Leenheer, Evy; Phillips, Amy; Davies, Joanne; Kakabadse, Dimitri; Miners, Kelly; Morgan, Peter; Wen, Li; Price, David A.

2016-01-01

NOD mice, a model strain for human type 1 diabetes, express proinsulin (PI) in the thymus. However, insulin-reactive T cells escape negative selection, and subsequent activation of the CD8+ T-cell clonotype G9C8, which recognizes insulin B15-23 via an αβ T-cell receptor (TCR) incorporating TRAV8-1/TRAJ9 and TRBV19/TRBJ2-3 gene rearrangements, contributes to the development of diabetes. In this study, we used fixed TRAV8-1/TRAJ9 TCRα-chain transgenic mice to assess the impact of PI isoform expression on the insulin-reactive CD8+ T-cell repertoire. The key findings were: 1) PI2 deficiency increases the frequency of insulin B15-23–reactive TRBV19+CD8+ T cells and causes diabetes; 2) insulin B15-23–reactive TRBV19+CD8+ T cells are more abundant in the pancreatic lymph nodes of mice lacking PI1 and/or PI2; 3) overexpression of PI2 decreases TRBV19 usage in the global CD8+ T-cell compartment; 4) a biased repertoire of insulin-reactive CD8+ T cells emerges in the periphery regardless of antigen exposure; and 5) low-avidity insulin-reactive CD8+ T cells are less affected by antigen exposure in the thymus than in the periphery. These findings inform our understanding of the diabetogenic process and reveal new avenues for therapeutic exploitation in type 1 diabetes. PMID:26953160

Enrichment of prostate cancer stem-like cells from human prostate cancer cell lines by culture in serum-free medium and chemoradiotherapy.

PubMed

Wang, Lei; Huang, Xing; Zheng, Xinmin; Wang, Xinghuan; Li, Shiwen; Zhang, Lin; Yang, Zhonghua; Xia, Zhiping

2013-01-01

The discovery of rare subpopulations of cancer stem cells (CSCs) has created a new focus in cancer research. As CSCs demonstrate resistance to chemoradiation therapy relative to other cancer cells, this allows the enrichment of CSC populations by killing apoptosis-susceptible cancer cells. In this study, three commonly used human prostate cancer (PCa) cell lines (DU145, PC-3 and LNCaP) were examined for their expression of the putative stem cell markers CD133 and CD44 via flow cytometric analysis. Under normal culture conditions, CD133(+)/CD44(+) cells were only present in the DU145 cell line, and comprised only a minor percentage (0.1% ± 0.01%) of the total population. However, the proportion of these CD133(+)/CD44(+) prostate CSCs could be increased in these cell lines via culture in serum-free medium (SFM), or through chemotherapy or radiotherapy. Indeed, after culture in SFM, the proportion of CD133(+)/CD44(+) cells in DU145 and PC-3 had increased to 10.3% and 3.0%, respectively. Moreover, the proportion had increased to 9.8% enriched by chemotherapy and 3.5% by radiotherapy in DU145. Colony-formation tests, cell invasion assays, and tumor xenografts in BALB/c nude mice were used to evaluate the stem cell properties of CD133(+)/CD44(+) PCa cells that were isolated via fluorescence-activated cell sorting (FACS). CD133(+)/CD44(+) cells had an enhanced colony-formation capability and invasive ability in vitro, and displayed greater tumorigenic properties in vivo. These results demonstrate the presence of CD133(+)/CD44(+) prostate CSCs in established PCa cell lines and that populations of these cells can be enriched by culture in SFM or chemoradiotherapy. Finding novel therapies to override chemoradiation resistance in the prostate CSCs is the key to improve long-term results in PCa management.

A small population of resident limb bud mesenchymal cells express few MSC-associated markers, but the expression of these markers is increased immediately after cell culture.

PubMed

Marín-Llera, Jessica Cristina; Chimal-Monroy, Jesús

2018-05-01

Skeletal progenitors are derived from resident limb bud mesenchymal cells of the vertebrate embryos. However, it remains poorly understood if they represent stem cells, progenitors, or multipotent mesenchymal stromal cells (MSC). Derived-MSC of different adult tissues under in vitro experimental conditions can differentiate into the same cellular lineages that are present in the limb. Here, comparing non-cultured versus cultured mesenchymal limb bud cells, we determined the expression of MSC-associated markers, the in vitro differentiation capacity and their gene expression profile. Results showed that in freshly isolated limb bud mesenchymal cells, the proportion of cells expressing Sca1, CD44, CD105, CD90, and CD73 is very low and a low expression of lineage-specific genes was observed. However, recently seeded limb bud mesenchymal cells acquired Sca1 and CD44 markers and the expression of the key differentiation genes Runx2 and Sox9, while Scx and Pparg genes decreased. Also, their chondrogenic differentiation capacity decreased through cellular passages while the osteogenic increased. Our findings suggest that the modification of the cell adhesion process through the in vitro method changed the limb mesenchymal cell immunophenotype leading to the expression and maintenance of common MSC-associated markers. These findings could have a significant impact on MSC study and isolation strategy because they could explain common variations observed in the MSC immunophenotype in different tissues. © 2018 International Federation for Cell Biology.

Associations Between Markers of Colorectal Cancer Stem Cells and Adenomas Among Ethnic Groups

PubMed Central

Leavell, Bonita J.; Van Buren, Eric; Antaki, Fadi; Axelrod, Bradley N.; Rambus, Mary Ann; Majumdar, Adhip P. N.

2013-01-01

Background and Purposes Most colorectal tumors develop from adenomatous polyps, which are detected by colonoscopy. African Americans (AAs) have higher incidence of colorectal cancer (CRC) and greater mortality from this disease than Caucasian Americans (CAs). We investigated whether differences in predisposition to CRC and its surrogate (colonic adenomas) between these ethnic groups were related to numbers of cancer stem or stem-like cells (CSCs) in colonocytes. Methods We analyzed colonic effluent from 11 AA and 14 CA patients who underwent scheduled colonoscopy examinations at the John D. Dingell Veterans Affairs Medical Center. We determined proportions of cells that expressed the CSC markers CD44 and CD166 by flow cytometry. Results The proportion of colonocytes that were CD44+CD166– in effluent from patients with adenomas was significantly greater than from patients without adenomas (P = 0.01); the proportion of CD44+CD166+ colonocytes was also greater (P = 0.07). Effluent from AAs with adenomas had 60 % more CD44+166– colonocytes than from CAs with adenomas. Using cutoff values of 8 % for AAs and 3 % for CAs, the proportion of CD44+166– colonocytes that had positive predictive value for detection of adenomas was 100 % for AAs and CAs, determined by receiver operator characteristic curve analysis. Conclusion The proportion of CD44+166– colonocytes in colonic effluent can be used to identify patients with adenoma. AAs with adenomas have a higher proportion of CD44+166– colonocytes than CA. The increased proportion of CSCs in colonic tissue from AA might be associated with the increased incidence of CRC in this population. PMID:22562538

Blocking the Adhesion Cascade at the Premetastatic Niche for Prevention of Breast Cancer Metastasis

PubMed Central

Kang, Shin-Ae; Hasan, Nafis; Mann, Aman P; Zheng, Wei; Zhao, Lichao; Morris, Lynsie; Zhu, Weizhu; Zhao, Yan D; Suh, K Stephen; Dooley, William C; Volk, David; Gorenstein, David G; Cristofanilli, Massimo; Rui, Hallgeir; Tanaka, Takemi

2015-01-01

Shear-resistant adhesion and extravasation of disseminated cancer cells at the target organ is a crucial step in hematogenous metastasis. We found that the vascular adhesion molecule E-selectin preferentially promoted the shear-resistant adhesion and transendothelial migration of the estrogen receptor (ER)–/CD44+ hormone-independent breast cancer cells, but not of the ER+/CD44-/low hormone-dependent breast cancer cells. Coincidentally, CD44+ breast cancer cells were abundant in metastatic lung and brain lesions in ER– breast cancer, suggesting that E-selectin supports hematogenous metastasis of ER–/CD44+ breast cancer. In an attempt to prevent hematogenous metastasis through the inhibition of a shear-resistant adhesion of CD44+ cancer cells to E-selectin-expressing blood vessels on the premetastatic niche, an E-selectin targeted aptamer (ESTA) was developed. We demonstrated that a single intravenous injection of ESTA reduced metastases to a baseline level in both syngeneic and xenogeneic forced breast cancer metastasis models without relocating the site of metastasis. The effect of ESTA was absent in E-selectin knockout mice, suggesting that E-selectin is a molecular target of ESTA. Our data highlight the potential application of an E-selectin antagonist for the prevention of hematogenous metastasis of ER–/CD44+ breast cancer. PMID:25815697

Blocking the adhesion cascade at the premetastatic niche for prevention of breast cancer metastasis.

PubMed

Kang, Shin-Ae; Hasan, Nafis; Mann, Aman P; Zheng, Wei; Zhao, Lichao; Morris, Lynsie; Zhu, Weizhu; Zhao, Yan D; Suh, K Stephen; Dooley, William C; Volk, David; Gorenstein, David G; Cristofanilli, Massimo; Rui, Hallgeir; Tanaka, Takemi

2015-06-01

Shear-resistant adhesion and extravasation of disseminated cancer cells at the target organ is a crucial step in hematogenous metastasis. We found that the vascular adhesion molecule E-selectin preferentially promoted the shear-resistant adhesion and transendothelial migration of the estrogen receptor (ER)(-)/CD44(+) hormone-independent breast cancer cells, but not of the ER(+)/CD44(-/low) hormone-dependent breast cancer cells. Coincidentally, CD44(+) breast cancer cells were abundant in metastatic lung and brain lesions in ER(-) breast cancer, suggesting that E-selectin supports hematogenous metastasis of ER(-)/CD44(+) breast cancer. In an attempt to prevent hematogenous metastasis through the inhibition of a shear-resistant adhesion of CD44(+) cancer cells to E-selectin-expressing blood vessels on the premetastatic niche, an E-selectin targeted aptamer (ESTA) was developed. We demonstrated that a single intravenous injection of ESTA reduced metastases to a baseline level in both syngeneic and xenogeneic forced breast cancer metastasis models without relocating the site of metastasis. The effect of ESTA was absent in E-selectin knockout mice, suggesting that E-selectin is a molecular target of ESTA. Our data highlight the potential application of an E-selectin antagonist for the prevention of hematogenous metastasis of ER(-)/CD44(+) breast cancer.

Notch1 inhibition alters the CD44hi/CD24lo population and reduces the formation of brain metastases from breast cancer.

PubMed

McGowan, Patricia M; Simedrea, Carmen; Ribot, Emeline J; Foster, Paula J; Palmieri, Diane; Steeg, Patricia S; Allan, Alison L; Chambers, Ann F

2011-07-01

Brain metastasis from breast cancer is an increasingly important clinical problem. Here we assessed the role of CD44(hi)/CD24(lo) cells and pathways that regulate them, in an experimental model of brain metastasis. Notch signaling (mediated by γ-secretase) has been shown to contribute to maintenance of the cancer stem cell (CSC) phenotype. Cells sorted for a reduced stem-like phenotype had a reduced ability to form brain metastases compared with unsorted or CD44(hi)/CD24(lo) cells (P < 0.05; Kruskal-Wallis). To assess the effect of γ-secretase inhibition, cells were cultured with DAPT and the CD44/CD24 phenotypes quantified. 231-BR cells with a CD44(hi)/CD24(lo) phenotype was reduced by about 15% in cells treated with DAPT compared with DMSO-treated or untreated cells (P = 0.001, ANOVA). In vivo, mice treated with DAPT developed significantly fewer micro- and macrometastases compared with vehicle treated or untreated mice (P = 0.011, Kruskal-Wallis). Notch1 knockdown reduced the expression of CD44(hi)/CD24(lo) phenotype by about 20%. In vitro, Notch1 shRNA resulted in a reduction in cellular growth at 24, 48, and 72 hours time points (P = 0.033, P = 0.002, and P = 0.009, ANOVA) and about 60% reduction in Matrigel invasion was observed (P < 0.001, ANOVA). Cells transfected with shNotch1 formed significantly fewer macrometastases and micrometastases compared with scrambled shRNA or untransfected cells (P < 0.001; Kruskal-Wallis). These data suggest that the CSC phenotype contributes to the development of brain metastases from breast cancer, and this may arise in part from increased Notch activity. ©2011 AACR.

Integrin alpha 10, CD44, PTEN, cadherin-11 and lactoferrin expressions are potential biomarkers for selecting patients in need of central nervous system prophylaxis in diffuse large B-cell lymphoma.

PubMed

Lemma, Siria A; Kuusisto, Milla; Haapasaari, Kirsi-Maria; Sormunen, Raija; Lehtinen, Tuula; Klaavuniemi, Tuula; Eray, Mine; Jantunen, Esa; Soini, Ylermi; Vasala, Kaija; Böhm, Jan; Salokorpi, Niina; Koivunen, Petri; Karihtala, Peeter; Vuoristo, Jussi; Turpeenniemi-Hujanen, Taina; Kuittinen, Outi

2017-08-01

Central nervous system (CNS) relapse is a devastating complication that occurs in about 5% of diffuse large B-cell lymphoma (DLBCL) patients. Currently, there are no predictive biological markers. We wanted to study potential biomarkers of CNS tropism that play a role in adhesion, migration and/or in the regulation of inflammatory responses. The expression levels of ITGA10, CD44, PTEN, cadherin-11, CDH12, N-cadherin, P-cadherin, lactoferrin and E-cadherin were studied with IHC and IEM. GEP was performed to see whether found expressional changes are regulated at DNA/RNA level. IHC included 96 samples of primary CNS lymphoma (PCNSL), secondary CNS lymphoma (sCNSL) and systemic DLBCL (sDLBCL). IEM included two PCNSL, one sCNSL, one sDLBCL and one reactive lymph node samples. GEP was performed on two DLBCL samples, one with and one without CNS relapse. CNS disease was associated with enhanced expression of cytoplasmic and membranous ITGA10 and nuclear PTEN (P < 0.0005, P = 0.002, P = 0.024, respectively). sCNSL presented decreased membranous CD44 and nuclear and cytoplasmic cadherin-11 expressions (P = 0.001, P = 0.006, P = 0.048, respectively). In PCNSL lactoferrin expression was upregulated (P < 0.0005). IEM results were mainly supportive of the IHC results. In GEP CD44, cadherin-11, lactoferrin and E-cadherin were under-expressed in CNS disease. Our results are in line with previous studies, where gene expressions in extracellular matrix and adhesion-related pathways are altered in CNS lymphoma. This study gives new information on the DLBCL CNS tropism. If further verified, these markers might become useful in predicting CNS relapses. © The Author 2017. Published by Oxford University Press.

CD45-mediated signaling pathway is involved in Rhizoctonia bataticola lectin (RBL)-induced proliferation and Th1/Th2 cytokine secretion in human PBMC

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pujari, Radha; Eligar, Sachin M.; Kumar, Natesh

2012-03-23

Highlights: Black-Right-Pointing-Pointer RBL, a potent mitogenic and complex N-glycan specific lectin binds to CD45 on PBMC. Black-Right-Pointing-Pointer RBL triggers CD45-mediated signaling involved in activation of p38MAPK and STAT-5. Black-Right-Pointing-Pointer Inhibition of CD45 PTPase signaling blocks RBL-induced ZAP70 phosphorylation. Black-Right-Pointing-Pointer RBL-CD45 mediated signaling is crucial for RBL-induced immunodulatory activities. -- Abstract: We earlier reported the mitogenic and immunostimulatory activities of Rhizoctonia bataticola lectin (RBL), purified from phytopathogenic fungus R. bataticola in human PBMC. The lectin demonstrates specificity towards glycoproteins containing complex N-glycans. Since CD45-protein tyrosine phosphatase that abundantly expresses N-glycans is important in T-cell signaling, the study aimed to investigate themore » involvement of CD45 in the immunomodulatory activities of RBL. Flowcytometry and confocal microscopy studies revealed that RBL exhibited binding to PBMC and colocalized with CD45. The binding was comparable in cells expressing different CD45 isoforms-RA, -RB and -RO. CD45 blocking antibody reduced the binding and proliferation of PBMC induced by RBL. CD45-PTPase inhibitor dephostatin inhibited RBL-induced proliferation, expression of CD25 and pZAP-70. RBL-induced secretion of Th1/Th2 cytokines were significantly inhibited in presence of dephostatin. Also, dephostatin blocked phosphorylation of p38MAPK and STAT-5 that was crucial for the biological functions of RBL. The study demonstrates the involvement of CD45-mediated signaling in RBL-induced PBMC proliferation and Th1/Th2 cytokine secretion through activation of p38MAPK and STAT-5.« less

[Markers of stromal invasion during background and precancerous changes of the glandular epithelium and in adenocarcinoma of the cervix uteri].

PubMed

Danilova, N V; Andreeva, Iu Iu; Zavalishina, L É; Mal'kov, P G

2012-01-01

It is very difficult to identify stromal invasion when the glandular epithelium of the cervix uteri is involved. It is necessary to draw a clear distinction between its glandular structures and adenocarcinoma in situ, involving the preexisting crypts and invasive glands. An attempt was made to assess the possibilities of using as markers of invasion the following stromal proteins and adhesion molecules: CD44, E-cadherin, beta-catenin, tenascin, and laminin. Fifty-three cases of benign glandular changes, 66 cases of dysplasias and adenocarcinomas in situ, and 47 cases of invasive adenocarcinoma were examined. An immunohistochemical study was performed according to the standard protocol using the antibodies to CD44, laminin, tenascin, E-cadherin, and beta-catenin and a semiquantitative assessment of results was made. CD44 was found to be redistributed from the cells to the tumor stroma. CD44 was not detected in the stroma surrounding the intact glands, so were benign epithelial changes. In the tumor environment, there was, on the contrary, a reaction with CD44 in 74.5% of invasive adenocarcinomas cases (p < 0.05). The expression of tenascin in the invasive adenocarcinomas and around the foci of early stromal invasion significantly exceeded that in the stroma around the intact glands and dysplastic changes (p < 0.05). All the study groups showed a membrane reaction with E-cadherin and beta-catenin, which probably suggested that changes were absent in the Wnt signaling pathway. In 70.2% of invasive adenocarcinomas, laminin demonstrated a significant cytoplasmic expression in 5-30% of the tumor cells predominantly located along the tumor invasion area or in the deepest tumor complexes (p > 0.05). CD44 and tenascin are of great diagnostic value in examining invasive and microinvasive adenocarcinomas of the cervix uteri. E-cadherin and beta-catenin are of no diagnostic value in the study groups of pathological processes. Laminin is a potential marker of stromal invasion; however, its expression calls for further investigation.

Basigin (CD147), a multifunctional transmembrane glycoprotein with various binding partners

PubMed Central

Muramatsu, Takashi

2016-01-01

Basigin, also called CD147 or EMMPRIN, is a transmembrane glycoprotein that belongs to the immunoglobulin superfamily. Basigin has isoforms; the common form (basigin or basigin-2) has two immunoglobulin domains, and the extended form (basigin-1) has three. Basigin is the receptor for cyclophilins, S100A9 and platelet glycoprotein VI, whereas basigin-1 serves as the receptor for the rod-derived cone viability factor. Basigin tightly associates with monocarboxylate transporters and is essential for their cell surface translocation and activities. In the same membrane plane, basigin also associates with other proteins including GLUT1, CD44 and CD98. The carbohydrate portion of basigin is recognized by lectins, such as galectin-3 and E-selectin. These molecular recognitions form the basis for the role of basigin in the transport of nutrients, migration of inflammatory leukocytes and induction of matrix metalloproteinases. Basigin is important in vision, spermatogenesis and other physiological phenomena, and plays significant roles in the pathogenesis of numerous diseases, including cancer. Basigin is also the receptor for an invasive protein RH5, which is present in malaria parasites. PMID:26684586

Organotypic culture of normal, dysplastic and squamous cell carcinoma-derived oral cell lines reveals loss of spatial regulation of CD44 and p75 NTR in malignancy.

PubMed

Dalley, Andrew J; AbdulMajeed, Ahmad A; Upton, Zee; Farah, Camile S

2013-01-01

Oral squamous cell carcinomas (OSCC) often arise from dysplastic lesions. The role of cancer stem cells in tumour initiation is widely accepted, yet the potential existence of pre-cancerous stem cells in dysplastic tissue has received little attention. Cell lines from oral diseases ranging in severity from dysplasia to malignancy provide opportunity to investigate the involvement of stem cells in malignant progression from dysplasia. Stem cells are functionally defined by their ability to generate hierarchical tissue structures in consortium with spatial regulation. Organotypic cultures readily display tissue hierarchy in vitro; hence, in this study, we compared hierarchical expression of stem cell-associated markers in dermis-based organotypic cultures of oral epithelial cells from normal tissue (OKF6-TERT2), mild dysplasia (DOK), severe dysplasia (POE-9n) and OSCC (PE/CA P J15). Expression of CD44, p75(NTR), CD24 and ALDH was studied in monolayers by flow cytometry and in organotypic cultures by immunohistochemistry. Spatial regulation of CD44 and p75(NTR) was evident for organotypic cultures of normal (OKF6-TERT2) and dysplasia (DOK and POE-9n) but was lacking for OSCC (PE/CA PJ15)-derived cells. Spatial regulation of CD24 was not evident. All monolayer cultures exhibited CD44, p75(NTR), CD24 antigens and ALDH activity (ALDEFLUOR(®) assay), with a trend towards loss of population heterogeneity that mirrored disease severity. In monolayer, increased FOXA1 and decreased FOXA2 expression correlated with disease severity, but OCT3/4, Sox2 and NANOG did not. We conclude that dermis-based organotypic cultures give opportunity to investigate the mechanisms that underlie loss of spatial regulation of stem cell markers seen with OSCC-derived cells. © 2012 John Wiley & Sons A/S.

Ex vivo expansion of canine cytotoxic large granular lymphocytes exhibiting characteristics of natural killer cells.

PubMed

Shin, Dong-Jun; Park, Ji-Yun; Jang, Youn-Young; Lee, Je-Jung; Lee, Youn-Kyung; Shin, Myung-Geun; Jung, Ji-Youn; Carson, William E; Cho, Duck; Kim, Sang-Ki

2013-06-15

Canine NK cells still are not well-characterized due to the lack of information concerning specific NK cell markers and the fact that NK cells are not an abundant cell population. In this study, we selectively expanded the canine cytotoxic large granular lymphocytes (CLGLs) that exhibit morphologic, genetic, and functional characteristics of NK cells from normal donor PBMCs. The cultured CLGLs were characterized by a high proportion of CD5(dim) expressing cells, of which the majority of cells co-expressed CD3 and CD8, but did not express TCRαβ and TCRγδ. The phenotype of the majority of the CLGLs was CD5(dim)CD3(+)CD8(+) TCRαβ(-)TCRγδ(-)CD4(-)CD21(-)CD11c(+/-)CD11d(+/-)CD44(+). The expression of mRNAs for NK cell-associated receptors (NKG2D, NKp30, NKp44, Ly49, perforin, and granzyme B) were highly upregulated in cultured CLGLs. Specifically, NKp46 was remarkably upregulated in the cultured CLGLs compared to PBMCs. The mRNAs for the NKT-associated iTCRα gene in CLGLs was present at a basal level. The cytotoxic activity of the CLGLs against canine NK cell-sensitive CTAC cells was remarkably elevated in a dose-dependent manner, and the CLGLs produced large amounts of IFN-γ. The antitumor activity of CLGLs extended to different types of canine tumor cells (CF41.Mg and K9TCC-pu-AXC) without specific antigen recognition. These results are consistent with prior reports, and strongly suggest that the selectively expanded CLGLs represent a population of canine NK cells. The results of this study will contribute to future research on canine NK cells as well as NK cell-based immunotherapy. Copyright © 2013 Elsevier B.V. All rights reserved.

Isolation of stem-like cells from spontaneous feline mammary carcinomas: phenotypic characterization and tumorigenic potential.

PubMed

Barbieri, Federica; Wurth, Roberto; Ratto, Alessandra; Campanella, Chiara; Vito, Guendalina; Thellung, Stefano; Daga, Antonio; Cilli, Michele; Ferrari, Angelo; Florio, Tullio

2012-04-15

Current carcinogenesis theory states that only a small subset of tumor cells, the cancer stem cells or tumor initiating cells (TICs), are responsible for tumor formation and progression. Human breast cancer-initiating cells have been identified as CD44-expressing cells, which retain tumorigenic activity and display stem cell-like properties. Spontaneous feline mammary carcinoma (FMC) is an aggressive cancer, which shows biological similarities to the human tumor counterpart. We report the isolation and phenotypic characterization of FMC-derived stem/progenitor cells, showing in vitro self-renewal, long-lasting proliferation and in vivo tumorigenicity. Twenty-one FMC samples were collected, histologically classified and characterized for the expression of Ki67, EGFR, ER-α and CD44, by immunohistochemistry. By culture in stem cell permissive conditions, we isolated, from 13 FMCs, a CD44-positive subpopulation able to survive and proliferate in vitro as mammospheres of different sizes and morphologies. When injected in NOD/SCID mice, FMC stem-like cells initiate tumors, generating cell heterogeneity and recapitulating the original histotype. In serum-containing medium, spheroid cells showed differentiation properties as shown by morphological changes, the loss of CD44 expression and tumorigenic potential. These data show that stem-defined culture of FMC enriches for TICs and validate the use of these cells as a suitable model for comparative oncology studies of mammary biology and testing therapeutic strategies aimed at eradicating TICs. Copyright © 2012 Elsevier Inc. All rights reserved.

[Differentiation of bone marrow derived from mesenchymal stem cells into cardiomyocyte-like cells induced by co-culture with rat myocardial cells].

PubMed

Zhang, Rong-Li; Jiang, Er-Lie; Wang, Mei; Zhou, Zheng; Zhai, Wen-Jing; Zhai, Wei-Hua; Wang, Hua; Wang, Zhi-Yong; Bao, Yu-Shi; DU, Hong; Han, Ming-Zhe

2008-10-01

The study was purposed to investigate the differentiation ability of mesenchymal stem cells (MSCs) into myocardial cells in vitro. Rat bone marrow-derived MSCs were labeled and co-cultured with neonatal rat cardiomyocytes (CM) for 5 - 7 days. The expression of cell surface antigens was detected by flow cytometry, and the expression of muscle-specific marker myosin and troponin T in labeled cells was detected by immunofluorescence. The results showed that in vitro cultured MSCs expressed CD90, CD44, CD105, CD54, not expressed CD34, CD45, CD31. After co-cultured with neonatal rat CM, labeled MSCs differentiated into cardiomyocyte-like cells expressing myosin and troponin T. It is concluded that MSCs can differentiate into cardiomyocyte-like cells when co-cultured with neonatal myocardial cells in vitro. In co-culture of two kind of cells in ratio of four to one showed obvious efficacy differentiating MSCs into CMs.

Estrogen Receptor-Alpha (ESR1) Governs the Lower Female Reproductive Tract Vulnerability to Candida albicans

PubMed Central

Salinas-Muñoz, Laura; Campos-Fernández, Raúl; Mercader, Enrique; Olivera-Valle, Irene; Fernández-Pacheco, Carlota; Matilla, Lara; García-Bordas, Julio; Brazil, Jennifer C.; Parkos, Charles A.; Asensio, Fernando; Muñoz-Fernández, Maria A.; Hidalgo, Andrés; Sánchez-Mateos, Paloma; Samaniego, Rafael; Relloso, Miguel

2018-01-01

Estradiol-based therapies predispose women to vaginal infections. Moreover, it has long been known that neutrophils are absent from the vaginal lumen during the ovulatory phase (high estradiol). However, the mechanisms that regulate neutrophil influx to the vagina remain unknown. We investigated the neutrophil transepithelial migration (TEM) into the vaginal lumen. We revealed that estradiol reduces the CD44 and CD47 epithelial expression in the vaginal ectocervix and fornix, which retain neutrophils at the apical epithelium through the estradiol receptor-alpha. In contrast, luteal progesterone increases epithelial expression of CD44 and CD47 to promote neutrophil migration into the vaginal lumen and Candida albicans destruction. Distinctive to vaginal mucosa, neutrophil infiltration is contingent to sex hormones to prevent sperm from neutrophil attack; although it may compromise immunity during ovulation. Thus, sex hormones orchestrate tolerance and immunity in the vaginal lumen by regulating neutrophil TEM. PMID:29881378

Estrogen Receptor-Alpha (ESR1) Governs the Lower Female Reproductive Tract Vulnerability to Candida albicans.

PubMed

Salinas-Muñoz, Laura; Campos-Fernández, Raúl; Mercader, Enrique; Olivera-Valle, Irene; Fernández-Pacheco, Carlota; Matilla, Lara; García-Bordas, Julio; Brazil, Jennifer C; Parkos, Charles A; Asensio, Fernando; Muñoz-Fernández, Maria A; Hidalgo, Andrés; Sánchez-Mateos, Paloma; Samaniego, Rafael; Relloso, Miguel

2018-01-01

Estradiol-based therapies predispose women to vaginal infections. Moreover, it has long been known that neutrophils are absent from the vaginal lumen during the ovulatory phase (high estradiol). However, the mechanisms that regulate neutrophil influx to the vagina remain unknown. We investigated the neutrophil transepithelial migration (TEM) into the vaginal lumen. We revealed that estradiol reduces the CD44 and CD47 epithelial expression in the vaginal ectocervix and fornix, which retain neutrophils at the apical epithelium through the estradiol receptor-alpha. In contrast, luteal progesterone increases epithelial expression of CD44 and CD47 to promote neutrophil migration into the vaginal lumen and Candida albicans destruction. Distinctive to vaginal mucosa, neutrophil infiltration is contingent to sex hormones to prevent sperm from neutrophil attack; although it may compromise immunity during ovulation. Thus, sex hormones orchestrate tolerance and immunity in the vaginal lumen by regulating neutrophil TEM.

Regulation of Leishmania (L.) amazonensis Protein Expression by Host T Cell Dependent Responses: Differential Expression of Oligopeptidase B, Tryparedoxin Peroxidase and HSP70 Isoforms in Amastigotes Isolated from BALB/c and BALB/c Nude Mice

PubMed Central

Teixeira, Priscila Camillo; Velasquez, Leonardo Garcia; Lepique, Ana Paula; de Rezende, Eloiza; Bonatto, José Matheus Camargo; Barcinski, Marcello Andre; Cunha-Neto, Edecio; Stolf, Beatriz Simonsen

2015-01-01

Leishmaniasis is an important disease that affects 12 million people in 88 countries, with 2 million new cases every year. Leishmania amazonensis is an important agent in Brazil, leading to clinical forms varying from localized (LCL) to diffuse cutaneous leishmaniasis (DCL). One interesting issue rarely analyzed is how host immune response affects Leishmania phenotype and virulence. Aiming to study the effect of host immune system on Leishmania proteins we compared proteomes of amastigotes isolated from BALB/c and BALB/c nude mice. The athymic nude mice may resemble patients with diffuse cutaneous leishmaniasis, considered T-cell hyposensitive or anergic to Leishmania´s antigens. This work is the first to compare modifications in amastigotes’ proteomes driven by host immune response. Among the 44 differentially expressed spots, there were proteins related to oxidative/nitrosative stress and proteases. Some correspond to known Leishmania virulence factors such as OPB and tryparedoxin peroxidase. Specific isoforms of these two proteins were increased in parasites from nude mice, suggesting that T cells probably restrain their posttranslational modifications in BALB/c mice. On the other hand, an isoform of HSP70 was increased in amastigotes from BALB/c mice. We believe our study may allow identification of potential virulence factors and ways of regulating their expression. PMID:25692783

Dose-escalation study for the targeting of CD44v+ cancer stem cells by sulfasalazine in patients with advanced gastric cancer (EPOC1205).

PubMed

Shitara, Kohei; Doi, Toshihiko; Nagano, Osamu; Imamura, Chiyo K; Ozeki, Takeshi; Ishii, Yuya; Tsuchihashi, Kenji; Takahashi, Shunji; Nakajima, Takako E; Hironaka, Shuichi; Fukutani, Miki; Hasegawa, Hiromi; Nomura, Shogo; Sato, Akihiro; Einaga, Yasuaki; Kuwata, Takeshi; Saya, Hideyuki; Ohtsu, Atsushi

2017-03-01

Cancer stem cells (CSCs) have enhanced mechanisms of protection from oxidative stress. A variant form of CD44 (CD44v), a major CSC marker, was shown to interact with xCT, a subunit of cystine-glutamate transporter, which maintains high levels of intracellular reduced glutathione (GSH) which defend the cell against oxidative stress. Sulfasalazine (SSZ) is an inhibitor of xCT and was shown to suppress the survival of CD44v-positive stem-like cancer cells both in vitro and in vivo. To find the dose of SSZ which can safely reduce the population of CD44v-positive cells in tumors, a dose-escalation study in patients with advanced gastric cancer was conducted. SSZ was given four times daily by oral administration with 2 weeks as one cycle. Tumor biopsies were obtained before and after 14 days of administration of SSZ to evaluate expression of CD44v and the intratumoral level of GSH. Eleven patients were enrolled and received a dosage from 8 to 12 g/day. Safety was confirmed up to a dosage of 12 g/day, which was considered the maximum tolerated dose. Among the eight patients with CD44v-positive cells in their pretreatment biopsy samples, the CD44v-positive cancer cell population appeared to be reduced in the posttreatment biopsy tissues of four patients. Intratumoral GSH levels were also decreased in two patients, suggesting biological effectiveness of SSZ at 8 g/day or greater. This is the first study of SSZ as an xCT inhibitor for targeting CSCs. Reduction of the levels of CD44v-positive cells and GSH was observed in some patients, consistent with the mode of action of SSZ in CSCs.

Pre-B cell leukemia homeobox 1 is associated with lupus susceptibility in mice and humans

PubMed Central

Cuda, Carla M.; Li, Shiwu; Liang, Shujuan; Yin, Yiming; Potula, Hari Hara S.K.; Xu, Zhiwei; Sengupta, Mayami; Chen, Yifang; Butfiloski, Edward; Baker, Henry; Chang, Lung-Ji; Dozmorov, Igor; Sobel, Eric S.; Morel, Laurence

2011-01-01

Sle1a.1 is part of the Sle1 susceptibility locus, which has the strongest association with lupus nephritis in the NZM2410 mouse model. Here we show that Sle1a.1 results in the production of activated and autoreactive CD4+ T cells. In addition, Sle1a.1 expression reduces the peripheral regulatory T cell (Treg) pool, as well as induces a defective response of CD4+ T cells to the retinoic acid (RA) expansion of TGFβ-induced Tregs. At the molecular level, Sle1a.1 corresponds to an increased expression of a novel splice isoform of Pbx1, Pbx1-d. Pbx1-d over-expression is sufficient to induce an activated/inflammatory phenotype in Jurkat T cells, and to decrease their apoptotic response to RA. PBX1-d is expressed more frequently in the CD4+ T cells from lupus patients than from healthy controls, and its presence correlates with an increased central memory T cell population. These findings indicate that Pbx1 is a novel lupus susceptibility gene that regulates T cell activation and tolerance. PMID:22180614

Trametinib plus 4-Methylumbelliferone Exhibits Antitumor Effects by ERK Blockade and CD44 Downregulation and Affects PD-1 and PD-L1 in Malignant Pleural Mesothelioma.

PubMed

Cho, Hiroyuki; Matsumoto, Seiji; Fujita, Yoshiko; Kuroda, Ayumi; Menju, Toshi; Sonobe, Makoto; Kondo, Nobuyuki; Torii, Ikuko; Nakano, Takashi; Lara, Primo N; Gandara, David R; Date, Hiroshi; Hasegawa, Seiki

2017-03-01

Malignant pleural mesothelioma (MPM) is a highly aggressive malignancy in which the mitogen-activated protein kinase pathway plays a critical role in the regulation of tumorigenesis. Hyaluronic acid (HA) is a major component of the extracellular matrix, and elevated HA levels with a concurrent increase in malignant properties are associated with MPM. We evaluated the effects of trametinib, a mitogen-activated protein kinase (MEK) inhibitor, and 4-methylumbelliferone (4-MU), an HA synthesis inhibitor, alone and in combination on MPM cells in vitro and in vivo. We studied the effects of trametinib, 4-MU, and their combination on MPM cells by using cell viability assays, Western blot analysis, and a mouse xenograft model. Trametinib and 4-MU exhibited antiproliferative activity in MPM cells. Trametinib blocked MEK-dependent extracellular signal-regulated kinase (ERK) phosphorylation and decreased CD44 expression in a concentration-dependent manner. Trametinib inhibited the expression of Fra-1 (the activator protein 1 [AP1] component), inhibited ERK phosphorylation, and decreased CD44 expression. 4-MU inhibited ERK phosphorylation but not CD44 expression. In a mouse xenograft model, trametinib and 4-MU alone suppressed tumor growth compared with a control. The combination had a greater inhibitory effect than either monotherapy. Immunohistochemical analysis showed that trametinib treatment alone significantly reduced expression of programmed cell death 1 ligand 1. Furthermore, the combination of trametinib and 4-MU resulted in higher expression of programmed cell death 1 and programmed cell death 1 ligand 1 than did the 4-MU treatment alone. Our results suggest that trametinib and 4-MU are promising therapeutic agents in MPM and that further study of the combination is warranted. Copyright © 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Preparation of Cytokine-activated NK Cells for Use in Adoptive Cell Therapy in Cancer Patients: Protocol Optimization and Therapeutic Potential.

PubMed

van Ostaijen-ten Dam, Monique M; Prins, Henk-Jan; Boerman, Gerharda H; Vervat, Carly; Pende, Daniela; Putter, Hein; Lankester, Arjan; van Tol, Maarten J D; Zwaginga, Jaap J; Schilham, Marco W

2016-01-01

Cell-based immunotherapy using donor-derived natural killer (NK) cells after allogeneic hematopoietic stem cell transplantation may be an attractive treatment of residual leukemia. This study aimed to optimize clinical grade production of a cytokine-activated NK-cell product. NK cells were isolated either by double depletion (CD3(-), CD19(-)) or by sequential depletion and enrichment (CD3(-,) CD56(+)) via CliniMACS from leukapheresis material and cultured in vitro with interleukin (IL)-2 or IL-15. Both NK cell isolation procedures yielded comparable recovery of NK cells and levels of T-cell contamination. After culture with cytokines, the CD3(-)CD56(+) procedure resulted in NK cells of higher purity, that is, less T cells and monocytes, higher viability, and a slightly higher yield than the CD3(-)CD19- procedure. CD69, NKp44, and NKG2A expression were higher on CD3(-)CD56(+) products, whereas lysis of Daudi cells was comparable. Five days of culture led to higher expression of CD69, NKp44, and NKp30 and lysis of K562 and Daudi cell lines. Although CD69 expression and lysis of Daudi cells were slightly higher in cultures with IL-2, T-cell contamination was lower with IL-15. Therefore, further experiments were performed with CD3(-)CD56(+) products cultured with IL-15. Cryopreservation of IL-15-activated NK cells resulted in a loss of cytotoxicity (>92%), whereas thawing of isolated, uncultured NK cells followed by culture with IL-15 yielded cells with about 43% of the original lytic activity. Five-day IL-15-activated NK cells lysed tumor target cell lines and primary leukemic blasts, providing the basis for NK cell–based immunotherapeutic strategies in a clinical setting.

Fulfilling Koch's postulates in glycoscience: HCELL, GPS and translational glycobiology.

PubMed

Sackstein, Robert

2016-06-01

Glycoscience-based research that is performed expressly to address medical necessity and improve patient outcomes is called "translational glycobiology". In the 19th century, Robert Koch proposed a set of postulates to rigorously establish causality in microbial pathogenesis, and these postulates can be reshaped to guide knowledge into how naturally-expressed glycoconjugates direct molecular processes critical to human well-being. Studies in the 1990s indicated that E-selectin, an endothelial lectin that binds sialofucosylated carbohydrate determinants, is constitutively expressed on marrow microvessels, and investigations in my laboratory indicated that human hematopoietic stem cells (HSCs) uniquely express high levels of a specialized glycoform of CD44 called "hematopoietic cell E-/L-selectin ligand" (HCELL) that functions as a highly potent E-selectin ligand. To assess the role of HCELL in directing HSC migration to marrow, a method called "glycosyltransferase-programmed stereosubstitution" (GPS) was developed to custom-modify CD44 glycans to enforce HCELL expression on viable cell surfaces. Human mesenchymal stem cells (MSCs) are devoid of E-selectin ligands, but GPS-based glycoengineering of CD44 on MSCs licenses homing of these cells to marrow in vivo, providing direct evidence that HCELL serves as a "bone marrow homing receptor". This review will discuss the molecular basis of cell migration in historical context, will describe the discovery of HCELL and its function as the bone marrow homing receptor, and will inform on how glycoengineering of CD44 serves as a model for adapting Koch's postulates to elucidate the key roles that glycoconjugates play in human biology and for realizing the immense impact of translational glycobiology in clinical medicine. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Expression of cancer stem markers could be influenced by silencing of p16 gene in HeLa cervical carcinoma cells.

PubMed

Wu, H; Zhang, J; Shi, H

2016-01-01

Effect of the tumor suppression gene p16 on the biological characteristics of HeLa cervical carcinoma cells was explored. The expression of p16 protein was increased in HeLa tumor sphere cells, and no significant difference in tumor spheres from the first to the fourth passages. Compared with those of parental HeLa cells, the proportion of CD44+/CD24- and ABCG2+ cells increased significantly in tumor spheres. However after the cells were silenced by the p16-sh289 vector, expression of P16 protein and the cell number of CD44+/CD24- and ABCG2+ decreased. Moreover, HeLa cells with p16 gene silencing showed decreased abilities of sphere formation and matrigel invasion. More HeLa cells with p16 gene silence were needed for tumor formation in nude mice. Tumor size and weight in mouse model established with p16 gene silenced HeLa cells were less than those with HeLa parental cell model. The present results indicate that silencing of the p16 gene inhibits expression of cancer stem cell markers and tumorigenic ability of HeLa cells.

CD44+ Cancer Stem-Like Cells in EBV-Associated Nasopharyngeal Carcinoma

PubMed Central

Lun, Samantha Wei-Man; Cheung, Siu Tim; Cheung, Phyllis Fung Yi; To, Ka-Fai; Woo, John Kong-Sang; Choy, Kwong-Wai; Chow, Chit; Cheung, Chartia Ching-Mei; Chung, Grace Tin-Yun; Cheng, Alice Suk-Hang; Ko, Chun-Wai; Tsao, Sai-Wah; Busson, Pierre; Ng, Margaret Heung-Ling; Lo, Kwok-Wai

2012-01-01

Nasopharyngeal carcinoma (NPC) is a unique EBV-associated epithelial malignancy, showing highly invasive and metastatic phenotype. Despite increasing evidence demonstrating the critical role of cancer stem-like cells (CSCs) in the maintenance and progression of tumors in a variety of malignancies, the existence and properties of CSC in EBV-associated NPC are largely unknown. Our study aims to elucidate the presence and role of CSCs in the pathogenesis of this malignant disease. Sphere-forming cells were isolated from an EBV-positive NPC cell line C666-1 and its tumor-initiating properties were confirmed by in vitro and in vivo assays. In these spheroids, up-regulation of multiple stem cell markers were found. By flow cytometry, we demonstrated that both CD44 and SOX2 were overexpressed in a majority of sphere-forming C666-1 cells. The CD44+SOX2+ cells was detected in a minor population in EBV-positive xenografts and primary tumors and considered as potential CSC in NPC. Notably, the isolated CD44+ NPC cells were resistant to chemotherapeutic agents and with higher spheroid formation efficiency, showing CSC properties. On the other hand, microarray analysis has revealed a number of differentially expressed genes involved in transcription regulation (e.g. FOXN4, GLI1), immune response (CCR7, IL8) and transmembrane transport (e.g. ABCC3, ABCC11) in the spheroids. Among these genes, increased expression of CCR7 in CD44+ CSCs was confirmed in NPC xenografts and primary tumors. Importantly, blocking of CCR7 abolished the sphere-forming ability of C666-1 in vitro. Expression of CCR7 was associated with recurrent disease and distant metastasis. The current study defined the specific properties of a CSC subpopulation in EBV-associated NPC. Our findings provided new insights into developing effective therapies targeting on CSCs, thereby potentiating treatment efficacy for NPC patients. PMID:23285037

Relationship of PCNA, C-erbB2 and CD44s expression with tumor grade and stage in urothelial carcinomas of the bladder

PubMed Central

Yıldırım, Ayhan; Kösem, Mustafa; Sayar, İlyas; Gelincik, İbrahim; Yavuz, Alparslan; Bozkurt, Aliseydi; Erkorkmaz, Ünal; Bayram, İrfan

2014-01-01

In the present study, the intention was to reveal the relationship of histological grade and stage with c-erbB2, CD44s, and PCNA immunoreactivity in bladder urothelial carcinomas (UC). In our study, we evaluated 46 items of transurethral resection material of patients submitted by YYU Faculty of Medicine, Main Department of Pathology, with a mass revealed in their bladder after clinical and radiological studies at our laboratories and who were diagnosed with urothelial carcinomas. PCNA, c-erbB2, and CD44s were applied in an immunohistochemical manner comprised from nine low-malignant potential papillary urothelial neoplasia, 23 low-grade papillary urothelial carcinoma, and 14 high-grade papillary urothelial carcinoma. Immunostaining was scored according to the percentage of positive cells. The immunohistochemical study demonstrated that the c-erbB2 and PCNA staining ratio increased when an increase occurred in stage and grade. The CD44s staining ratio decreased. C-erbB2, PCNA, and CD44s appear to be a useful marker in the assessment of the prognosis and treatment options in urothelial carcinomas. PMID:25035774

Two widely expressed plasma membrane H(+)-ATPase isoforms of Nicotiana tabacum are differentially regulated by phosphorylation of their penultimate threonine.

PubMed

Bobik, Krzysztof; Duby, Geoffrey; Nizet, Yannick; Vandermeeren, Caroline; Stiernet, Patrick; Kanczewska, Justyna; Boutry, Marc

2010-04-01

The plasma membrane H(+)-ATPases PMA2 and PMA4 are the most widely expressed in Nicotiana plumbaginifolia, and belong to two different subfamilies. Both are activated by phosphorylation of a Thr at the penultimate position and the subsequent binding of 14-3-3 proteins. Their expression in Saccharomyces cerevisiae revealed functional and regulatory differences. To determine whether different regulatory properties between PMA2 and PMA4 exist in plants, we generated two monoclonal antibodies able to detect phosphorylation of the penultimate Thr of either PMA2 or PMA4 in a total protein extract. We also raised Nicotiana tabacum transgenic plants expressing 6-His-tagged PMA2 or PMA4, enabling their individual purification. Using these tools we showed that phosphorylation of the penultimate Thr of both PMAs was high during the early exponential growth phase of an N. tabacum cell culture, and then progressively declined. This decline correlated with decreased 14-3-3 binding and decreased plasma membrane ATPase activity. However, the rate and extent of the decrease differed between the two isoforms. Cold stress of culture cells or leaf tissues reduced the Thr phosphorylation of PMA2, whereas no significant changes in Thr phosphorylation of PMA4 were seen. These results strongly suggest that PMA2 and PMA4 are differentially regulated by phosphorylation. Analysis of the H(+)-ATPase phosphorylation status in leaf tissues indicated that no more than 44% (PMA2) or 32% (PMA4) was in the activated state under normal growth conditions. Purification of either isoform showed that, when activated, the two isoforms did not form hetero-oligomers, which is further support for these two H(+)-ATPase subfamilies having different properties.

Sensitivity of GBM cells to cAMP agonist-mediated apoptosis correlates with CD44 expression and agonist resistance with MAPK signaling.

PubMed

Daniel, Paul M; Filiz, Gulay; Mantamadiotis, Theo

2016-12-01

In some cell types, activation of the second messenger cAMP leads to increased expression of proapoptotic Bim and subsequent cell death. We demonstrate that suppression of the cAMP pathway is a common event across many cancers and that pharmacological activation of cAMP in glioblastoma (GBM) cells leads to enhanced BIM expression and apoptosis in specific GBM cell types. We identified the MAPK signaling axis as the determinant of cAMP agonist sensitivity in GBM cells, with high MAPK activity corresponding to cAMP resistance and low activity corresponding to sensitization to cAMP-induced apoptosis. Sensitive cells were efficiently killed by cAMP agonists alone, while targeting both the cAMP and MAPK pathways in resistant GBM cells resulted in efficient apoptosis. We also show that CD44 is differentially expressed in cAMP agonist-sensitive and -resistant cells. We thus propose that CD44 may be a useful biomarker for distinguishing tumors that may be sensitive to cAMP agonists alone or cAMP agonists in combination with other pathway inhibitors. This suggests that using existing chemotherapeutic compounds in combination with existing FDA-approved cAMP agonists may fast track trials toward improved therapies for difficult-to-treat cancers, such as GBM.

A functional glycoproteomics approach identifies CD13 as a novel E-selectin ligand in breast cancer.

PubMed

Carrascal, M A; Silva, M; Ferreira, J A; Azevedo, R; Ferreira, D; Silva, A M N; Ligeiro, D; Santos, L L; Sackstein, R; Videira, P A

2018-05-17

The glycan moieties sialyl-Lewis-X and/or -A (sLe X/A ) are the primary ligands for E-selectin, regulating subsequent tumor cell extravasation into distant organs. However, the nature of the glycoprotein scaffolds displaying these glycans in breast cancer remains unclear and constitutes the focus of the present investigation. We isolated glycoproteins that bind E-selectin from the CF1_T breast cancer cell line, derived from a patient with ductal carcinoma. Proteins were identified using bottom-up proteomics approach by nanoLC-orbitrap LTQ-MS/MS. Data were curated using bioinformatics tools to highlight clinically relevant glycoproteins, which were validated by flow cytometry, Western blot, immunohistochemistry and in-situ proximity ligation assays in clinical samples. We observed that the CF1_T cell line expressed sLe X , but not sLe A and the E-selectin reactivity was mainly on N-glycans. MS and bioinformatics analysis of the targeted glycoproteins, when narrowed down to the most clinically relevant species in breast cancer, identified CD44 glycoprotein (HCELL) and CD13 as key E-selectin ligands. Additionally, the co-expression of sLe X -CD44 and sLe X -CD13 was confirmed in clinical breast cancer tissue samples. Both CD44 and CD13 glycoforms display sLe X in breast cancer and bind E-selectin, suggesting a key role in metastasis development. Such observations provide a novel molecular rationale for developing targeted therapeutics. While HCELL expression in breast cancer has been previously reported, this is the first study indicating that CD13 functions as an E-selectin ligand in breast cancer. This observation supports previous associations of CD13 with metastasis and draws attention to this glycoprotein as an anti-cancer target. Copyright © 2018 Elsevier B.V. All rights reserved.

Manufacture of endothelial colony-forming progenitor cells from steady-state peripheral blood leukapheresis using pooled human platelet lysate.

PubMed

Siegel, Georg; Fleck, Erika; Elser, Stefanie; Hermanutz-Klein, Ursula; Waidmann, Marc; Northoff, Hinnak; Seifried, Erhard; Schäfer, Richard

2018-05-01

Endothelial colony-forming progenitor cells (ECFCs) are promising candidates for cell therapies. However, ECFC translation to the clinic requires optimized isolation and manufacture technologies according to good manufacturing practice (GMP). ECFCs were manufactured from steady-state peripheral blood (PB) leukapheresis (11 donors), using GMP-compliant technologies including pooled human platelet (PLT) lysate, and compared to human umbilical cord endothelial cells, human aortic endothelial cells, and human cerebral microvascular endothelial cells. Specific variables assessed were growth kinetics, phenotype, trophic factors production, stimulation of tube formation, and Dil-AcLDL uptake. ECFCs could be isolated from PB leukapheresis units with mean processed volume of 5411 mL and mean white blood cell (WBC) concentration factor of 8.74. The mean frequency was 1.44 × 10 -8 ECFCs per WBC, corresponding to a mean of 177.8 ECFCs per apheresis unit. Expandable for up to 12 cumulative population doublings, calculated projection showed that approximately 730 × 10 3 ECFCs could be manufactured from 1 apheresis unit. ECFCs produced epidermal growth factor, hepatocyte growth factor, vascular endothelial growth factor (VEGF)-A, PLT-derived growth factor-B, interleukin-8, and monocyte chemoattractant protein-1, featured high potential for capillary-like tubes formation, and showed no telomerase activity. They were characterized by CD29, CD31, CD44, CD105, CD117, CD133, CD144, CD146, and VEGF-R2 expression, with the most common subpopulation CD34+CD117-CD133-. Compared to controls, ECFCs featured greater Dil-AcLDL uptake and higher expression of CD29, CD31, CD34, CD44, CD144, and VEGF-R2. Here we show that isolation of ECFCs with proangiogenic profile from steady-state PB leukapheresis is feasible, marking a first step toward ECFC product manufacture according to GMP. © 2018 AABB.

HBx drives alpha fetoprotein expression to promote initiation of liver cancer stem cells through activating PI3K/AKT signal pathway.

PubMed

Zhu, Mingyue; Li, Wei; Lu, Yan; Dong, Xu; Lin, Bo; Chen, Yi; Zhang, Xueer; Guo, Junli; Li, Mengsen

2017-03-15

Hepatitis B virus (HBV)-X protein (HBx) plays critical role in inducing the malignant transformation of liver cells. Alpha fetoprotein (AFP) expression is closely related to hepatocarcinogenesis. We report that Oct4, Klf4, Sox2 and c-myc expression positively associated with AFP(+)/HBV(+) hepatocellular carcinoma(HCC) tissues, and the expression of the stemness markers CD44, CD133 and EpCAM was significantly higher in AFP(+)/HBV(+) HCC tissues compared to normal liver tissues or AFP (-)/HBV(-) HCC tissues. AFP expression turned on prior to expression of Oct4, Klf4, Sox2 and c-myc, and the stemness markers CD44, CD133 and EpCAM in the normal human liver L-02 cell line or CHL cell lines upon transfection with MCV-HBx vectors. Stem-like cells generated more tumour colonies compared to primary cells, and xenografts induced tumourigenesis in nude mice. Expression of reprogramming-related proteins was significantly enhanced in HLE cells while transfected with pcDNA3.1-afp vectors. The specific PI3K inhibitor Ly294002 inhibited the effects of pcDNA3.1-afp vectors. AFP-siRNA vectors were able to inhibit tumour colony formation and reprogramming-related gene expression. Altogether, HBx stimulates AFP expression to induce natural reprogramming of liver cells, and AFP plays a critical role in promoting the initiation of HCC progenitor/stem cells. AFP may be a potential novel biotarget for combating HBV-induced hepatocarcinogenesis. © 2016 UICC.

Effects of extracellular modulation through hypoxia on the glucose metabolism of human breast cancer stem cells

NASA Astrophysics Data System (ADS)

Yustisia, I.; Jusman, S. W. A.; Wanandi, S. I.

2017-08-01

Cancer stem cells have been reported to maintain stemness under certain extracellular changes. This study aimed to analyze the effect of extracellular O2 level modulation on the glucose metabolism of human CD24-/CD44+ breast cancer stem cells (BCSCs). The primary BCSCs (CD24-/CD44+ cells) were cultured under hypoxia (1% O2) for 0.5, 4, 6, 24 and 48 hours. After each incubation period, HIF1α, GLUT1 and CA9 expressions, as well as glucose metabolism status, including glucose consumption, lactate production, O2 consumption and extracellular pH (pHe) were analyzed using qRT-PCR, colorimetry, fluorometry, and enzymatic reactions, respectively. Hypoxia caused an increase in HIF1α mRNA expressions and protein levels and shifted the metabolic states to anaerobic glycolysis, as demonstrated by increased glucose consumption and lactate production, as well as decreased O2 consumption and pHe. Furthermore, we demonstrated that GLUT1 and CA9 mRNA expressions simultaneously increased, in line with HIF1α expression. In conclusion, modulation of the extracellular environment of human BCSCs through hypoxia shifedt the metabolic state of BCSCs to anaerobic glycolysis, which might be associated with GLUT1 and CA9 expressions regulated by HIFlα transcription factor.

Directing stem cell trafficking via GPS.

PubMed

Sackstein, Robert

2010-01-01

The success of stem-cell-based regenerative therapeutics critically hinges on delivering relevant stem/progenitor cells to sites of tissue injury. To achieve adequate parenchymal infiltration following intravascular administration, it is first necessary that circulating cells bind to target tissue endothelium with sufficient strength to overcome the prevailing forces of hemodynamic shear. The principal mediators of these shear-resistant binding interactions consist of a family of C-type lectins known as "selectins" that bind discrete sialofucosylated glycans on their respective ligands. One member of this family, E-selectin, is an endothelial molecule that is inducibly expressed on postcapillary venules at all sites of tissue injury, but is also constitutively expressed on the luminal surface of bone marrow and dermal microvascular endothelium. Most stem/progenitor cells express high levels of CD44, and, in particular, human hematopoietic stem cells express a specialized sialofucosylated glycoform of CD44 known as "hematopoietic cell E-/L-selectin ligand" (HCELL) that functions as a potent E-selectin ligand. This chapter describes a method called "glycosyltransferase-programmed stereosubstitution" (GPS) for custom-modifying CD44 glycans to create HCELL on the surface of living cells that natively lack HCELL. Ex vivo glycan engineering of HCELL via GPS licenses trafficking of infused cells to endothelial beds that express E-selectin, thereby enabling efficient vascular delivery of stem/progenitor cells to sites where they are needed. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Effects of chronic Akt/mTOR inhibition by rapamycin on mechanical overload-induced hypertrophy and myosin heavy chain transition in masseter muscle.

PubMed

Umeki, Daisuke; Ohnuki, Yoshiki; Mototani, Yasumasa; Shiozawa, Kouichi; Fujita, Takayuki; Nakamura, Yoshiki; Saeki, Yasutake; Okumura, Satoshi

2013-01-01

To examine the effects of the Akt/mammalian target of rapamycin (mTOR) pathway on masseter muscle hypertrophy and myosin heavy chain (MHC) transition in response to mechanical overload, we analyzed the effects of bite-opening (BO) on the hypertrophy and MHC composition of masseter muscle of BO-rats treated or not treated with rapamycin (RAPA), a selective mTOR inhibitor. The masseter muscle weight in BO-rats was significantly greater than that in controls, and this increase was attenuated by RAPA treatment. Expression of slow-twitch MHC isoforms was significantly increased in BO-rats with/without RAPA treatment, compared with controls, but the magnitude of the increase was much smaller in RAPA-treated BO-rats. Phosphorylation of p44/42 MAPK (ERK1/2), which preserves fast-twitch MHC isoforms in skeletal muscle, was significantly decreased in BO-rats, but the decrease was abrogated by RAPA treatment. Calcineurin signaling is known to be important for masseter muscle hypertrophy and fast-to-slow MHC isoform transition, but expression of known calcineurin activity modulators was unaffected by RAPA treatment. Taken together, these results indicate that the Akt/mTOR pathway is involved in both development of masseter muscle hypertrophy and fast-to-slow MHC isoform transition in response to mechanical overload with inhibition of the ERK1/2 pathway and operates independently of the calcineurin pathway.

The Selective Centrifugation Ensures a Better In Vitro Isolation of ASCs and Restores a Soft Tissue Regeneration In Vivo.

PubMed

De Francesco, Francesco; Guastafierro, Antonio; Nicoletti, Gianfranco; Razzano, Sergio; Riccio, Michele; Ferraro, Giuseppe A

2017-05-12

Autologous fat grafting procedures in plastic surgery have been extensively used to reinforce soft tissue in congenital or acquired tissue impairments. With this background, the aim of this study is firstly to examine the impact of a selective centrifugation on existing adipose stem cells (ASCs) in terms of stemness profile maintenance and, secondly, to investigate the effect of restoring volume in reconstruction on patients affected by soft tissue damage. After centrifugation, the fat graft products were separated into two layers and subsequently examined in vitro for the expression of CD34, CD90, CD117, CD105, CD29, CD31, CD44, CD73, CD133, CD14 and CD45 markers by flow cytometry and gene expression analyses were performed for Sox2, WNT3A, END, CD44, FUT4, COLL1, CTNNB1, hbEGF, KRTLG, MMP2 and VIM genes. The results showed that in the middle-high density (MHD) layer there was a peak concentration of ASCs, compared to another layer obtained after centrifugation. Research carried out on patients under treatment for soft tissue regeneration using cells obtained from MHD layer selection will be fundamental in comparative analysis. These studies will lead to an adequate standardization of outcomes, provided that treatment is performed through cell selection. Therefore, a unique procedure in tissue reconstruction and regeneration through fat grafting is presented here.

High-Throughput Sequencing of Islet-Infiltrating Memory CD4+ T Cells Reveals a Similar Pattern of TCR Vβ Usage in Prediabetic and Diabetic NOD Mice

PubMed Central

Marrero, Idania; Hamm, David E.; Davies, Joanna D.

2013-01-01

Autoreactive memory CD4+ T cells play a critical role in the development of type 1 diabetes, but it is not yet known how the clonotypic composition and TCRβ repertoire of the memory CD4+ T cell compartment changes during the transition from prediabetes to diabetes. In this study, we used high-throughput sequencing to analyze the TCRβ repertoire of sorted islet-infiltrating memory CD4+CD44high T cells in 10-week-old prediabetic and recently diabetic NOD mice. We show that most clonotypes of islet-infiltrating CD4+CD44high T cells were rare, but high-frequency clonotypes were significantly more common in diabetic than in prediabetic mice. Moreover, although the CD4+CD44high TCRβ repertoires were highly diverse at both stages of disease development, dominant use of TRBV1 (Vβ2), TRBV13-3 (Vβ8.1), and TRBV19 (Vβ6) was evident in both prediabetic and diabetic mice. Our findings strongly suggest that therapeutic targeting of cells specifically expressing the dominant TCRβ might reduce pancreatic infiltration in prediabetic mice and attenuate the progression to diabetes. PMID:24146886

Expression of VEGF₁₆₅b, VEGFR1, VEGFR2 and CD34 in benign and malignant tumors of parotid glands.

PubMed

Błochowiak, Katarzyna J; Sokalski, Jerzy; Bodnar, Magdalena B; Trzybulska, Dorota; Marszałek, Andrzej K; Witmanowski, Henryk

2018-01-01

Vascular endothelial growth factor (VEGF) is an angiogenic factor and could be involved in the pathogenesis of salivary gland tumors. VEGF exerts its biological function by binding to its receptors, VEGFR1 and VEGFR2. An alternative splice variant of VEGF (VEGFxxxb) is an anti-angiogenic factor. Binding VEGF165b with VEGFR2 results in an impaired angiogenic response. The imbalance of VEGFxxx and VEGFxxxb isoforms can underpin pathological angiogenesis. The purpose of this study was to evaluate and compare the expression of VEGF165b, VEGFR1, VEGFR2, and CD34 in benign and malignant parotid gland tumors and to explore the possible correlations between their expression and clinicopathological features of tumors. The study was performed on archived paraffin-embedded tissue samples derived from 70 patients with benign and malignant parotid gland tumors (25 with malignant tumors, 23 with pleomorphic adenoma and 22 with Warthin's tumor). Immunohistochemical staining of selected tissue sections was performed using monoclonal antibodies. Immunohistochemical staining of selected molecules was used for evaluation of their expression in tissue sections. There were no statistically significant differences in the expression of the selected proteins localized in the tumor and surgical margin taken from the same patient. Expression of VEGFR2 correlated with VEGF165b in mixed tumors. There was a statistically significant difference in the expression of VEGFR1 in malignant tumors between females and males, and between the expression of VEGFR1 and the score of T classification in malignant tumors. VEGF165b cannot be treated as a prognostic factor. VEGF receptors correlated with selected clinicopathological data of malignant tumors, indicating their possible role as a prognostic marker. The balance of VEGF isoforms have a limited influence on the development of parotid glands tumors. The correlation between VEGF165b and VEGFR2 in mixed tumors suggests the existence of an additional antiangiogenic pathway in poorly vascularized mixed tumors.

Phosphoinositide 3-Kinase p110δ Mediates Estrogen- and FSH-Stimulated Ovarian Follicle Growth

PubMed Central

Li, Qian; He, Hui; Zhang, Yin-Li; Li, Xiao-Meng; Guo, Xuejiang; Huo, Ran; Bi, Ye; Li, Jing

2013-01-01

In the mammalian ovary, primordial follicles are generated early in life and remain dormant for prolonged periods. Their growth resumes via primordial follicle activation, and they continue to grow until the preovulatory stage under the regulation of hormones and growth factors, such as estrogen, FSH, and IGF-1. Both FSH and IGF-1 activate the phosphatidylinositol-3 kinase (PI3K)/Akt (acute transforming retrovirus thymoma protein kinase) signaling pathway in granulosa cells (GCs), yet it remains inconclusive whether the PI3K pathway is crucial for follicle growth. In this study, we investigated the p110δ isoform (encoded by the Pik3cd gene) of PI3K catalytic subunit expression in the mouse ovary and its function in fertility. Pik3cd-null females were subfertile, exhibited fewer growing follicles and more atretic antral follicles in the ovary, and responded poorly to exogenous gonadotropins compared with controls. Ovary transplantation showed that Pik3cd-null ovaries responded poorly to FSH stimulation in vitro; this confirmed that the follicle growth defect was intrinsically ovarian. In addition, estradiol (E2)-stimulated follicle growth and GC proliferation in preantral follicles was impaired in Pik3cd-null ovaries. FSH and E2 substantially activated the PI3K/Akt pathway in GCs of control mice but not in those of Pik3cd-null mice. However, primordial follicle activation and oocyte meiotic maturation were not affected by Pik3cd knockout. Taken together, our findings indicate that the p110δ isoform of the PI3K catalytic subunit is a key component of the PI3K pathway for both FSH and E2-stimulated follicle growth in ovarian GCs; however, it is not required for primordial follicle activation and oocyte development. PMID:23820902

Does the liposuction method influence the phenotypic characteristic of human adipose-derived stem cells?

PubMed

Bajek, Anna; Gurtowska, Natalia; Gackowska, Lidia; Kubiszewska, Izabela; Bodnar, Magdalena; Marszałek, Andrzej; Januszewski, Rafał; Michalkiewicz, Jacek; Drewa, Tomasz

2015-05-14

Adipose-derived stem cells (ASCs) possess a high differentiation and proliferation potential. However, the phenotypic characterization of ASCs is still difficult. Until now, there is no extensive analysis of ASCs markers depending on different liposuction methods. Therefore, the aim of the present study was to analyse 242 surface markers and determine the differences in the phenotypic pattern between ASCs obtained during mechanical and ultrasound-assisted liposuction. ASCs were isolated from healthy donors, due to mechanical and ultrasound-assisted liposuction and cultured in standard medium to the second passage. Differentiation potential and markers expression was evaluated to confirm the mesenchymal nature of cells. Then, the BD LyoplateTM Human Cell Surface Marker Screening Panel was used. Results shown that both population of ASCs are characterized by high expression of markers specific for ASCs: cluster of differentiation (CD)9, CD10, CD34, CD44, CD49d, CD54, CD55, CD59, CD71 and low expression of CD11a, CD11c and CD144. Moreover, we have noticed significant differences in antigen expression in 58 markers from the 242 studied. Presented study shows for the first time that different liposuction methods are not a significant factor which can influence the expression of human ASCs surface markers. © 2015 The Authors.

The retinal specific CD147 Ig0 domain: from molecular structure to biological activity

PubMed Central

Redzic, Jasmina S.; Armstrong, Geoffrey S.; Isern, Nancy. G.; Jones, David N.M.; Kieft, Jeffrey S.; Eisenmesser, Elan Zohar

2011-01-01

CD147 is a type I transmembrane protein that is involved in inflammatory diseases, cancer progression, and multiple human pathogens utilize CD147 for efficient infection. In several cancers, CD147 expression is so high that it is now used as a prognostic marker. The two primary isoforms of CD147 that are related to cancer progression have been identified, differing in their number of immunoglobulin (Ig)-like domains. These include CD147 Ig1-Ig2 that is ubiquitously expressed in most tissues and CD147 Ig0-Ig1-Ig2 that is retinal specific and implicated in retinoblastoma. However, little is known in regard to the retinal specific CD147 Ig0 domain despite its potential role in retinoblastoma. We present the first crystal structure of the human CD147 Ig0 domain and show that the CD147 Ig0 domain is a crystallographic dimer with an I-type domain structure, which is maintained in solution. Furthermore, we have utilized our structural data together with mutagenesis to probe the biological activity of CD147-containing proteins both with and without the CD147 Ig0 domain within several model cell lines. Our findings reveal that the CD147 Ig0 domain is a potent stimulator of interleukin-6 and suggest that the CD147 Ig0 domain has its own receptor distinct from that of the other CD147 Ig-like domains, CD147 Ig1-Ig2. Finally, we show that the CD147 Ig0 dimer is the functional unit required for activity and can be disrupted by a single point mutation. PMID:21620857

Whole-Transcriptome Analysis of CD133+CD144+ Cancer Stem Cells Derived from Human Laryngeal Squamous Cell Carcinoma Cells.

PubMed

Wu, Yongyan; Zhang, Yuliang; Niu, Min; Shi, Yong; Liu, Hongliang; Yang, Dongli; Li, Fei; Lu, Yan; Bo, Yunfeng; Zhang, Ruiping; Li, Zhenyu; Luo, Hongjie; Cui, Jiajia; Sang, Jiangwei; Xiang, Caixia; Gao, Wei; Wen, Shuxin

2018-06-27

CD133+CD44+ cancer stem cells previously isolated from laryngeal squamous cell carcinoma (LSCC) cell lines showed strong malignancy and tumorigenicity. However, the molecular mechanism underlying the enhanced malignancy remained unclear. Cell proliferation assay, spheroid-formation experiment, RNA sequencing (RNA-seq), miRNA-seq, bioinformatic analysis, quantitative real-time PCR, migration assay, invasion assay, and luciferase reporter assay were used to identify differentially expressed mRNAs, lncRNAs, circRNAs and miRNAs, construct transcription regulatory network, and investigate functional roles and mechanism of circRNA in CD133+CD44+ laryngeal cancer stem cells. Differentially expressed genes in TDP cells were mainly enriched in the biological processes of cell differentiation, regulation of autophagy, negative regulation of cell death, regulation of cell growth, response to hypoxia, telomere maintenance, cellular response to gamma radiation, and regulation of apoptotic signaling, which are closely related to the malignant features of tumor cells. We constructed the regulatory network of differentially expressed circRNAs, miRNAs and mRNAs. qPCR findings for the expression of key genes in the network were consistent with the sequencing data. Moreover, our data revealed that circRNA hg19_circ_0005033 promotes proliferation, migration, invasion, and chemotherapy resistance of laryngeal cancer stem cells. This study provides potential biomarkers and targets for LSCC diagnosis and therapy, and provide important evidences for the heterogeneity of LSCC cells at the transcription level. © 2018 The Author(s). Published by S. Karger AG, Basel.

Passage-dependent morphological and phenotypical changes of a canine histiocytic sarcoma cell line (DH82 cells).

PubMed

Heinrich, Franziska; Contioso, Vanessa Bono; Stein, Veronika M; Carlson, Regina; Tipold, Andrea; Ulrich, Reiner; Puff, Christina; Baumgärtner, Wolfgang; Spitzbarth, Ingo

2015-01-15

DH82 cells represent a permanent macrophage cell line isolated from a dog with histiocytic sarcoma (HS) and are commonly used in various fields of research upon infection and cancer, respectively. Despite its frequent use, data on cell surface antigen expression of this cell line are fragmentary and in part inconsistent. We therefore aimed at a detailed morphological and antigenic characterization of DH82 cells with respect to passage-dependent differences. Cellular morphology of early (≤ 13) and late (≥ 66) passages of DH82 cells was evaluated via scanning electron microscopy. Moreover, cells were labelled with 10 monoclonal antibodies directed against CD11c, CD14, CD18, CD44, CD45, CD80, CD86, MHC-I, MHC-II, and ICAM-1 for flow cytometric analysis. Early passage cells were characterized by round cell bodies with abundant small cytoplasmic projections whereas later passages exhibited a spindle-shaped morphology with large processes. The percentage of CD11c-, CD14-, CD18-, CD45-, and CD80 positive cells significantly decreased in late passages whereas the expression of CD44, CD86, MHC-I, MHC-II and ICAM-1 remained unchanged. DH82 cells represent a remarkably heterogeneous cell line with divergent antigenic and morphologic properties. The present findings have important implications for future studies, which should consider distinct characteristics with regard to the used passage. Copyright © 2014 Elsevier B.V. All rights reserved.

The effects of CD147 on the cell proliferation, apoptosis, invasion, and angiogenesis in glioma.

PubMed

Yin, Haoyuan; Shao, Ying; Chen, Xuan

2017-01-01

To analyze the effects of extracellular matrix metalloproteinase inducer (CD147) on glioma proliferation, apoptosis, invasion, and angiogenesis. Tissue samples were obtained from 101 glioma cases while normal brain tissues were obtained from 30 brain injury cases. Immunohistochemical assay was performed to detect the expressions of CD147, CD34, and VEGF in tissue samples. QRT-PCR was performed to detect the relative expression of CD147 mRNA in human glioma cell lines. CD147 siRNA was transfected into glioma cell line U251. Cell proliferation, apoptosis, invasion, and angiogenesis were tested by MTT, flow cytometry, Transwell assay, and vasculogenic mimicry assay, respectively. Expressions of relative proteins were analyzed with western blot. CD147 was positively expressed with the percentage of 0, 37.5, 44.8, 67.9, and 85.7 % in normal tissues and glioma tissues with WHO grades I-IV, respectively, and the scores of MVDand VEGF were associated with the expression of CD147. CD147 was significantly upregulated in the human glioma cell lines (P < 0.05). Downregulated the expression of CD147 suppressed cell proliferation, blocked cell cycle, induced apoptosis, inhibited cell invasion and angiogenesis in glioma cells in vitro. The expression of CD147 was significantly associated with WHO tumor grade and angiogenesis; silencing of CD147 contributed to inhibition of glioma proliferation, invasion, and angiogenesis. Our study provided firm evidence that CD 147 is a potential glioma target for anti-angiogenic therapies.

HnRNP L and L-like cooperate in multiple-exon regulation of CD45 alternative splicing

PubMed Central

Preußner, Marco; Schreiner, Silke; Hung, Lee-Hsueh; Porstner, Martina; Jäck, Hans-Martin; Benes, Vladimir; Rätsch, Gunnar; Bindereif, Albrecht

2012-01-01

CD45 encodes a trans-membrane protein-tyrosine phosphatase expressed in diverse cells of the immune system. By combinatorial use of three variable exons 4–6, isoforms are generated that differ in their extracellular domain, thereby modulating phosphatase activity and immune response. Alternative splicing of these CD45 exons involves two heterogeneous ribonucleoproteins, hnRNP L and its cell-type specific paralog hnRNP L-like (LL). To address the complex combinatorial splicing of exons 4–6, we investigated hnRNP L/LL protein expression in human B-cells in relation to CD45 splicing patterns, applying RNA-Seq. In addition, mutational and RNA-binding analyses were carried out in HeLa cells. We conclude that hnRNP LL functions as the major CD45 splicing repressor, with two CA elements in exon 6 as its primary target. In exon 4, one element is targeted by both hnRNP L and LL. In contrast, exon 5 was never repressed on its own and only co-regulated with exons 4 and 6. Stable L/LL interaction requires CD45 RNA, specifically exons 4 and 6. We propose a novel model of combinatorial alternative splicing: HnRNP L and LL cooperate on the CD45 pre-mRNA, bridging exons 4 and 6 and looping out exon 5, thereby achieving full repression of the three variable exons. PMID:22402488

Granulocyte and monocyte adsorption apheresis for refractory skin diseases due to activated neutrophils, psoriasis, and associated arthropathy.

PubMed

Sakanoue, Masanao; Takeda, Koichiro; Kawai, Kazuhiro; Kanekura, Takuro

2013-10-01

Granulocyte and monocyte adsorption apheresis (GMA), an extracorporeal apheresis instrument whose column contains cellulose acetate (CA) beads, is designed to remove activated granulocytes and monocytes. We previously demonstrated that GMA was useful for treating neutrophilic dermatoses and associated arthropathy as it adsorbs Mac-1 (CD11b/CD18)-expressing neutrophils to the CA beads by the binding of complement component (iC3b) and CD11b expressed on activated neutrophils. The objective of this study is to further assess the clinical effectiveness of GMA in the treatment of neutrophilic dermatoses and associated arthropathy. The effect of GMA for skin lesions and joint lesions was assessed in 44 and 23 patients, respectively. Mac-1 expression on peripheral neutrophils was measured by flow cytometry. Skin lesions and arthropathy improved in 39 of 44 patients (88.6%) and 22 of 23 (95.6%), respectively. Mac-1 (CD11b/CD18) expression on the peripheral neutrophils, 27.1 ± 6.66 MFI (mean fluorescence intensity) before treatment, was reduced to 17.9 ± 3.02 MFI by GMA (P < 0.05). Clinical effectiveness of GMA for the treatment of intractable neutrophilic dermatoses and associated arthropathy was further confirmed. © 2013 The Authors. Therapeutic Apheresis and Dialysis © 2013 International Society for Apheresis.

The retinal specific CD147 Ig0 domain: from molecular structure to biological activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Redzic, Jasmina S.; Armstrong, Geoffrey S.; Isern, Nancy G.

2011-06-18

CD147 is a type I transmembrane protein that is involved in inflammatory diseases, cancer progression, and multiple human pathogens utilize CD147 for efficient infection. In several cancers, CD147 expression is so high that it is now used as a prognostic marker. The two primary isoforms of CD147 that are related to cancer progression have been identified, differing in their number of immunoglobulin (Ig)-like domains. These include CD147 Ig1-Ig2 that is ubiquitously expressed in most tissues and CD147 Ig0-Ig1-Ig2 that is retinal specific and implicated in retinoblastoma. However, little is known in regard to the retinal specific CD147 Ig0 domain despitemore » its potential role in retinoblastoma. Thus, here we have extensively characterized the CD147 Ig0 domain by elucidating its three-dimensional structure through crystallography and its solution behavior through several biophysical methods that include nuclear magnetic resonance. Furthermore, we have utilized this data together with mutagenesis to probe the biological activity of CD147-containing proteins both with and without the CD147 Ig0 domain within several model cell lines. Our findings reveal that the CD147 Ig0 domain is a potent stimulator of interleukin-6, which is a well-known contributor to retinoblastoma and suggest that the CD147 Ig0 domain has its own receptor distinct from that of the other CD147 Ig-like domains, CD147 Ig1-Ig2. Furthermore, we show that the CD147 Ig0 dimer is the functional unit required for activity and can be disrupted by a single point mutation.« less

CD44-positive cells are candidates for astrocyte precursor cells in developing mouse cerebellum.

PubMed

Cai, Na; Kurachi, Masashi; Shibasaki, Koji; Okano-Uchida, Takayuki; Ishizaki, Yasuki

2012-03-01

Neural stem cells are generally considered to be committed to becoming precursor cells before terminally differentiating into either neurons or glial cells during neural development. Neuronal and oligodendrocyte precursor cells have been identified in several areas in the murine central nervous system. The presence of astrocyte precursor cells (APCs) is not so well understood. The present study provides several lines of evidence that CD44-positive cells are APCs in the early postnatal mouse cerebellum. In developing mouse cerebellum, CD44-positive cells, mostly located in the white matter, were positive for the markers of the astrocyte lineage, but negative for the markers of mature astrocytes. CD44-positive cells were purified from postnatal cerebellum by fluorescence-activated cell sorting and characterized in vitro. In the absence of any signaling molecule, many cells died by apoptosis. The surviving cells gradually expressed glial fibrillary acidic protein, a marker for mature astrocytes, indicating that differentiation into mature astrocytes is the default program for these cells. The cells produced no neurospheres nor neurons nor oligodendrocytes under any condition examined, indicating these cells are not neural stem cells. Leukemia inhibitory factor greatly promoted astrocytic differentiation of CD44-positive cells, whereas bone morphogenetic protein 4 (BMP4) did not. Fibroblast growth factor-2 was a potent mitogen for these cells, but was insufficient for survival. BMP4 inhibited activation of caspase-3 and greatly promoted survival, suggesting a novel role for BMP4 in the control of development of astrocytes in cerebellum. We isolated and characterized only CD44 strongly positive large cells and discarded small and/or CD44 weakly positive cells in this study. Further studies are necessary to characterize these cells to help determine whether CD44 is a selective and specific marker for APCs in the developing mouse cerebellum. In conclusion, we succeeded in preparing APC candidates from developing mouse cerebellum, characterized them in vitro, and found that BMPs are survival factors for these cells.

The loss-of-function disease-mutation G301R in the Na+/K+-ATPase α2 isoform decreases lesion volume and improves functional outcome after acute spinal cord injury in mice.

PubMed

Ellman, Ditte Gry; Isaksen, Toke Jost; Lund, Minna Christiansen; Dursun, Safinaz; Wirenfeldt, Martin; Jørgensen, Louise Helskov; Lykke-Hartmann, Karin; Lambertsen, Kate Lykke

2017-09-08

The Na + /K + -ATPases are transmembrane ion pumps important for maintenance of ion gradients across the plasma membrane that serve to support multiple cellular functions, such as membrane potentials, regulation of cellular volume and pH, and co-transport of signaling transmitters in all animal cells. The α 2 Na + /K + -ATPase subunit isoform is predominantly expressed in astrocytes, which us the sharp Na + -gradient maintained by the sodium pump necessary for astroglial metabolism. Prolonged ischemia induces an elevation of [Na + ] i , decreased ATP levels and intracellular pH owing to anaerobic metabolism and lactate accumulation. During ischemia, Na + /K + -ATPase-related functions will naturally increase the energy demand of the Na + /K + -ATPase ion pump. However, the role of the α 2 Na + /K + -ATPase in contusion injury to the spinal cord remains unknown. We used mice heterozygous mice for the loss-of-function disease-mutation G301R in the Atp1a2 gene (α 2 +/G301R ) to study the effect of reduced α 2 Na + /K + -ATPase expression in a moderate contusion spinal cord injury (SCI) model. We found that α 2 +/G301R mice display significantly improved functional recovery and decreased lesion volume compared to littermate controls (α 2 +/+ ) 7 days after SCI. The protein level of the α 1 isoform was significantly increased, in contrast to the α 3 isoform that significantly decreased 3 days after SCI in both α 2 +/G301R and α 2 +/+ mice. The level of the α 2 isoform was significantly decreased in α 2 +/G301R mice both under naïve conditions and 3 days after SCI compared to α 2 +/+ mice. We found no differences in astroglial aquaporin 4 levels and no changes in the expression of chemokines (CCL2, CCL5 and CXCL1) and cytokines (TNF, IL-6, IL-1β, IL-10 and IL-5) between genotypes, just as no apparent differences were observed in location and activation of CD45 and F4/80 positive microglia and infiltrating leukocytes. Our proof of concept study demonstrates that reduced expression of the α 2 isoform in the spinal cord is protective following SCI. Importantly, the BMS and lesion volume were assessed at 7 days after SCI, and longer time points after SCI were not evaluated. However, the α 2 isoform is a potential possible target of therapeutic strategies for the treatment of SCI.

The sensitivity of human mesenchymal stem cells to vibration and cold storage conditions representative of cold transportation

PubMed Central

Nikolaev, N. I.; Liu, Y.; Hussein, H.; Williams, D. J.

2012-01-01

In the current study, the mechanical and hypothermic damage induced by vibration and cold storage on human mesenchymal stem cells (hMSCs) stored at 2–8°C was quantified by measuring the total cell number and cell viability after exposure to vibration at 50 Hz (peak acceleration 140 m s−2 and peak displacement 1.4 mm), 25 Hz (peak acceleration 140 m s−2, peak displacement 5.7 mm), 10 Hz (peak acceleration 20 m s−2, peak displacement 5.1 mm) and cold storage for several durations. To quantify the viability of the cells, in addition to the trypan blue exclusion method, the combination of annexin V-FITC and propidium iodide was applied to understand the mode of cell death. Cell granularity and a panel of cell surface markers for stemness, including CD29, CD44, CD105 and CD166, were also evaluated for each condition. It was found that hMSCs were sensitive to vibration at 25 Hz, with moderate effects at 50 Hz and no effects at 10 Hz. Vibration at 25 Hz also increased CD29 and CD44 expression. The study further showed that cold storage alone caused a decrease in cell viability, especially after 48 h, and also increased CD29 and CD44 and attenuated CD105 expressions. Cell death would most likely be the consequence of membrane rupture, owing to necrosis induced by cold storage. The sensitivity of cells to different vibrations within the mechanical system is due to a combined effect of displacement and acceleration, and hMSCs with a longer cold storage duration were more susceptible to vibration damage, indicating a coupling between the effects of vibration and cold storage. PMID:22628214

Purification of human adipose-derived stem cells from fat tissues using PLGA/silk screen hybrid membranes.

PubMed

Chen, Da-Chung; Chen, Li-Yu; Ling, Qing-Dong; Wu, Meng-Hsueh; Wang, Ching-Tang; Suresh Kumar, S; Chang, Yung; Munusamy, Murugan A; Alarfajj, Abdullah A; Wang, Han-Chow; Hsu, Shih-Tien; Higuchi, Akon

2014-05-01

The purification of human adipose-derived stem cells (hADSCs) from human adipose tissue cells (stromal vascular fraction) was investigated using membrane filtration through poly(lactide-co-glycolic acid)/silk screen hybrid membranes. Membrane filtration methods are attractive in regenerative medicine because they reduce the time required to purify hADSCs (i.e., less than 30 min) compared with conventional culture methods, which require 5-12 days. hADSCs expressing the mesenchymal stem cell markers CD44, CD73, and CD90 were concentrated in the permeation solution from the hybrid membranes. Expression of the surface markers CD44, CD73, and CD99 on the cells in the permeation solution from the hybrid membranes, which were obtained using 18 mL of feed solution containing 50 × 10⁴ cells, was statistically significantly higher than that of the primary adipose tissue cells, indicating that the hADSCs can be purified in the permeation solution by the membrane filtration method. Cells expressing the stem cell-associated marker CD34 could be successfully isolated in the permeation solution, whereas CD34⁺ cells could not be purified by the conventional culture method. The hADSCs in the permeation solution demonstrated a superior capacity for osteogenic differentiation based on their alkali phosphatase activity, their osterix gene expression, and the results of mineralization analysis by Alizarin Red S and von Kossa staining compared with the cells from the suspension of human adipose tissue. These results suggest that the hADSCs capable of osteogenic differentiation preferentially permeate through the hybrid membranes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Apigenin: Selective CK2 inhibitor increases Ikaros expression and improves T cell homeostasis and function in murine pancreatic cancer

PubMed Central

Nelson, Nadine; Szekeres, Karoly; Iclozan, Cristina; Rivera, Ivannie Ortiz; McGill, Andrew; Johnson, Gbemisola; Nwogu, Onyekachi

2017-01-01

Pancreatic cancer (PC) evades immune destruction by favoring the development of regulatory T cells (Tregs) that inhibit effector T cells. The transcription factor Ikaros is critical for lymphocyte development, especially T cells. We have previously shown that downregulation of Ikaros occurs as a result of its protein degradation by the ubiquitin-proteasome system in our Panc02 tumor-bearing (TB) mouse model. Mechanistically, we observed a deregulation in the balance between Casein Kinase II (CK2) and protein phosphatase 1 (PP1), which suggested that increased CK2 activity is responsible for regulating Ikaros’ stability in our model. We also showed that this loss of Ikaros expression is associated with a significant decrease in CD4+ and CD8+ T cell percentages but increased CD4+CD25+ Tregs in TB mice. In this study, we evaluated the effects of the dietary flavonoid apigenin (API), on Ikaros expression and T cell immune responses. Treatment of splenocytes from naïve mice with (API) stabilized Ikaros expression and prevented Ikaros downregulation in the presence of murine Panc02 cells in vitro, similar to the proteasome inhibitor MG132. In vivo treatment of TB mice with apigenin (TB-API) improved survival, reduced tumor weights and prevented splenomegaly. API treatment also restored protein expression of some Ikaros isoforms, which may be attributed to its moderate inhibition of CK2 activity from splenocytes of TB-API mice. This partial restoration of Ikaros expression was accompanied by a significant increase in CD4+ and CD8+ T cell percentages and a reduction in Treg percentages in TB-API mice. In addition, CD8+ T cells from TB-API mice produced more IFN-γ and their splenocytes were better able to prime allogeneic CD8+ T cell responses compared to TB mice. These results provide further evidence that Ikaros is regulated by CK2 in our pancreatic cancer model. More importantly, our findings suggest that API may be a possible therapeutic agent for stabilizing Ikaros expression and function to maintain T cell homeostasis in murine PC. PMID:28152014

Constitutive expression of the promyelocytic leukemia-associated oncogene PML-RARalpha in TF1 cells: isoform-specific and retinoic acid-dependent effects on growth, bcl-2 expression, and apoptosis.

PubMed

Slack, J L; Yu, M

1998-05-01

Two major isoforms of PML-RARalpha are associated with (15;17)-positive acute promyelocytic leukemia (APL); however, functional differences between these isoforms have been difficult to define, and the molecular mechanism by which each isoform contributes to the pathogenesis of APL is not fully understood. To address these issues, the 'short' (S) and 'long' (L) isoforms of PML-RARalpha were constitutively expressed in the factor-dependent human erythroleukemia cell line, TF1. Expression of the L, but not the S, isoform inhibited growth of these cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). In the absence of GM-CSF, the S isoform partially protected against apoptosis, while the L isoform accelerated cell death. Treatment with all-trans retinoic acid (ATRA) inhibited cell growth and caused apoptosis only in PML-RARalpha-expressing cells, and these effects of ATRA were more marked in cells expressing the L isoform. ATRA treatment also led to downregulation of bcl-2 and endogenous RARalpha in PML-RARalpha-expressing cells, but had little effect on the level of exogenously expressed PML-RARalpha. We conclude that (1) subtle differences exist in the biologic activities of the L and S isoforms of PML-RARalpha, and (2) both isoforms are capable of transducing an ATRA-mediated signal that leads to downregulation of bcl-2 and induction of programmed cell death.

CD133(+)/CD44(+)/Oct4(+)/Nestin(+) stem-like cells isolated from Panc-1 cell line may contribute to multi-resistance and metastasis of pancreatic cancer.

PubMed

Wang, Dongqing; Zhu, Haitao; Zhu, Ying; Liu, Yanfang; Shen, Huiling; Yin, Ruigen; Zhang, Zhijian; Su, Zhaoliang

2013-05-01

Pancreatic cancer is an aggressive malignant disease. Owing to the lack of early symptoms, accompanied by extensive metastasis and high resistance to chemotherapy, pancreatic adenocarcinoma becomes the fourth leading cause of cancer-related deaths. In this study, we identified a subpopulation of cells isolated from the Panc-1 cell line and named pancreatic cancer stem-like cells. These Panc-1 stem-like cells expressed high levels of CD133/CD44/Oct4/Nestin. Compared to Panc-1 cells, Panc-1 stem-like cells were resistant to gemcitabine and expressed high levels of MDR1; furthermore, Panc-1 stem-like cells have high anti-apoptotic, but weak proliferative potential. These results indicated that Panc-1 stem-like cells, as a novel group, may be a potential major cause of pancreatic cancer multidrug resistance and extensive metastasis. Copyright © 2012 Elsevier GmbH. All rights reserved.

Basigin (CD147), a multifunctional transmembrane glycoprotein with various binding partners.

PubMed

Muramatsu, Takashi

2016-05-01

Basigin, also called CD147 or EMMPRIN, is a transmembrane glycoprotein that belongs to the immunoglobulin superfamily. Basigin has isoforms; the common form (basigin or basigin-2) has two immunoglobulin domains, and the extended form (basigin-1) has three. Basigin is the receptor for cyclophilins, S100A9 and platelet glycoprotein VI, whereas basigin-1 serves as the receptor for the rod-derived cone viability factor. Basigin tightly associates with monocarboxylate transporters and is essential for their cell surface translocation and activities. In the same membrane plane, basigin also associates with other proteins including GLUT1, CD44 and CD98. The carbohydrate portion of basigin is recognized by lectins, such as galectin-3 and E-selectin. These molecular recognitions form the basis for the role of basigin in the transport of nutrients, migration of inflammatory leukocytes and induction of matrix metalloproteinases. Basigin is important in vision, spermatogenesis and other physiological phenomena, and plays significant roles in the pathogenesis of numerous diseases, including cancer. Basigin is also the receptor for an invasive protein RH5, which is present in malaria parasites. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society.

Distinct CPT-induced deaths in lung cancer cells caused by clathrin-mediated internalization of CP micelles

NASA Astrophysics Data System (ADS)

Liu, Yu-Sheng; Cheng, Ru-You; Lo, Yu-Lun; Hsu, Chin; Chen, Su-Hwei; Chiu, Chien-Chih; Wang, Li-Fang

2016-02-01

We previously synthesized a chondroitin sulfate-graft-poly(ε-caprolactone) copolymer (H-CP) with a high content of poly(ε-caprolactone) (18.7 mol%), which self-assembled in water into a rod-like micelle to encapsulate hydrophobic camptothecin (CPT) in the core (micelle/CPT) for tumor-targeted drug delivery. As a result of the recognition of the micelle by CD44, the micelle/CPT entered CRL-5802 cells efficiently and released CPT efficaciously, resulting in higher tumor suppression than commercial CPT-11. In this study, H1299 cells were found to have a higher CD44 expression than CRL-5802 cells. However, the lower CD44-expressing CRL-5802 cells had a higher percentage of cell death and higher cellular uptake of the micelle/CPT than the higher CD44-expressing H1299 cells. Examination of the internalization pathway of the micelle/CPT in the presence of different endocytic chemical inhibitors showed that the CRL-5802 cells involved clathrin-mediated endocytosis, which was not found in the H1299 cells. Analysis of the cell cycle of the two cell lines exposed to the micelle/CPT revealed that the CRL-5802 cells arrested mainly in the S phase and the H1299 cells arrested mainly in the G2-M phase. A consistent result was also found in the evaluation of γ-H2AX expression, which was about three-fold higher in the CRL-5802 cells than in the H1299 cells. A near-infrared dye, IR780, was encapsulated into the micelle to observe the in vivo biodistribution of the micelle/IR780 in tumor-bearing mice. The CRL-5802 tumor showed a higher fluorescence intensity than the H1299 tumor at any tracing time after 1 h. Thus we tentatively concluded that CRL-5802 cells utilized the clathrin-mediated internalization pathway and arrested in the S phase on exposure to the micelle/CPT; all are possible reasons for the better therapeutic outcome in CRL-5802 cells than in H1299 cells.We previously synthesized a chondroitin sulfate-graft-poly(ε-caprolactone) copolymer (H-CP) with a high content of poly(ε-caprolactone) (18.7 mol%), which self-assembled in water into a rod-like micelle to encapsulate hydrophobic camptothecin (CPT) in the core (micelle/CPT) for tumor-targeted drug delivery. As a result of the recognition of the micelle by CD44, the micelle/CPT entered CRL-5802 cells efficiently and released CPT efficaciously, resulting in higher tumor suppression than commercial CPT-11. In this study, H1299 cells were found to have a higher CD44 expression than CRL-5802 cells. However, the lower CD44-expressing CRL-5802 cells had a higher percentage of cell death and higher cellular uptake of the micelle/CPT than the higher CD44-expressing H1299 cells. Examination of the internalization pathway of the micelle/CPT in the presence of different endocytic chemical inhibitors showed that the CRL-5802 cells involved clathrin-mediated endocytosis, which was not found in the H1299 cells. Analysis of the cell cycle of the two cell lines exposed to the micelle/CPT revealed that the CRL-5802 cells arrested mainly in the S phase and the H1299 cells arrested mainly in the G2-M phase. A consistent result was also found in the evaluation of γ-H2AX expression, which was about three-fold higher in the CRL-5802 cells than in the H1299 cells. A near-infrared dye, IR780, was encapsulated into the micelle to observe the in vivo biodistribution of the micelle/IR780 in tumor-bearing mice. The CRL-5802 tumor showed a higher fluorescence intensity than the H1299 tumor at any tracing time after 1 h. Thus we tentatively concluded that CRL-5802 cells utilized the clathrin-mediated internalization pathway and arrested in the S phase on exposure to the micelle/CPT; all are possible reasons for the better therapeutic outcome in CRL-5802 cells than in H1299 cells. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr08345a

CD147 expression predicts biochemical recurrence after prostatectomy independent of histologic and pathologic features.

PubMed

Bauman, Tyler M; Ewald, Jonathan A; Huang, Wei; Ricke, William A

2015-07-25

CD147 is an MMP-inducing protein often implicated in cancer progression. The purpose of this study was to investigate the expression of CD147 in prostate cancer (PCa) progression and the prognostic ability of CD147 in predicting biochemical recurrence after prostatectomy. Plasma membrane-localized CD147 protein expression was quantified in patient samples using immunohistochemistry and multispectral imaging, and expression was compared to clinico-pathological features (pathologic stage, Gleason score, tumor volume, preoperative PSA, lymph node status, surgical margins, biochemical recurrence status). CD147 specificity and expression were confirmed with immunoblotting of prostate cell lines, and CD147 mRNA expression was evaluated in public expression microarray datasets of patient prostate tumors. Expression of CD147 protein was significantly decreased in localized tumors (pT2; p = 0.02) and aggressive PCa (≥pT3; p = 0.004), and metastases (p = 0.001) compared to benign prostatic tissue. Decreased CD147 was associated with advanced pathologic stage (p = 0.009) and high Gleason score (p = 0.02), and low CD147 expression predicted biochemical recurrence (HR 0.55; 95 % CI 0.31-0.97; p = 0.04) independent of clinico-pathologic features. Immunoblot bands were detected at 44 kDa and 66 kDa, representing non-glycosylated and glycosylated forms of CD147 protein, and CD147 expression was lower in tumorigenic T10 cells than non-tumorigenic BPH-1 cells (p = 0.02). Decreased CD147 mRNA expression was associated with increased Gleason score and pathologic stage in patient tumors but is not associated with recurrence status. Membrane-associated CD147 expression is significantly decreased in PCa compared to non-malignant prostate tissue and is associated with tumor progression, and low CD147 expression predicts biochemical recurrence after prostatectomy independent of pathologic stage, Gleason score, lymph node status, surgical margins, and tumor volume in multivariable analysis.

Light- and transmission-electron-microscopic investigations on distribution of CD44, connexin 43 and actin cytoskeleton during the foreign body reaction to a nanoparticular hydroxyapatite in mini-pigs.

PubMed

Wenisch, Sabine; Cavalcanti-Adam, E Ada; Tryankowski, Eva; Raabe, Oksana; Kilian, Olaf; Heiss, Christian; Alt, Volker; Arnhold, Stefan; Schnettler, Reinhard

2012-07-01

Foreign body giant cells (FBGCs) are formed by fusion of mononucleated macrophages during the foreign body response to a nanoparticulate hydroxyapatite (HA) implanted in defects of mini-pig femura. The molecular mechanisms underlying the formation of FBGCs are still largely obscure. Here we propose connexin 43 (cx43) and CD44 as candidate molecules involved in the fusion process. Immunohistochemistry and ultrastructural immunogold labeling indicated that cx43 is present within the ruffled border of FBGCs and is the main component of gap junctions formed between fusing macrophages. CD44 was strongly expressed during clustering and fusion of mononucleated macrophages. FBGCs adhering apically at the implanted HA showed CD44 reactivity only along the basolateral aspects of the plasma membranes, while podosome formation was observed within the sealing zone and ruffled border. Taken together, these findings demonstrate that cx43 and CD44 are part of the fusion machinery responsible for the formation of FBGCs. Furthermore, the results of microfilament and cx43 labeling suggest a functional role for podosomes and hemi-channels in biomaterial degradation. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Expression of phosphoinositide-specific phospholipase C isoforms in native endothelial cells.

PubMed

Béziau, Delphine M; Toussaint, Fanny; Blanchette, Alexandre; Dayeh, Nour R; Charbel, Chimène; Tardif, Jean-Claude; Dupuis, Jocelyn; Ledoux, Jonathan

2015-01-01

Phospholipase C (PLC) comprises a superfamily of enzymes that play a key role in a wide array of intracellular signalling pathways, including protein kinase C and intracellular calcium. Thirteen different mammalian PLC isoforms have been identified and classified into 6 families (PLC-β, γ, δ, ε, ζ and η) based on their biochemical properties. Although the expression of PLC isoforms is tissue-specific, concomitant expression of different PLC has been reported, suggesting that PLC family is involved in multiple cellular functions. Despite their critical role, the PLC isoforms expressed in native endothelial cells (ECs) remains undetermined. A conventional PCR approach was initially used to elucidate the mRNA expression pattern of PLC isoforms in 3 distinct murine vascular beds: mesenteric (MA), pulmonary (PA) and middle cerebral arteries (MCA). mRNA encoding for most PLC isoforms was detected in MA, MCA and PA with the exception of η2 and β2 (only expressed in PA), δ4 (only expressed in MCA), η1 (expressed in all but MA) and ζ (not detected in any vascular beds tested). The endothelial-specific PLC expression was then sought in freshly isolated ECs. Interestingly, the PLC expression profile appears to differ across the investigated arterial beds. While mRNA for 8 of the 13 PLC isoforms was detected in ECs from MA, two additional PLC isoforms were detected in ECs from PA and MCA. Co-expression of multiple PLC isoforms in ECs suggests an elaborate network of signalling pathways: PLC isoforms may contribute to the complexity or diversity of signalling by their selective localization in cellular microdomains. However in situ immunofluorescence revealed a homogeneous distribution for all PLC isoforms probed (β3, γ2 and δ1) in intact endothelium. Although PLC isoforms play a crucial role in endothelial signal transduction, subcellular localization alone does not appear to be sufficient to determine the role of PLC in the signalling microdomains found in the native endothelium.

Effects of Lycium barbarum Polysaccharides on Apoptosis, Cellular Adhesion, and Oxidative Damage in Bone Marrow Mononuclear Cells of Mice Exposed to Ionizing Radiation Injury

PubMed Central

Zhou, Jing; Pang, Hua; Li, Wenbo; Liu, Qiong; Xu, Lu; Liu, Qian; Liu, Ying

2016-01-01

Lycium barbarum has been used for more than 2500 years as a traditional herb and food in China. We investigated the effects of Lycium barbarum polysaccharides (LBP) on apoptosis, oxidative damage, and expression of adhesion molecules in bone marrow mononuclear cells (BMNC) of mice injured by ionizing radiation. Kunming mice were exposed to X-rays; then mice in the LBP groups were continuously injected with various concentrations of LBP intraperitoneally for 14 days. Mice in the control group were continuously injected with normal saline (NS) by the same route for 14 days. A normal group was set up. After 1, 7, and 14 days of treatment, mice were killed and BMNC were extracted. Cell cycle, apoptosis, and the expression of adhesion molecules CD44 and CD49d were detected by flow cytometry. The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were identified by colorimetric analyses. LBP significantly decreased the percentage of G0/G1 phase, apoptosis, MDA level, and expression of CD44 and CD49d and distinctly increased the activity of SOD. LBP showed a protective effect on BMNC against ionizing radiation-induced apoptosis and oxidative damage and altered the expression of adhesion molecule. PMID:27314019

Emergence of dendritic cells in the myocardium after acute myocardial infarction - implications for inflammatory myocardial damage.

PubMed

Yilmaz, Atilla; Dietel, Barbara; Cicha, Iwona; Schubert, Katja; Hausmann, Roland; Daniel, Werner G; Garlichs, Christoph D; Stumpf, Christian

2010-03-01

Dendritic cells (DC) are crucial for T cell mediated immune responses. Recently, we observed a significant decrease in circulating myeloid DC precursors in patients with acute myocardial infarction (AMI). The aim of the present study was to investigate whether myeloid DC are present in infarcted myocardium. Myocardial specimens of 10 patients with AMI and 7 accident victims (controls) were collected after autopsy. In immunostainings the presence of DC (CD209(+), fascin(+)), T cells (CD3(+)), macrophages (CD68(+)), and HLA-DR expression was analyzed. Significantly higher numbers of CD209(+)-DC (97 vs. 44 cells/0.25 mm(2), p=0.03), fascin(+)-DC (54 vs. 8 cells/0.25 mm(2), p=0.02), T cells (27 vs. 6 cells/0.25 mm(2), p=0.02), and macrophages (44 vs. 6 cells/0.25 mm(2), p=0.01) associated with high HLA-DR expression were detected in infarcted myocardium. Frequent colocalizations of DC and T cells were observed. In occluded coronary arteries numerous DC, T cells, macrophages and high HLA-DR expression were found. We show that DC are present in infarcted myocardium after AMI. High HLA-DR expression and the colocalization with T cells suggest that they might trigger an immune response leading to further myocardial damage.

Functional characterization of mouse spinal cord infiltrating CD8+ lymphocytes

PubMed Central

Deb, Chandra; Howe, Charles L

2011-01-01

Understanding the immunopathogenesis of neuroimmunological diseases of the CNS requires a robust method for isolating and characterizing the immune effector cells that infiltrate the spinal cord in animal models. We have developed a simple and rapid isolation method that produces high yields of spinal cord infiltrating leukocytes from a single demyelinated spinal cord and which maintains high surface expression of key immunophenotyping antigens. Using this method and the Theiler’s virus model of chronic demyelination, we report the presence of spinal cord infiltrating acute effector CD8+ lymphocytes that are CD45hiCD44loCD62L− and a population of spinal cord infiltrating target effector memory CD8+ lymphocytes that are CD45hiCD44hiCD62L−. These cells respond robustly to ex vivo stimulation by producing interferon γ but do not exhibit specificity for Theiler’s virus in a cytotoxicity assay. We conclude that target-derived lymphocytes in a mouse model of chronic spinal cord demyelination may have unique functional specificities. PMID:19596449

The Selective Centrifugation Ensures a Better In Vitro Isolation of ASCs and Restores a Soft Tissue Regeneration In Vivo

PubMed Central

De Francesco, Francesco; Guastafierro, Antonio; Nicoletti, Gianfranco; Razzano, Sergio; Riccio, Michele; Ferraro, Giuseppe A.

2017-01-01

Autologous fat grafting procedures in plastic surgery have been extensively used to reinforce soft tissue in congenital or acquired tissue impairments. With this background, the aim of this study is firstly to examine the impact of a selective centrifugation on existing adipose stem cells (ASCs) in terms of stemness profile maintenance and, secondly, to investigate the effect of restoring volume in reconstruction on patients affected by soft tissue damage. After centrifugation, the fat graft products were separated into two layers and subsequently examined in vitro for the expression of CD34, CD90, CD117, CD105, CD29, CD31, CD44, CD73, CD133, CD14 and CD45 markers by flow cytometry and gene expression analyses were performed for Sox2, WNT3A, END, CD44, FUT4, COLL1, CTNNB1, hbEGF, KRTLG, MMP2 and VIM genes. The results showed that in the middle-high density (MHD) layer there was a peak concentration of ASCs, compared to another layer obtained after centrifugation. Research carried out on patients under treatment for soft tissue regeneration using cells obtained from MHD layer selection will be fundamental in comparative analysis. These studies will lead to an adequate standardization of outcomes, provided that treatment is performed through cell selection. Therefore, a unique procedure in tissue reconstruction and regeneration through fat grafting is presented here. PMID:28498335

Expression patterns of the aquaporin gene family during renal development: influence of genetic variability.

PubMed

Parreira, Kleber S; Debaix, Huguette; Cnops, Yvette; Geffers, Lars; Devuyst, Olivier

2009-08-01

High-throughput analyses have shown that aquaporins (AQPs) belong to a cluster of genes that are differentially expressed during kidney organogenesis. However, the spatiotemporal expression patterns of the AQP gene family during tubular maturation and the potential influence of genetic variation on these patterns and on water handling remain unknown. We investigated the expression patterns of all AQP isoforms in fetal (E13.5 to E18.5), postnatal (P1 to P28), and adult (9 weeks) kidneys of inbred (C57BL/6J) and outbred (CD-1) mice. Using quantitative polymerase chain reaction (PCR), we evidenced two mRNA patterns during tubular maturation in C57 mice. The AQPs 1-7-11 showed an early (from E14.5) and progressive increase to adult levels, similar to the mRNA pattern observed for proximal tubule markers (Megalin, NaPi-IIa, OAT1) and reflecting the continuous increase in renal cortical structures during development. By contrast, AQPs 2-3-4 showed a later (E15.5) and more abrupt increase, with transient postnatal overexpression. Most AQP genes were expressed earlier and/or stronger in maturing CD-1 kidneys. Furthermore, adult CD-1 kidneys expressed more AQP2 in the collecting ducts, which was reflected by a significant delay in excreting a water load. The expression patterns of proximal vs. distal AQPs and the earlier expression in the CD-1 strain were confirmed by immunoblotting and immunostaining. These data (1) substantiate the clustering of important genes during tubular maturation and (2) demonstrate that genetic variability influences the regulation of the AQP gene family during tubular maturation and water handling by the mature kidney.

The mouse tumor cell lines EL4 and RMA display mosaic expression of NK-related and certain other surface molecules and appear to have a common origin.

PubMed

Gays, F; Unnikrishnan, M; Shrestha, S; Fraser, K P; Brown, A R; Tristram, C M; Chrzanowska-Lightowlers, Z M; Brooks, C G

2000-05-15

As a potential means for facilitating studies of NK cell-related molecules, we examined the expression of these molecules on a range of mouse tumor cell lines. Of the lines we initially examined, only EL4 and RMA expressed such molecules, both lines expressing several members of the Ly49 and NKRP1 families. Unexpectedly, several of the NK-related molecules, together with certain other molecules including CD2, CD3, CD4, CD32, and CD44, were often expressed in a mosaic manner, even on freshly derived clones, indicating frequent switching in expression. In each case examined, switching was controlled at the mRNA level, with expression of CD3zeta determining expression of the entire CD3-TCR complex. Each of the variable molecules was expressed independently, with the exception that CD3 was restricted to cells that also expressed CD2. Treatment with drugs that affect DNA methylation and histone acetylation could augment the expression of at least some of the variable molecules. The striking phenotypic similarity between EL4 and RMA led us to examine the state of their TCRbeta genes. Both lines had identical rearrangements on both chromosomes, indicating that RMA is in fact a subline of EL4. Overall, these findings suggest that EL4 is an NK-T cell tumor that may have retained a genetic mechanism that permits the variable expression of a restricted group of molecules involved in recognition and signaling.

Chitosan promotes cancer progression and stem cell properties in association with Wnt signaling in colon and hepatocellular carcinoma cells

PubMed Central

Chang, Po-Hsiang; Sekine, Keisuke; Chao, Hsiao-Mei; Hsu, Shan-hui; Chern, Edward

2017-01-01

Cancer stem cells (CSCs), a small population of cancer cells, have been considered to be the origin of cancer initiation, recurrence, and metastasis. Tumor microenvironment provides crucial signals for CSCs to maintain stem cell properties and promotes tumorigenesis. Therefore, establishment of an appropriate cell culture system to mimic the microenvironment for CSC studies is an important issue. In this study, we grew colon and hepatocellular carcinoma (HCC) cells on chitosan membranes and evaluated the tumor progression and the CSC properties. Experimental results showed that culturing cancer cells on chitosan increased cell motility, drug resistance, quiescent population, self-renewal capacity, and the expression levels of stemness and CSC marker genes, such as OCT4, NANOG, CD133, CD44, and EpCAM. Furthermore, we demonstrated that chitosan might activate canonical Wnt/β-catenin-CD44 axis signaling in CD44positive colon cancer cells and noncanonical Wnt-STAT3 signaling in CD44negative HCC cells. In conclusion, chitosan as culture substrates activated the essential signaling of CSCs and promoted CSC properties. The chitosan culture system provides a convenient platform for the research of CSC biology and screening of anticancer drugs. PMID:28367998

Enhancing Adoptive Cell Therapy of Cancer through Targeted Delivery of Small-Molecule Immunomodulators to Internalizing or Noninternalizing Receptors.

PubMed

Zheng, Yiran; Tang, Li; Mabardi, Llian; Kumari, Sudha; Irvine, Darrell J

2017-03-28

Adoptive cell therapy (ACT) has achieved striking efficacy in B-cell leukemias, but less success treating other cancers, in part due to the rapid loss of ACT T-cell effector function in vivo due to immunosuppression in solid tumors. Transforming growth factor-β (TGF-β) signaling is an important mechanism of immune suppression in the tumor microenvironment, but systemic inhibition of TGF-β is toxic. Here we evaluated the potential of targeting a small molecule inhibitor of TGF-β to ACT T-cells using PEGylated immunoliposomes. Liposomes were prepared that released TGF-β inhibitor over ∼3 days in vitro. We compared the impact of targeting these drug-loaded vesicles to T-cells via an internalizing receptor (CD90) or noninternalizing receptor (CD45). When lymphocytes were preloaded with immunoliposomes in vitro prior to adoptive therapy, vesicles targeted to both CD45 and CD90 promoted enhanced T-cell expression of granzymes relative to free systemic drug administration, but only targeting to CD45 enhanced accumulation of granzyme-expressing T-cells in tumors, which correlated with the greatest enhancement of T-cell antitumor activity. By contrast, when administered i.v. to target T-cells in vivo, only targeting of a CD90 isoform expressed exclusively by the donor T-cells led to greater tumor regression over equivalent doses of free systemic drug. These results suggest that in vivo, targeting of receptors uniquely expressed by donor T-cells is of paramount importance for maximal efficacy. This immunoliposome strategy should be broadly applicable to target exogenous or endogenous T-cells and defines parameters to optimize delivery of supporting (or suppressive) drugs to these important immune effectors.

Enhancing Adoptive Cell Therapy of Cancer through Targeted Delivery of Small-Molecule Immunomodulators to Internalizing or Non-Internalizing Receptors

PubMed Central

Zheng, Yiran; Tang, Li; Mabardi, Llian; Kumari, Sudha; Irvine, Darrell J.

2017-01-01

Adoptive cell therapy (ACT) has achieved striking efficacy in B-cell leukemias, but less success treating other cancers, in part due to the rapid loss of ACT T-cell effector function in vivo due to immunosuppression in solid tumors. Transforming growth factor-β (TGF-β) signaling is an important mechanism of immune suppression in the tumor microenvironment, but systemic inhibition of TGF-β is toxic. Here we evaluated the potential of targeting a small molecule inhibitor of TGF-β to ACT T-cells using PEGylated immunoliposomes. Liposomes were prepared that released TGF-β inhibitor over ~3 days in vitro. We compared the impact of targeting these drug-loaded vesicles to T-cells via an internalizing receptor (CD90) or non-internalizing receptor (CD45). When lymphocytes were pre-loaded with immunoliposomes in vitro prior to adoptive therapy, vesicles targeted to both CD45 and CD90 promoted enhanced T-cell expression of granzymes relative to free systemic drug administration, but only targeting to CD45 enhanced accumulation of granzyme-expressing T-cells in tumors, which correlated with the greatest enhancement of T-cell anti-tumor activity. By contrast, when administered i.v. to target T-cells in vivo, only targeting of a CD90 isoform expressed exclusively by the donor T-cells led to greater tumor regression over equivalent doses of free systemic drug. These results suggest that in vivo, targeting of receptors uniquely expressed by donor T-cells is of paramount importance for maximal efficacy. This immunoliposome strategy should be broadly applicable to target exogenous or endogenous T-cells and defines parameters to optimize delivery of supporting (or suppressive) drugs to these important immune effectors. PMID:28231431

Adhesion-mediated self-renewal abilities of Ph+ blastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Funayama, Keiji; Saito-Kurimoto, Yumi; Ebihara, Yasuhiro

2010-05-28

The Philadelphia chromosome-positive blastoma, maintained by serial subcutaneous transplantation in nude mice, is a highly proliferating biological mass consisting of homogenous CD34{sup +}CD38{sup -} myeloblastoid cells. These cells newly evolved from pluripotent leukemia stem cells of chronic myeloid leukemia in the chronic phase. Therefore, this mass may provide a unique tool for better understanding cellular and molecular mechanisms of self-renewal of leukemia stem cells. In this paper, we demonstrated that intravenously injected blastoma cells can cause Ph+ blastic leukemia with multiple invasive foci in NOD/SCID mice but not in nude mice. In addition, using an in vitro culture system, wemore » clearly showed that blastoma cell adhesion to OP9 stromal cells accelerates blastoma cell proliferation that is associated with up-regulation of BMI1 gene expression; increased levels of {beta}-catenin and the Notch1 intra-cellular domain; and changed the expression pattern of variant CD44 forms, which are constitutively expressed in these blastoma cells. These findings strongly suggest that adhesion of leukemic stem cells to stromal cells via CD44 might be indispensable for their cellular defense against attack by immune cells and for maintenance of their self-renewal ability.« less

Hyaluronic Acid-Based pH-Sensitive Polymer-Modified Liposomes for Cell-Specific Intracellular Drug Delivery Systems.

PubMed

Miyazaki, Maiko; Yuba, Eiji; Hayashi, Hiroshi; Harada, Atsushi; Kono, Kenji

2018-01-17

For the enhancement of therapeutic effects and reduction of side effects derived from anticancer drugs in cancer chemotherapy, it is imperative to develop drug delivery systems with cancer-specificity and controlled release function inside cancer cells. pH-sensitive liposomes are useful as an intracellular drug delivery system because of their abilities to transfer their contents into the cell interior through fusion or destabilization of endosome, which has weakly acidic environment. We earlier reported liposomes modified with various types of pH-sensitive polymers based on synthetic polymers and biopolymers as vehicles for intracellular drug delivery systems. In this study, hyaluronic acid (HA)-based pH-sensitive polymers were designed as multifunctional polymers having not only pH-sensitivity but also targeting properties to cells expressing CD44, which is known as a cancer cell surface marker. Carboxyl group-introduced HA derivatives of two types, MGlu-HA and CHex-HA, which have a more hydrophobic side chain structure than that of MGlu-HA, were synthesized by reaction with various dicarboxylic anhydrides. These polymer-modified liposomes were stable at neutral pH, but showed content release under weakly acidic conditions. CHex-HA-modified liposomes delivered their contents into CD44-expressing cells more efficiently than HA-modified or MGlu-HA-modified liposomes or unmodified liposomes, whereas the same liposomes were taken up only slightly by cells expressing CD44 proteins less. Competition assay using free HA or other polymers revealed that HA derivative-modified liposomes might be recognized by CD44. Therefore, HA-derivative-modified liposomes are useful as cell-specific intracellular drug delivery systems.

ERK1 is important for Th2 differentiation and development of experimental asthma

PubMed Central

Goplen, Nicholas; Karim, Zunayet; Guo, Lei; Zhuang, Yonghua; Huang, Hua; Gorska, Magdalena M.; Gelfand, Erwin; Pagés, Gilles; Pouysségur, Jacques; Alam, Rafeul

2012-01-01

The ERK1/2 signaling pathway regulates a variety of T-cell functions. We observed dynamic changes in the expression of ERK1/2 during T-helper cell differentiation. Specifically, the expression of ERK1/2 was decreased and increased by IL-12 and IL-4, respectively. To address this subject further, we examined the specific role of ERK1 in Th2 differentiation and development of experimental asthma using ERK1−/− mice. ERK1−/− mice were unable to mount airway inflammation and hyperreactivity in two different models of asthma, acute and chronic. ERK1−/− mice had reduced expression of Th2 cytokines IL-4 and IL-5 but not IL-17A or IFN-γ. They had reduced levels of allergen-specific IgE and blood eosinophils. T cells from immunized ERK1−/− mice manifested reduced proliferation in response to the sensitizing allergen. ERK1−/− T cells had reduced and short-lived expression of JunB following TCR stimulation, which likely contributed to their impaired Th2 differentiation. Immunized ERK1−/− mice showed reduced numbers of CD44high CD4 T cells in the spleen. In vitro studies demonstrated that Th2 but not Th1 cells from ERK1−/− mice had reduced numbers of CD44high cells. Finally, CD4 T cells form ERK1−/− mice expressed higher levels of BIM under growth factor-deprived conditions and reduced Mcl-1 on stimulation. As a result, the survival of CD4 T cells, especially CD44high Th2 cells, was much reduced in ERK1−/− mice. We conclude that ERK1 plays a nonredundant role in Th2 differentiation and development of experimental asthma. ERK1 controls Th2 differentiation and survival through its effect on JunB and BIM, respectively.—Goplen, N., Karim, Z., Guo, L., Zhuang, Y., Huang, H., Gorska, M. M., Gelfand, E., Pagés, G., Pouysségur, J., Alam, R. ERK1 is important for Th2 differentiation and development of experimental asthma. PMID:22262639

Purification and Characterization of Recombinant Darbepoetin Alfa from Leishmania tarentolae.

PubMed

Kianmehr, Anvarsadat; Mahrooz, Abdolkarim; Oladnabi, Morteza; Safdari, Yaghoub; Ansari, Javad; Veisi, Kamal; Evazalipour, Mehdi; Shahbazmohammadi, Hamid; Omidinia, Eskandar

2016-09-01

Darbepoetin alfa is a biopharmaceutical glycoprotein that stimulates erythropoiesis and is used to treat anemia, which associated with renal failure and cancer chemotherapy. We herein describe the structural characterization of recombinant darbepoetin alfa produced by Leishmania tarentolae T7-TR host. The DNA expression cassette was integrated into the L. tarentolae genome through homologous recombination. Transformed clones were selected by antibiotic resistance, diagnostic PCRs, and protein expression analysis. The structure of recombinant darbepoetin alfa was analyzed by isoelectric focusing, ultraviolet-visible spectrum, and circular dichroism (CD) spectroscopy. Expression analysis showed the presence of a protein band at 40 kDa, and its expression level was 51.2 mg/ml of culture medium. Darbepoetin alfa have 5 isoforms with varying degree of sialylation. The UV absorption and CD spectra were analogous to original drug (Aranesp), which confirmed that the produced protein was darbepoetin alfa. Potency test results revealed that the purified protein was biologically active. In brief, the structural and biological characteristics of expressed darbepoetin alfa were very similar to Aranesp which has been normally expressed in CHO. Our data also suggest that produced protein has potential to be developed for clinical use.

Mass Cytometry Identifies Distinct Lung CD4+ T Cell Patterns in Löfgren’s Syndrome and Non-Löfgren’s Syndrome Sarcoidosis

PubMed Central

Kaiser, Ylva; Lakshmikanth, Tadepally; Chen, Yang; Mikes, Jaromir; Eklund, Anders; Brodin, Petter; Achour, Adnane; Grunewald, Johan

2017-01-01

Sarcoidosis is a granulomatous disorder of unknown etiology, characterized by accumulation of activated CD4+ T cells in the lungs. Disease phenotypes Löfgren’s syndrome (LS) and “non-LS” differ in terms of clinical manifestations, genetic background, HLA association, and prognosis, but the underlying inflammatory mechanisms largely remain unknown. Bronchoalveolar lavage fluid cells from four HLA-DRB1*03+ LS and four HLA-DRB1*03− non-LS patients were analyzed by mass cytometry, using a panel of 33 unique markers. Differentially regulated CD4+ T cell populations were identified using the Citrus algorithm, and t-stochastic neighborhood embedding was applied for dimensionality reduction and single-cell data visualization. We identified 19 individual CD4+ T cell clusters differing significantly in abundance between LS and non-LS patients. Seven clusters more frequent in LS patients were characterized by significantly higher expression of regulatory receptors CTLA-4, PD-1, and ICOS, along with low expression of adhesion marker CD44. In contrast, 12 clusters primarily found in non-LS displayed elevated expression of activation and effector markers HLA-DR, CD127, CD39, as well as CD44. Hierarchical clustering further indicated functional heterogeneity and diverse origins of T cell receptor Vα2.3/Vβ22-restricted cells in LS. Finally, a near-complete overlap of CD8 and Ki-67 expression suggested larger influence of CD8+ T cell activity on sarcoid inflammation than previously appreciated. In this study, we provide detailed characterization of pulmonary T cells and immunological parameters that define separate disease pathways in LS and non-LS. With direct association to clinical parameters, such as granuloma persistence, resolution, or chronic inflammation, these results provide a valuable foundation for further exploration and potential clinical application. PMID:28955342

Spontaneous and natural cytotoxicity receptor-mediated cytotoxicity are effector functions of distinct natural killer subsets in hepatitis C virus-infected chimpanzees.

PubMed

Verstrepen, B E; Nieuwenhuis, I G; Mooij, P; Bogers, W M; Boonstra, A; Koopman, G

2016-07-01

In humans, CD16 and CD56 are used to identify functionally distinct natural killer (NK) subsets. Due to ubiquitous CD56 expression, this marker cannot be used to distinguish between NK cell subsets in chimpanzees. Therefore, functional analysis of distinct NK subsets during hepatitis C virus (HCV) infection has never been performed in these animals. In the present study an alternative strategy was used to identify four distinct NK subsets on the basis of the expression of CD16 and CD94. The expression of activating and inhibiting surface receptors showed that these subsets resemble human NK subsets. CD107 expression was used to determine degranulation of the different subsets in naive and HCV-infected chimpanzees. In HCV-infected chimpanzees increased spontaneous cytotoxicity was observed in CD94(high/dim) CD16(pos) and CD94(low) CD16(pos) subsets. By contrast, increased natural cytotoxicity receptor (NCR)- mediated degranulation after NKp30 and NKp44 triggering was demonstrated in the CD94(dim) CD16(neg) subset. Our findings suggest that spontaneous and NCR-mediated cytotoxicity are effector functions of distinct NK subsets in HCV-infected chimpanzees. © 2016 British Society for Immunology.

Blockade of Notch3 inhibits the stem-like property and is associated with ALDH1A1 and CD44 via autophagy in non-small lung cancer.

PubMed

Ma, Yuanyuan; Li, Mingzhen; Si, Jiahui; Xiong, Ying; Lu, Fangliang; Zhang, Jianzhi; Zhang, Liyi; Zhang, Panpan; Yang, Yue

2016-06-01

Acquired resistance to standard chemotherapy causes treatment failure in patients with local advanced and advanced non-small lung cancer (NSCLC). Cancer stem cells (CSCs) are a small subpopulation within cancer that is thought to be resistant to conventional chemotherapy. The Notch pathway is one of the most intensively studied for putative therapeutic targets of CSCs in solid tumors. In our study, suppression of Notch3 decreased colony and sphere formation of stem-like property in lung cancer cells. In addition, Notch3 expression was demonstrated to be upregulated in the patients with chemoresistance and related to poor prognosis of NSCLC patients. Our results also showed that CSC markers ALDH1A1 and CD44 were highly expressed in NSCLC patients with chemoresistance and these two markers were positively correlated with Notch3 expression in lung cancer specimens from TCGA database. Furthermore, the lung cancer cells with drug resistance were shown to be associated with activation of autophagy. All the data support a crucial role of Notch3 in the increase of stem-like property in NSCLC cells that might be associated with upregulation of ALDH1A1 and CD44 and activation of autophagy.

Insulin restores gestational diabetes mellitus-reduced adenosine transport involving differential expression of insulin receptor isoforms in human umbilical vein endothelium.

PubMed

Westermeier, Francisco; Salomón, Carlos; González, Marcelo; Puebla, Carlos; Guzmán-Gutiérrez, Enrique; Cifuentes, Fredi; Leiva, Andrea; Casanello, Paola; Sobrevia, Luis

2011-06-01

To determine whether insulin reverses gestational diabetes mellitus (GDM)-reduced expression and activity of human equilibrative nucleoside transporters 1 (hENT1) in human umbilical vein endothelium cells (HUVECs). Primary cultured HUVECs from full-term normal (n = 44) and diet-treated GDM (n = 44) pregnancies were used. Insulin effect was assayed on hENT1 expression (protein, mRNA, SLC29A1 promoter activity) and activity (initial rates of adenosine transport) as well as endothelial nitric oxide (NO) synthase activity (serine(1177) phosphorylation, l-citrulline formation). Adenosine concentration in culture medium and umbilical vein blood (high-performance liquid chromatography) as well as insulin receptor A and B expression (quantitative PCR) were determined. Reactivity of umbilical vein rings to adenosine and insulin was assayed by wire myography. Experiments were in the absence or presence of l-N(G)-nitro-l-arginine methyl ester (l-NAME; NO synthase inhibitor) or ZM-241385 (an A(2A)-adenosine receptor antagonist). Umbilical vein blood adenosine concentration was higher, and the adenosine- and insulin-induced NO/endothelium-dependent umbilical vein relaxation was lower in GDM. Cells from GDM exhibited increased insulin receptor A isoform expression in addition to the reported NO-dependent inhibition of hENT1-adenosine transport and SLC29A1 reporter repression, and increased extracellular concentration of adenosine and NO synthase activity. Insulin reversed all these parameters to values in normal pregnancies, an effect blocked by ZM-241385 and l-NAME. GDM and normal pregnancy HUVEC phenotypes are differentially responsive to insulin, a phenomenon where insulin acts as protecting factor for endothelial dysfunction characteristic of this syndrome. Abnormal adenosine plasma levels, and potentially A(2A)-adenosine receptors and insulin receptor A, will play crucial roles in this phenomenon in GDM.

Estrogen directly and specifically downregulates NaPi-IIa through the activation of both estrogen receptor isoforms (ERα and ERβ) in rat kidney proximal tubule

PubMed Central

Burris, Dara; Webster, Rose; Sheriff, Sulaiman; Faroqui, Rashma; Levi, Moshe; Hawse, John R.

2015-01-01

We have previously demonstrated that estrogen (E2) downregulates phosphate transporter NaPi-IIa and causes phosphaturia and hypophosphatemia in ovariectomized rats. In the present study, we examined whether E2 directly targets NaPi-IIa in the proximal tubule (PT) and studied the respective roles of estrogen receptor isoforms (ERα and ERβ) in the downregulation of NaPi-IIa using both in vivo and an in vitro expression systems. We found that estrogen specifically downregulates NaPi-IIa but not NaPi-IIc or Pit2 in the kidney cortex. Proximal tubules incubated in a “shake” suspension with E2 for 24 h exhibited a dose-dependent decrease in NaPi-IIa protein abundance. Results from OVX rats treated with specific agonists for either ERα [4,4′,4″;-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol, PPT] or ERβ [4,4′,4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol, DPN] or both (PPT + DPN), indicated that only the latter caused a sharp downregulation of NaPi-IIa, along with significant phosphaturia and hypophosphatemia. Lastly, heterologous expression studies demonstrated that estrogen downregulated NaPi-IIa only in U20S cells expressing both ERα and ERβ, but not in cells expressing either receptor alone. In conclusion, these studies demonstrate that rat PT cells express both ERα and ERβ and that E2 induces phosphaturia by directly and specifically targeting NaPi-IIa in the PT cells. This effect is mediated via a mechanism involving coactivation of both ERα and ERβ, which likely form a functional heterodimer complex in the rat kidney proximal tubule. PMID:25608964

Transient receptor potential vanilloid-type 2 targeting on stemness in liver cancer.

PubMed

Hu, Zecheng; Cao, Xiaocheng; Fang, Yu; Liu, Guoxing; Xie, Chengzhi; Qian, Ke; Lei, Xiaohua; Cao, Zhenyu; Du, Huihui; Cheng, Xiangding; Xu, Xundi

2018-06-12

The malignant phenotype of the cells resulting from human liver cancer is driven by liver cancer stem-like cells (LCSLCs). Transient Receptor Potential Vanilloid-type 2 channel (TRPV2) contributes to the progression of different tumor types, including liver cancer. In the current study, the TRPV2 expression levels give rise to the effect on stemness in liver cancer cell lines. TRPV2 knockdown in HepG2 cells enhanced spheroid and colony formation, and expression levels of CD133, CD44 and ALDH1 whereas the opposite effects were observed in TRPV2 enforced expression in SMMC-7721 cells. Furthermore, TRPV2 overexpression restored inhibition of spheroid and colony formation, and stem cell markers expression in HepG2 cells with TRPV2 silencing. The addition of the TRPV2 agonist probenecid and the TRPV2 antagonist tranilast suppressed and/or increased in vitro spheroid and colony formation, and stem cell marker expression of LCSLCs and/or liver cancer cell lines, respectively. Notably, probenecid and tranilast significantly inhibited or promoted tumor growth of HepG2 xenografts in the severe combined immunodeficiency (SCID) mouse model, respectively. TRPV2 expression at protein levels revealed converse correlation with those of CD133 and CD44 in human hepatocellular carcinoma (HCC) tissue. Collectively, the data demonstrate that TRPV2 exert effects on stemness of liver cancer and is a potential target in the treatment of human liver cancer patients. Copyright © 2018. Published by Elsevier Masson SAS.

Prostate Cancer Stem Cell-Targeted Efficacy of a New-Generation Taxoid, SBT-1214 and Novel Polyenolic Zinc-Binding Curcuminoid, CMC2.24

PubMed Central

Botchkina, Galina I.; Zuniga, Edison S.; Rowehl, Rebecca H.; Park, Rosa; Bhalla, Rahuldev; Bialkowska, Agnieszka B.; Johnson, Francis; Golub, Lorne M.; Zhang, Yu; Ojima, Iwao; Shroyer, Kenneth R.

2013-01-01

Background Prostate cancer is the second leading cause of cancer death among men. Multiple evidence suggests that a population of tumor-initiating, or cancer stem cells (CSCs) is responsible for cancer development and exceptional drug resistance, representing a highly important therapeutic target. The present study evaluated CSC-specific alterations induced by new-generation taxoid SBT-1214 and a novel polyenolic zinc-binding curcuminoid, CMC2.24, in prostate CSCs. Principal Findings The CD133high/CD44high phenotype was isolated from spontaneously immortalized patient-derived PPT2 cells and highly metastatic PC3MM2 cells. Weekly treatment of the NOD/SCID mice bearing PPT2- and PC3MM3-induced tumors with the SBT-1214 led to dramatic suppression of tumor growth. Four of six PPT2 and 3 of 6 PC3MM2 tumors have shown the absence of viable cells in residual tumors. In vitro, SBT-1214 (100nM-1µM; for 72 hr) induced about 60% cell death in CD133high/CD44+/high cells cultured on collagen I in stem cell medium (in contrast, the same doses of paclitaxel increased proliferation of these cells). The cytotoxic effects were increased when SBT-1214 was combined with the CMC2.24. A stem cell-specific PCR array assay revealed that this drug combination mediated massive inhibition of multiple constitutively up-regulated stem cell-related genes, including key pluripotency transcription factors. Importantly, this drug combination induced expression of p21 and p53, which were absent in CD133high/CD44high cells. Viable cells that survived this treatment regimen were no longer able to induce secondary spheroids, exhibited significant morphological abnormalities and died in 2-5 days. Conclusions We report here that the SBT-1214 alone, or in combination with CMC2.24, possesses significant activity against prostate CD133high/CD44+/high tumor-initiating cells. This drug combination efficiently inhibits expression of the majority of stem cell-related genes and pluripotency transcription factors. In addition, it induces a previously absent expression of p21 and p53 (“gene wake-up”), which can potentially reverse drug resistance by increasing sensitivity to anti-cancer drugs. PMID:24086245

Altered expression of pectoral myosin heavy chain isoforms corresponds to migration status in the white-crowned sparrow (Zonotrichia leucophrys gambelii)

PubMed Central

Welch, Kenneth C.; Ramenofsky, Marilyn

2016-01-01

Birds undergo numerous changes as they progress through life-history stages, yet relatively few studies have examined how birds adapt to both the dynamic energetic and mechanical demands associated with such transitions. Myosin heavy chain (MyHC) expression, often linked with muscle fibre type, is strongly correlated with a muscle's mechanical power-generating capability, thus we examined several morphological properties, including MyHC expression of the pectoralis, in a long-distance migrant, the white-crowned sparrow (Zonotrichia leucophrys gambelii) throughout the progression from winter, spring departure and arrival on breeding grounds. White-crowned sparrows demonstrated significant phenotypic flexibility throughout the seasonal transition, including changes in prealternate moult status, lipid fuelling, body condition and flight muscle morphology. Pectoral MyHC expression also varied significantly over the course of the study. Wintering birds expressed a single, newly classified adult fast 2 isoform. At spring departure, pectoral isoform expression included two MyHC isoforms: the adult fast 2 isoform along with a smaller proportion of a newly present adult fast 1 isoform. By spring arrival, both adult fast isoforms present at departure remained, yet expression had shifted to a greater relative proportion of the adult fast 1 isoform. Altering pectoral MyHC isoform expression in preparation for and during spring migration may represent an adaptation to modulate muscle mechanical output to support long-distance flight. PMID:28018664

Regulation of Head and Neck Squamous Cancer Stem Cells by PI3K and SOX2

PubMed Central

Keysar, Stephen B.; Le, Phuong N.; Miller, Bettina; Jackson, Brian C.; Eagles, Justin R.; Nieto, Cera; Kim, Jihye; Tang, Binwu; Glogowska, Magdalena J.; Morton, J. Jason; Padilla-Just, Nuria; Gomez, Karina; Warnock, Emily; Reisinger, Julie; Arcaroli, John J.; Messersmith, Wells A.; Wakefield, Lalage M.; Gao, Dexiang; Tan, Aik-Choon; Serracino, Hilary

2017-01-01

Background: We have an incomplete understanding of the differences between cancer stem cells (CSCs) in human papillomavirus–positive (HPV-positive) and –negative (HPV-negative) head and neck squamous cell cancer (HNSCC). The PI3K pathway has the most frequent activating genetic events in HNSCC (especially HPV-positive driven), but the differential signaling between CSCs and non-CSCs is also unknown. Methods: We addressed these unresolved questions using CSCs identified from 10 HNSCC patient-derived xenografts (PDXs). Sored populations were serially passaged in nude mice to evaluate tumorigenicity and tumor recapitulation. The transcription profile of HNSCC CSCs was characterized by mRNA sequencing, and the susceptibility of CSCs to therapy was investigated using an in vivo model. SOX2 transcriptional activity was used to follow the asymmetric division of PDX-derived CSCs. All statistical tests were two-sided. Results: CSCs were enriched by high aldehyde dehydrogenase (ALDH) activity and CD44 expression and were similar between HPV-positive and HPV-negative cases (percent tumor formation injecting ≤ 1x103 cells: ALDH+CD44high = 65.8%, ALDH-CD44high = 33.1%, ALDH+CD44high = 20.0%; and injecting 1x105 cells: ALDH-CD44low = 4.4%). CSCs were resistant to conventional therapy and had PI3K/mTOR pathway overexpression (GSEA pathway enrichment, P < .001), and PI3K inhibition in vivo decreased their tumorigenicity (40.0%–100.0% across cases). PI3K/mTOR directly regulated SOX2 protein levels, and SOX2 in turn activated ALDH1A1 (P < .001 013C and 067C) expression and ALDH activity (ALDH+ [%] empty-control vs SOX2, 0.4% ± 0.4% vs 14.5% ± 9.8%, P = .03 for 013C and 1.7% ± 1.3% vs 3.6% ± 3.4%, P = .04 for 067C) in 013C and 067 cells. SOX2 enhanced sphere and tumor growth (spheres/well, 013C P < .001 and 067C P = .04) and therapy resistance. SOX2 expression prompted mesenchymal-to-epithelial transition (MET) by inducing CDH1 (013C P = .002, 067C P = .01), followed by asymmetric division and proliferation, which contributed to tumor formation. Conclusions: The molecular link between PI3K activation and CSC properties found in this study provides insights into therapeutic strategies for HNSCC. Constitutive expression of SOX2 in HNSCC cells generates a CSC-like population that enables CSC studies. PMID:27634934

Co-expression of CD147 and GLUT-1 indicates radiation resistance and poor prognosis in cervical squamous cell carcinoma.

PubMed

Huang, Xin-Qiong; Chen, Xiang; Xie, Xiao-Xue; Zhou, Qin; Li, Kai; Li, Shan; Shen, Liang-Fang; Su, Juan

2014-01-01

The aim of this study was to investigate the association of CD147 and GLUT-1, which play important roles in glycolysis in response to radiotherapy and clinical outcomes in patients with locally advanced cervical squamous cell carcinoma (LACSCC). The records of 132 female patients who received primary radiation therapy to treat LACSCC at FIGO stages IB-IVA were retrospectively reviewed. Forty-seven patients with PFS (progression-free survival) of less than 36 months were regarded as radiation-resistant. Eighty-five patients with PFS longer than 36 months were regarded as radiation-sensitive. Using pretreatment paraffin-embedded tissues, we evaluated CD147 and GLUT-1 expression by immunohistochemistry. Overexpression of CD147, GLUT-1, and CD147 and GLUT-1 combined were 44.7%, 52.9% and 36.5%, respectively, in the radiation-sensitive group, and 91.5%, 89.4% and 83.0%, respectively, in the radiation-resistant group. The 5-year progress free survival (PFS) rates in the CD147-low, CD147-high, GLUT-1-low, GLUT-1-high, CD147- and/or GLUT-1-low and CD147- and GLUT-1- dual high expression groups were 66.79%, 87.10%, 52.78%, 85.82%, 55.94%, 82.90% and 50.82%, respectively. CD147 and GLUT-1 co-expression, FIGO stage and tumor diameter were independent poor prognostic factors for patients with LACSCC in multivariate Cox regression analysis. Patients with high expression of CD147 alone, GLUT-1 alone or co-expression of CD147 and GLUT-1 showed greater resistance to radiotherapy and a shorter PFS than those with low expression. In particular, co-expression of CD147 and GLUT-1 can be considered as a negative independent prognostic factor.

Co-expression of CD147 and GLUT-1 indicates radiation resistance and poor prognosis in cervical squamous cell carcinoma

PubMed Central

Huang, Xin-Qiong; Chen, Xiang; Xie, Xiao-Xue; Zhou, Qin; Li, Kai; Li, Shan; Shen, Liang-Fang; Su, Juan

2014-01-01

The aim of this study was to investigate the association of CD147 and GLUT-1, which play important roles in glycolysis in response to radiotherapy and clinical outcomes in patients with locally advanced cervical squamous cell carcinoma (LACSCC). The records of 132 female patients who received primary radiation therapy to treat LACSCC at FIGO stages IB-IVA were retrospectively reviewed. Forty-seven patients with PFS (progression-free survival) of less than 36 months were regarded as radiation-resistant. Eighty-five patients with PFS longer than 36 months were regarded as radiation-sensitive. Using pretreatment paraffin-embedded tissues, we evaluated CD147 and GLUT-1 expression by immunohistochemistry. Overexpression of CD147, GLUT-1, and CD147 and GLUT-1 combined were 44.7%, 52.9% and 36.5%, respectively, in the radiation-sensitive group, and 91.5%, 89.4% and 83.0%, respectively, in the radiation-resistant group. The 5-year progress free survival (PFS) rates in the CD147-low, CD147-high, GLUT-1-low, GLUT-1-high, CD147- and/or GLUT-1-low and CD147- and GLUT-1- dual high expression groups were 66.79%, 87.10%, 52.78%, 85.82%, 55.94%, 82.90% and 50.82%, respectively. CD147 and GLUT-1 co-expression, FIGO stage and tumor diameter were independent poor prognostic factors for patients with LACSCC in multivariate Cox regression analysis. Patients with high expression of CD147 alone, GLUT-1 alone or co-expression of CD147 and GLUT-1 showed greater resistance to radiotherapy and a shorter PFS than those with low expression. In particular, co-expression of CD147 and GLUT-1 can be considered as a negative independent prognostic factor. PMID:24817962

Augmentation of the expression of the eotaxin receptor on duodenal neutrophils by IL-21.

PubMed

Takeda, Yuji; Kato, Tomoyuki; Nemoto, Nobuhito; Araki, Akemi; Gazi, Mohammad Yeashin; Nara, Hidetoshi; Asao, Hironobu

2018-05-16

Inflammation can occur via different mechanisms, such as via acute and chronic responses, on numerous occasions and function accordingly through various roles. There are more than five subsets of neutrophils; neutrophilic heterogeneity is modulated by the inflammatory condition. To understand the characteristics of inflammation, identification of atypical neutrophils is important. In this study, we found that the expression of eotaxin receptor (CD193) on atypical neutrophils in the duodenum is augmented in IL-21 isoform transgenic (Tg) mice. In a series of studies, we have established a Tg mouse strain to further investigate the functions of IL-21 in vivo. Interestingly, Tg mice immunized with ovalbumin (OVA) were more sensitive to OVA-induced systemic anaphylaxis as compared with wild type mice with duodenal and splenic gross congestion. Further analysis conducted in the duodenum of Tg mice revealed that only the number of neutrophils migrating into the duodenum was significantly increased prior to immunization. Previous studies have shown that the gastrointestinal compartment and the spleen constantly produce eotaxin, which regulates basal levels of tissue eosinophils. Therefore, we analyzed CD193 expression on neutrophils and eosinophils. As expected, its expression by duodenal neutrophils was upregulated in Tg mice. Furthermore, the addition of IL-21 into bone marrow cell culture increased the number of CD193 + neutrophils, which easily migrated into the duodenum. These observations suggested that CD193 + neutrophils increase in number under inflammatory conditions due to chronic IL-21 production. Copyright © 2018 Elsevier Ltd. All rights reserved.

Cd²⁺-induced alteration of the global proteome of human skin fibroblast cells.

PubMed

Prins, John M; Fu, Lijuan; Guo, Lei; Wang, Yinsheng

2014-03-07

Cadmium (Cd(2+)) is a toxic heavy metal and a well-known human carcinogen. The toxic effects of Cd(2+) on biological systems are diverse and thought to be exerted through a complex array of mechanisms. Despite the large number of studies aimed to elucidate the toxic mechanisms of action of Cd(2+), few have been targeted toward investigating the ability of Cd(2+) to disrupt multiple cellular pathways simultaneously and the overall cellular responses toward Cd(2+) exposure. In this study, we employed a quantitative proteomic method, relying on stable isotope labeling by amino acids in cell culture (SILAC) and LC-MS/MS, to assess the Cd(2+)-induced simultaneous alterations of multiple cellular pathways in cultured human skin fibroblast cells. By using this approach, we were able to quantify 2931 proteins, and 400 of them displayed significantly changed expression following Cd(2+) exposure. Our results unveiled that Cd(2+) treatment led to the marked upregulation of several antioxidant enzymes (e.g., metallothionein-1G, superoxide dismutase, pyridoxal kinase, etc.), enzymes associated with glutathione biosynthesis and homeostasis (e.g., glutathione S-transferases, glutathione synthetase, glutathione peroxidase, etc.), and proteins involved in cellular energy metabolism (e.g., glycolysis, pentose phosphate pathway, and the citric acid cycle). Additionally, we found that Cd(2+) treatment resulted in the elevated expression of two isoforms of dimethylarginine dimethylaminohydrolase (DDAH I and II), enzymes known to play a key role in regulating nitric oxide biosynthesis. Consistent with these findings, we observed elevated formation of nitric oxide in human skin (GM00637) and lung (IMR-90) fibroblast cells following Cd(2+) exposure. The upregulation of DDAH I and II suggests a role of nitric oxide synthesis in Cd(2+)-induced toxicity in human cells.

NCR1 Expression Identifies Canine Natural Killer Cell Subsets with Phenotypic Similarity to Human Natural Killer Cells

PubMed Central

Foltz, Jennifer A.; Somanchi, Srinivas S.; Yang, Yanwen; Aquino-Lopez, Arianexys; Bishop, Erin E.; Lee, Dean A.

2016-01-01

Canines spontaneously develop many cancers similar to humans – including osteosarcoma, leukemia, and lymphoma – offering the opportunity to study immune therapies in a genetically heterogeneous and immunocompetent environment. However, a lack of antibodies recognizing canine NK cell markers has resulted in suboptimal characterization and unknown purity of NK cell products, hindering the development of canine models of NK cell adoptive immunotherapy. To this end, we generated a novel antibody to canine NCR1 (NKp46), the putative species-wide marker of NK cells, enabling purification of NK cells for further characterization. We demonstrate that CD3−/NKp46+ cells in healthy and osteosarcoma-bearing canines have phenotypic similarity to human CD3−/NKp46+ NK cells, expressing mRNA for CD16 and the natural cytotoxicity receptors NKp30, NKp44, and NKp80. Functionally, we demonstrate with the calcein release assay that canine CD3−/NKp46+ cells kill canine tumor cell lines without prior sensitization and secrete IFN-γ, TNF-α, IL-8, IL-10, and granulocyte-macrophage colony-stimulating factor as measured by Luminex. Similar to human NK cells, CD3−/NKp46+ cells expand rapidly on feeder cells expressing 4-1BBL and membrane-bound IL-21 (median = 20,283-fold in 21 days). Furthermore, we identify a minor Null population (CD3−/CD21−/CD14−/NKp46−) with reduced cytotoxicity against osteosarcoma cells, but similar cytokine secretion as CD3−/NKp46+ cells. Null cells in canines and humans have reduced expression of NKG2D, NKp44, and CD16 compared to NKp46+ NK cells and can be induced to express NKp46 with further expansion on feeder cells. In conclusion, we have identified and characterized canine NK cells, including an NKp46− subset of canine and human NK cells, using a novel anti-canine NKp46 antibody, and report robust ex vivo expansion of canine NK cells sufficient for adoptive immunotherapy. PMID:27933061

MIA PaCa-2 and PANC-1 - pancreas ductal adenocarcinoma cell lines with neuroendocrine differentiation and somatostatin receptors.

PubMed

Gradiz, Rui; Silva, Henriqueta C; Carvalho, Lina; Botelho, Maria Filomena; Mota-Pinto, Anabela

2016-02-17

Studies using cell lines should always characterize these cells to ensure that the results are not distorted by unexpected morphological or genetic changes possibly due to culture time or passage number. Thus, the aim of this study was to describe those MIA PaCa-2 and PANC-1 cell line phenotype and genotype characteristics that may play a crucial role in pancreatic cancer therapeutic assays, namely neuroendocrine chemotherapy and peptide receptor radionuclide therapy. Epithelial, mesenchymal, endocrine and stem cell marker characterization was performed by immunohistochemistry and flow cytometry, and genotyping by PCR, gene sequencing and capillary electrophoresis. MIA PaCa-2 (polymorphism) expresses CK5.6, AE1/AE3, E-cadherin, vimentin, chromogranin A, synaptophysin, SSTR2 and NTR1 but not CD56. PANC-1 (pleomorphism) expresses CK5.6, MNF-116, vimentin, chromogranin A, CD56 and SSTR2 but not E-cadherin, synaptophysin or NTR1. MIA PaCA-1 is CD24(-), CD44(+/++), CD326(-/+) and CD133/1(-), while PANC-1 is CD24(-/+), CD44(+), CD326(-/+) and CD133/1(-). Both cell lines have KRAS and TP53 mutations and homozygous deletions including the first 3 exons of CDKN2A/p16(INK4A), but no SMAD4/DPC4 mutations or microsatellite instability. Both have neuroendocrine differentiation and SSTR2 receptors, precisely the features making them suitable for the therapies we propose to assay in future studies.

MicroRNA-126 deficiency enhanced the activation and function of CD4+ T cells by elevating IRS-1 pathway.

PubMed

Chu, F; Hu, Y; Zhou, Y; Guo, M; Lu, J; Zheng, W; Xu, H; Zhao, J; Xu, L

2018-02-01

Recent evidence has shown that microRNA-126 (miR-126) has been involved in the development and function of immune cells, which contributed to the pathogenesis of related clinical diseases. However, the potential role of miR-126 in the development and function of CD4 + T cells remains largely unknown. Here we first found that the activation and proliferation, as well as the expression of interferon (IFN)-γ, of CD4 + T cells from miR-126 knock-down (KD) mice using the miRNA-sponge technique were enhanced significantly in vitro, compared with those in CD4 + T cells from wild-type (WT) mice. To monitor further the possible effect of miR-126 deficiency on the function of CD4 + T cells in vivo, we used dextran sulphate sodium (DSS)-induced murine model of acute autoimmune colitis and found that miR-126 deficiency could elevate the pathology of colitis. Importantly, the proportion of CD4 + T cells in splenocytes increased significantly in miR-126KD mice. Moreover, the expression levels of CD69 and CD44 on CD4 + T cells increased significantly and the expression level of CD62L decreased significantly. Of note, adoptive cell transfer assay showed that the pathology of colitis was more serious in carboxyfluorescein succinimidyl ester (CFSE)-labelled miR-126KD CD4 + T cell-transferred group, compared with that in the CFSE-labelled WT CD4 + T cells transferred group. Consistently, the expression levels of CD69 and CD44 on CFSE + cells increased significantly. Furthermore, both the proliferation and IFN-γ secretion of CFSE + cells also increased significantly in the CFSE-labelled miR-126KD CD4 + T cell-transferred group. Mechanistic evidence showed that the expression of insulin receptor substrate 1 (IRS-1), as a functional target of miR-126, was elevated in CD4 + T cells from miR-126KD mice, accompanied by altered transduction of the extracellular regulated kinase, protein B (AKT) and nuclear factor kappa B (NF-κB) pathway. Our data revealed a novel role in which miR-126 was an intrinsic regulator in the function of CD4 + T cells, which provided preliminary basis for exploring further the role of miR-126 in the development, function of CD4 + T cells and related clinical diseases. © 2017 British Society for Immunology.

Biomarker-specific conjugated nanopolyplexes for the active coloring of stem-like cancer cells

NASA Astrophysics Data System (ADS)

Hong, Yoochan; Lee, Eugene; Choi, Jihye; Haam, Seungjoo; Suh, Jin-Suck; Yang, Jaemoon

2016-06-01

Stem-like cancer cells possess intrinsic features and their CD44 regulate redox balance in cancer cells to survive under stress conditions. Thus, we have fabricated biomarker-specific conjugated polyplexes using CD44-targetable hyaluronic acid and redox-sensible polyaniline based on a nanoemulsion method. For the most sensitive recognition of the cellular redox at a single nanoparticle scale, a nano-scattering spectrum imaging analyzer system was introduced. The conjugated polyplexes showed a specific targeting ability toward CD44-expressing cancer cells as well as a dramatic change in its color, which depended on the redox potential in the light-scattered images. Therefore, these polyaniline-based conjugated polyplexes as well as analytical processes that include light-scattering imaging and measurements of scattering spectra, clearly establish a systematic method for the detection and monitoring of cancer microenvironments.

The Na, K-ATPase β-Subunit Isoforms Expression in Glioblastoma Multiforme: Moonlighting Roles

PubMed Central

Rotoli, Deborah; Cejas, Mariana-Mayela; Maeso, María-del-Carmen; Pérez-Rodríguez, Natalia-Dolores; Morales, Manuel; Ávila, Julio

2017-01-01

Glioblastoma multiforme (GBM) is the most common form of malignant glioma. Recent studies point out that gliomas exploit ion channels and transporters, including Na, K-ATPase, to sustain their singular growth and invasion as they invade the brain parenchyma. Moreover, the different isoforms of the β-subunit of Na, K-ATPase have been implicated in regulating cellular dynamics, particularly during cancer progression. The aim of this study was to determine the Na, K-ATPase β subunit isoform subcellular expression patterns in all cell types responsible for microenvironment heterogeneity of GBM using immunohistochemical analysis. All three isoforms, β1, β2/AMOG (Adhesion Molecule On Glia) and β3, were found to be expressed in GBM samples. Generally, β1 isoform was not expressed by astrocytes, in both primary and secondary GBM, although other cell types (endothelial cells, pericytes, telocytes, macrophages) did express this isoform. β2/AMOG and β3 positive expression was observed in the cytoplasm, membrane and nuclear envelope of astrocytes and GFAP (Glial Fibrillary Acidic Protein) negative cells. Interestingly, differences in isoforms expression have been observed between primary and secondary GBM: in secondary GBM, β2 isoform expression in astrocytes was lower than that observed in primary GBM, while the expression of the β3 subunit was more intense. These changes in β subunit isoforms expression in GBM could be related to a different ionic handling, to a different relationship between astrocyte and neuron (β2/AMOG) and to changes in the moonlighting roles of Na, K-ATPase β subunits as adaptor proteins and transcription factors. PMID:29117147

Osteoblast gene expression is differentially regulated by TGF-beta isoforms.

PubMed

Fagenholz, P J; Warren, S M; Greenwald, J A; Bouletreau, P J; Spector, J A; Crisera, F E; Longaker, M T

2001-03-01

The transforming growth factor beta (TGF-beta) superfamily encompasses a number of important growth factors including several TGF-beta isoforms, the bone morphogenetic proteins, activins, inhibins, and growth and differentiation factors. TGF-beta 1, -beta 2, and -beta 3 are three closely related isoforms that are widely expressed during skeletal morphogenesis and bone repair. Numerous studies suggest that each isoform has unique in vivo functions; however, the effects of these TGF-beta isoforms on osteoblast gene expression and maturation have never been directly compared. In the current study, we treated undifferentiated neonatal rat calvaria osteoblast-enriched cell cultures with 2.5 ng/ml of each TGF-beta isoform and analyzed gene expression at 0, 3, 6, and 24 hours. We demonstrated unique isoform-specific regulation of endogenous TGF-beta 1 and type I collagen mRNA transcription. To assess the effects of extended TGF-beta treatment on osteoblast maturation, we differentiated osteoblast cultures in the presence of 2.5 ng/ml of each TGF-beta isoform. Analysis of collagen I, alkaline phosphatase, and osteocalcin demonstrated that each TGF-beta isoform uniquely suppressed the transcription of these osteoblast differentiation markers. Interestingly, TGF-beta isoform treatment increased osteopontin expression in primary osteoblasts after 4 and 10 days of differentiation. To our knowledge, these data provide the first direct comparison of the effects of the TGF-beta isoforms on osteoblast gene expression in vitro. Furthermore, these data suggest that TGF-beta isoforms may exert their unique in vivo effects by differentially regulating osteoblast cytokine secretion, extracellular matrix production, and the rate of cellular maturation.

Articular cartilage-derived cells hold a strong osteogenic differentiation potential in comparison to mesenchymal stem cells in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Salamon, Achim, E-mail: achim.salamon@med.uni-rostock.de; Jonitz-Heincke, Anika, E-mail: anika.jonitz@med.uni-rostock.de; Adam, Stefanie, E-mail: stefanie.adam@med.uni-rostock.de

Cartilaginous matrix-degenerative diseases like osteoarthritis (OA) are characterized by gradual cartilage erosion, and also by increased presence of cells with mesenchymal stem cell (MSC) character within the affected tissues. Moreover, primary chondrocytes long since are known to de-differentiate in vitro and to be chondrogenically re-differentiable. Since both findings appear to conflict with each other, we quantitatively assessed the mesenchymal differentiation potential of OA patient cartilage-derived cells (CDC) towards the osteogenic and adipogenic lineage in vitro and compared it to that of MSC isolated from adipose tissue (adMSC) of healthy donors. We analyzed expression of MSC markers CD29, CD44, CD105, andmore » CD166, and, following osteogenic and adipogenic induction in vitro, quantified their expression of osteogenic and adipogenic differentiation markers. Furthermore, CDC phenotype and proliferation were monitored. We found that CDC exhibit an MSC CD marker expression pattern similar to adMSC and a similar increase in proliferation rate during osteogenic differentiation. In contrast, the marked reduction of proliferation observed during adipogenic differentiation of adMSC was absent in CDC. Quantification of differentiation markers revealed a strong osteogenic differentiation potential for CDC, however almost no capacity for adipogenic differentiation. Since in the pathogenesis of OA, cartilage degeneration coincides with high bone turnover rates, the high osteogenic differentiation potential of OA patient-derived CDC may affect clinical therapeutic regimens aiming at autologous cartilage regeneration in these patients. - Highlights: • We analyze the mesenchymal differentiation capacity of cartilage-derived cells (CDC). • CDC express mesenchymal stem cell (MSC) markers CD29, CD44, CD105, and CD166. • CDC and MSC proliferation is reduced in adipogenesis and increased in osteogenesis. • Adipogenic differentiation is virtually absent in CDC, but strong in MSC. • Osteogenic differentiation is significantly stronger for CDC than for MSC.« less

NHS-A isoform of the NHS gene is a novel interactor of ZO-1.

PubMed

Sharma, Shiwani; Koh, Katrina S Y; Collin, Caitlin; Dave, Alpana; McMellon, Amy; Sugiyama, Yuki; McAvoy, John W; Voss, Anne K; Gécz, Jozef; Craig, Jamie E

2009-08-15

Mutations in the NHS (Nance-Horan Syndrome) gene lead to severe congenital cataracts, dental defects and sometimes mental retardation. NHS encodes two protein isoforms, NHS-A and -1A that display cell-type dependent differential expression and localization. Here we demonstrate that of these two isoforms, the NHS-A isoform associates with the cell membrane in the presence of intercellular contacts and it immunoprecipitates with the tight junction protein ZO-1 in MDCK (Madin Darby Canine Kidney) epithelial cells and in neonatal rat lens. The NHS-1A isoform however is a cytoplasmic protein. Both Nhs isoforms are expressed during mouse development. Immunolabelling of developing mouse with the anti-NHS antibody that detects both isoforms revealed the protein in the developing head including the eye and brain. It was primarily expressed in epithelium including neural epithelium and certain vascular endothelium but only weakly expressed in mesenchymal cells. In the epithelium and vascular endothelium the protein associated with the cell membrane and co-localized with ZO-1, which indirectly indicates expression of the Nhs-A isoform in these structures. Membrane localization of the protein in the lens vesicle similarly supports Nhs-A expression. In conclusion, the NHS-A isoform of NHS is a novel interactor of ZO-1 and may have a role at tight junctions. This isoform is important in mammalian development especially of the organs in the head.

Identification of alternatively spliced isoforms of interleukin-2/15 receptor β chain in ducks.

PubMed

Jeong, Jipseol; Kim, Woo H; Yeo, Jaeseung; Fernandez, Cherry P; Kim, Suk; Lee, Youn-Jeong; Lillehoj, Hyun S; Min, Wongi

2014-12-15

Interleukin (IL)-2 and IL-15 receptor β (IL-2/15Rβ, CD122) play important roles in signal transduction for biological functions of IL-2 and IL-15. We found that ducks possess three different IL-2/15Rβ transcripts, a conventional form (duIL-2/15Rβ) and two variants. Comparisons between the cDNA and genomic sequences revealed that the two variants, duIL-2/15Rβ-d7 and duIL-2/15Rβ-d9, were novel spliced transcripts resulting from skipping exons 7 and 9, respectively. Expression profiles of duIL-2/15Rβ and its isoforms were examined in healthy tissues, concanavalin A (ConA)-stimulated splenic lymphocytes and in livers and spleens of Riemerella anatipestifer-infected ducks using quantitative real-time PCR (qRT-PCR). Generally, duIL-2/15Rβ-d9 expression was undetectable in healthy tissues, ConA-activated samples, and R. anatipestifer-infected ducks. Expression levels of duIL-2/15Rβ transcript were relatively high to moderate in all healthy tissues tested, while duIL-2/15Rβ-d7 expression was low. Compared to untreated controls, expression levels of duIL-2/15Rβ were elevated in ConA-activated splenic lymphocytes and in livers on day 7 in R. anatipestifer-infected ducks, while duIL-2/15Rβ-d7 expression was unchanged. Additionally, COS-7 cells transfected with duIL-2/15Rβ, duIL-2/15Rβ-d7, or duIL-2/15Rβ-d9 constructs generated 73 kilodalton (kDa), 31kDa, and 40kDa proteins, respectively. This study identified three different IL-2/15Rβ transcripts, including two isoforms generated by alternative splicing and their gene expression patterns in stimulated conditions. Copyright © 2014 Elsevier B.V. All rights reserved.

Cellular Fibronectin Expression in Human Trabecular Meshwork and Induction by Transforming Growth Factor-β2

PubMed Central

Medina-Ortiz, Wanda E.; Belmares, Ricardo; Neubauer, Sandra; Wordinger, Robert J.; Clark, Abbot F.

2013-01-01

Purpose. Levels of TGF-β2 are higher in POAG aqueous humor, causing deposition of extracellular matrix (ECM) proteins, including fibronectin (FN), in the glaucomatous human trabecular meshwork (HTM) that may be responsible for elevated IOP. The purpose of this study was to identify the expression of cellular FN (cFN) isoforms (EDA and EDB) in HTM cells and tissues, and to determine whether TGF-β2 can induce cFN expression and fibril formation in cultured HTM cells. Methods. Expression of cFN mRNA isoforms and induction by recombinant TGF-β2 (5 ng/mL) were determined by quantitative RT-PCR. The TGF-β2 induction of EDA isoform protein expression and FN fibril formation were analyzed using Western immunoblots and immunocytochemistry (ICC), respectively. Immunohistochemistry (IHC) analysis was used to examine total FN and EDA isoform expression in normal (NTM) and glaucomatous (GTM) trabecular meshwork (TM) tissues. Results. Both cFN mRNA isoforms were expressed in cultured HTM cells and were induced by TGF-β2 after 2, 4, and 7 days (P < 0.05). Similarly, EDA isoform protein and fibril formation were increased after 4 and 7 days of TGF-β2 treatment. Finally, GTM tissues had significantly greater EDA isoform protein levels (1.7-fold, P < 0.05) compared to NTM tissues. Conclusions. This study demonstrated that cFN isoforms are expressed and induced in HTM cells by TGF-β2. Also, increased EDA isoform protein levels were seen in GTM tissues. Our findings suggest that induction of cFN isoform expression in the TM ECM may be a novel pathologic mechanism involved in the TM changes associated with glaucoma. PMID:24030464

Three-Dimensional Cellular Arrangement in Epithelial Ovarian Cancer Cell Lines TOV-21G and SKOV-3 is Associated with Apoptosis-Related miRNA Expression Modulation.

PubMed

de Lima, Aline Brito; Silva, Luciana Maria; Gonçales, Nikole Gontijo; Carvalho, Maria Raquel Santos; da Silva Filho, Agnaldo Lopes; da Conceição Braga, Letícia

2018-01-06

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy, and the lack of chemoresistance biomarkers contributes to the poor prognosis. Cancer stem cells (CSC) have been investigated in EOC to understand its relationship with chemoresistance and recurrence. In this context, in vitro cultivation-models are important tools for CSC studies. MicroRNAs (miRNAs) play key roles in cancer, CSC regulation and apoptosis. Thus, this study aims to evaluate the tumorsphere model as CSC-enrichment method in EOC studies and investigate apoptosis-related miRNAs in tumorspheres-derived EOC cell lines. TOV-21G and SKOV-3 were cultured in monolayer and tumorspheres. Genetic profiles of cell lines were obtained using COSMIC database. CD24/CD44/CD146/CD177 and ALDH1 markers were evaluated in cell lines and tumorspheres-derived by flow cytometry. Eleven miRNAs were selected by in silico analysis for qPCR analysis. According to COSMIC, TOV-21G and SKOV-3 have eight and nine cancer-related mutations, respectively. TOV-21G showed a CD44 +/high /CD24 -/low /CD117 -/low /CD146 -/low /ALDH1 low profile in both culture models; thus, no significant difference between cultivation models was identified. SKOV-3 showed a CD44 +/high /CD24 +/high / CD117 -/low /CD146 -/low /ALDH1 low profile in both culture models, although the tumorsphere model showed a significant increase in CD24 +/high subpopulation (ovarian CSC-like). Among eleven miRNAs, we observed differences in miRNA expression between culture models. MiR-26a was overexpressed in TOV-21G tumorspheres, albeit downregulated in SKOV-3 tumorspheres. MiR-125b-5p, miR-17-5p and miR-221 was downregulated in tumorsphere model in both cell lines. Given that tumorsphere-derived SKOV-3 had a higher ratio of CD24 +/high cells, we suggest that miR-26a, miR-125b-5p, miR-17-5p and miR-221 downregulation could be related to poor EOC prognosis.

Flow cytometric characterization of culture expanded multipotent mesenchymal stromal cells (MSCs) from horse adipose tissue: towards the definition of minimal stemness criteria.

PubMed

Pascucci, L; Curina, G; Mercati, F; Marini, C; Dall'Aglio, C; Paternesi, B; Ceccarelli, P

2011-12-15

In the last decades, multipotent mesenchymal progenitor cells have been isolated from many adult tissues of different species. The International Society for Cellular Therapy (ISCT) has recently established that multipotent mesenchymal stromal cells (MSCs) is the currently recommended designation. In this study, we used flow cytometry to evaluate the expression of several molecules related to stemness (CD90, CD44, CD73 and STRO-1) in undifferentiated, early-passaged MSCs isolated from adipose tissue of four donor horses (AdMSCs). The four populations unanimously expressed high levels of CD90 and CD44. On the contrary, they were unexpectedly negative to CD73. A small percentage of the cells, finally, showed the expression of STRO-1. This last result might be due to the existence of a small subpopulation of STRO-1+ cells or to a poor cross-reactivity of the antibody. A remarkable donor-to-donor consistency and reproducibility of these findings was demonstrated. The data presented herein support the idea that equine AdMSCs may be easily isolated and selected by adherence to tissue culture plastic and exhibit a surface profile characterized by some peculiar differences in comparison to those described in other species. Continued characterization of these cells will help to clarify several aspects of their biology and may ultimately enable the isolation of specific, purified subpopulations. Copyright © 2011 Elsevier B.V. All rights reserved.

Notch-1 induces Epithelial-mesenchymal transition consistent with cancer stem cell phenotype in pancreatic cancer cells

PubMed Central

Bao, Bin; Wang, Zhiwei; Ali, Shadan; Kong, Dejuan; Li, Yiwei; Ahmad, Aamir; Banerjee, Sanjeev; Azmi, Asfar S.; Miele, Lucio; Sarkar, Fazlul H.

2011-01-01

Activation of Notch-1 is known to be associated with the development and progression of human malignancies including pancreatic cancer. Emerging evidence suggest that the acquisition of epithelial-mesenchymal transition (EMT) phenotype and induction of cancer stem cell (CSC) or cancer stem-like cell phenotype are interrelated and contributes to tumor recurrence and drug resistance. The molecular mechanism(s) by which Notch-1 contributes to the acquisition of EMT phenotype and CSC self-renewal capacity has not been fully elucidated. Here we show that forced over-expression of Notch-1 leads to increased cell growth, clonogenicity, migration and invasion of AsPC-1 cells. Moreover, over-expression of Notch-1 led to the induction of EMT phenotype by activation of mesenchymal cell markers such as ZEB1, CD44, EpCAM, and Hes 1. Here we also report, for the first time, that over-expression of Notch-1 leads to increased expression of miR-21, and decreased expression of miR-200b, miR-200c, let-7a, let-7b, and let-7c. Re-expression of miR-200b led to decreased expression of ZEB1, and vimentin, and increased expression of E-cadherin. Over-expression of Notch-1 also increased the formation of pancreatospheres consistent with expression of CSC surface markers CD44 and EpCAM. Finally, we found that genistein, a known natural anti-tumor agent inhibited cell growth, clonogenicity, migration, invasion, EMT phenotype, formation of pancreatospheres and expression of CD44 and EpCAM. These results suggest that the activation of Notch-1 signaling contributes to the acquisition of EMT phenotype, which is in part mediated through the regulation of miR-200b and CSC self-renewal capacity, and these processes could be attenuated by genistein treatment. PMID:21463919

Isoform-selective induction of human p110δ PI3K expression by TNFα: identification of a new and inducible PIK3CD promoter

PubMed Central

Whitehead, Maria A.; Bombardieri, Michele; Pitzalis, Costantino; Vanhaesebroeck, Bart

2012-01-01

PI3Ks (phosphoinositide 3-kinases) are signalling molecules and drug targets with important biological functions, yet the regulation of PI3K gene expression is poorly understood. Key PI3Ks are the class IA PI3Ks that consist of a catalytic subunit (p110α, p110β and p110δ) in complex with a p85 regulatory subunit. Whereas p110α and p110β are ubiquitously expressed, high levels of p110δ are mainly found in white blood cells, with most non-leucocytes expressing low levels of p110δ. In the present paper we report that TNFα (tumour necrosis factor α) stimulation induces p110δ expression in human ECs (endothelial cells) and synovial fibroblasts, but not in leucocytes, through transcription start sites located in a novel promoter region in the p110δ gene (PIK3CD). This promoter is used in all cell types, including solid tumour cell lines that express p110δ, and is activated by TNFα in ECs and synovial fibroblasts. We further present a detailed biochemical and bioinformatic characterization of p110δ gene regulation, demonstrating that PIK3CD has distinct promoters, some of which can be dynamically activated by pro-inflammatory mediators. This is the first molecular identification of a PI3K promoter under the control of acute extracellular stimulation. PMID:22375552

P1 promoter-driven HNF4α isoforms are specifically repressed by β-catenin signaling in colorectal cancer cells.

PubMed

Babeu, Jean-Philippe; Jones, Christine; Geha, Sameh; Carrier, Julie C; Boudreau, François

2018-06-13

HNF4α is a key nuclear receptor for regulating gene expression in the gut. While both P1 and P2 isoform classes of HNF4α are expressed in colonic epithelium, specific inhibition of P1 isoforms is commonly found in colorectal cancer. Previous studies have suggested that P1 and P2 isoforms may regulate different cellular functions. Despite these advances, it remains unclear whether these isoform classes are functionally divergent in the context of human biology. Here, the consequences of specific inhibition of P1 or P2 isoform expression was measured in a human colorectal cancer cell transcriptome. Results indicate that P1 isoforms were specifically associated with the control of cell metabolism while P2 isoforms globally supported aberrant oncogenic signalization, promoting cancer cell survival and progression. P1 promoter-driven isoform expression was found to be repressed by β-catenin, one of the earliest oncogenic pathways to be activated during colon tumorigenesis. These findings identify a novel cascade by which the expression of P1 isoforms are rapidly shut down in the early stages of colon tumorigenesis, allowing a change in HNF4α-dependent transcriptome thereby promoting colorectal cancer progression. © 2018. Published by The Company of Biologists Ltd.

miR-125b Functions as a Key Mediator for Snail-induced Stem Cell Propagation and Chemoresistance*

PubMed Central

Liu, Zixing; Liu, Hao; Desai, Shruti; Schmitt, David C.; Zhou, Ming; Khong, Hung T.; Klos, Kristine S.; McClellan, Steven; Fodstad, Oystein; Tan, Ming

2013-01-01

Chemoresistance is a major obstacle in cancer treatment. Our previous studies have shown that miR-125b plays an important role in chemoresistance. Here we report a novel mechanism that up-regulation of miR-125b through Wnt signaling by Snail enriches cancer stem cells. Overexpression of Snail dramatically increases the expression of miR-125b through the Snail-activated Wnt/β-catenin/TCF4 axis. Snail confers chemoresistance by repressing Bak1 through up-regulation of miR-125b. Restoring the expression of Bak1 or depleting miR-125b re-sensitizes Snail-expressing cancer cells to Taxol, indicating that miR-125b is critical in Snail-induced chemoresistance. Moreover, overexpression of miR-125b significantly increases the cancer stem cell population (CD24-CD44+), while depletion of miR-125b or rescue of the expression of Bak1 increases the non-stem cell population (CD24+CD44+) in Snail-overexpressing cells. These findings strongly support that miR-125b functions as a key mediator in Snail-induced cancer stem cell enrichment and chemoresistance. This novel mechanism for Snail-induced stem cell propagation and chemoresistance may have important implications in the development of strategies for overcoming cancer cell resistance to chemotherapy. PMID:23255607

SHBG Is an Important Factor in Stemness Induction of Cells by DHT In Vitro and Associated with Poor Clinical Features of Prostate Carcinomas

PubMed Central

Ma, Yuanyuan; Liang, Dongming; Liu, Jian; Wen, Jian-Guo; Servoll, Einar; Waaler, Gudmund; Sæter, Thorstein; Axcrona, Karol; Vlatkovic, Ljiljana; Axcrona, Ulrika; Paus, Elisabeth; Yang, Yue; Zhang, Zhiqian; Kvalheim, Gunnar; Nesland, Jahn M.; Suo, Zhenhe

2013-01-01

Androgen plays a vital role in prostate cancer development. However, it is not clear whether androgens influence stem-like properties of prostate cancer, a feature important for prostate cancer progression. In this study, we show that upon DHT treatment in vitro, prostate cancer cell lines LNCaP and PC-3 were revealed with higher clonogenic potential and higher expression levels of stemness related factors CD44, CD90, Oct3/4 and Nanog. Moreover, sex hormone binding globulin (SHBG) was also simultaneously upregulated in these cells. When the SHBG gene was blocked by SHBG siRNA knock-down, the induction of Oct3/4, Nanog, CD44 and CD90 by DHT was also correspondingly blocked in these cells. Immunohistochemical evaluation of clinical samples disclosed weakly positive, and areas negative for SHBG expression in the benign prostate tissues, while most of the prostate carcinomas were strongly positive for SHBG. In addition, higher levels of SHBG expression were significantly associated with higher Gleason score, more seminal vesicle invasions and lymph node metastases. Collectively, our results show a role of SHBG in upregulating stemness of prostate cancer cells upon DHT exposure in vitro, and SHBG expression in prostate cancer samples is significantly associated with poor clinicopathological features, indicating a role of SHBG in prostate cancer progression. PMID:23936228

FGFR signaling regulates resistance of head and neck cancer stem cells to cisplatin.

PubMed

McDermott, Sarah C; Rodriguez-Ramirez, Christie; McDermott, Sean P; Wicha, Max S; Nör, Jacques E

2018-05-18

Patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) have poor prognosis with less than 1-year median survival. Platinum-based chemotherapy remains the first-line treatment for HNSCC. The cancer stem cell (CSC) hypothesis postulates that tumors are maintained by a self-renewing CSC population that is also capable of differentiating into non-self renewing cell populations that constitute the bulk of the tumor. A small population of CSC exists within HNSCC that are relatively resistant to chemotherapy and clinically predicted to contribute to tumor recurrence. These head and neck CSCs (HNCSC) are identified by high cell-surface expression of CD44 and high intracellular activity of aldehyde dehydrogenase (ALDH) and termed ALDH high CD44 high . Here, we performed microarray analysis in two HNSCC cell lines (UM-SCC-1, UM-SCC-22B) to investigate molecular pathways active in untreated and cisplatin-resistant ALDH high CD44 high cells. Gene set enrichment analysis and iPathway analysis identified signaling pathways with major implications to the pathobiology of cancer (e.g. TNFα, IFN, IL6/STAT, NF-κB) that are enriched in cisplatin-resistant ALDH high CD44 high cells, when compared to control cells. FGF2 was also enriched in cisplatin-resistant ALDH high CD44 high , which was confirmed by ELISA analysis. Inhibition of FGF signaling using BGJ398, a pan-FGF receptor (FGFR) small-molecule inhibitor, decreased ALDH high CD44 high alone in UM-SCC-1 and preferentially targeted cisplatin-resistant ALDH high CD44 high cells in UM-SCC-22B. These findings suggest that FGFR signaling might play an important role in the resistance of head and neck CSC to cisplatin. Collectively, this work suggests that some head and neck cancer patients might benefit from the combination of cisplatin and a FGFR inhibitor.

[Correlation of CD82 and hTERT expressions and HPV infection with penile cancer].

PubMed

Zhai, Jian-Po; Li, Ming; Wang, Qi-Yan; Wei, Dong; Xu, Ke-Xin

2011-09-01

To study the correlation of the expressions of CD82 and hTERT and HPV infection with the clinical pathological features of penile cancer and identify their prognostic significance in the lymphatic metastasis of the disease. A total of 44 patients underwent partial or radical penectomy and lymph node dissection. The expressions of CD82 and hTERT were determined by immunohistochemistry, and HPV infection was detected by PCR. The positive rates of CD82, hTERT, and HPV DNA in penile carcinoma were 47.7%, 38.6% and 25.9%, respectively. The amplified HPV DNA was HPV-16. The pathological stage and hTERT expression were positively correlated with inguinal lymph node metastasis of penile cancer (P = 0.032, P = 0.041), and so was the pathological stage with the expression of CD82 (P = 0.045), but neither the pathological stage, nor the expression of CD82 or the positive rate of HPV DNA showed any correlation with lymph node metastasis (P = 0.627, P = 0.094, P = 0.633). The pathological grade and hTERT expression are independent prognostic factors for lymph node metastasis in penile carcinoma. These features help the prognosis and identification of the patient at the risk of nodal metastasis.

First Trimester Pregnancy Loss and the Expression of Alternatively Spliced NKp30 Isoforms in Maternal Blood and Placental Tissue

PubMed Central

Shemesh, Avishai; Tirosh, Dan; Sheiner, Eyal; Benshalom-Tirosh, Neta; Brusilovsky, Michael; Segev, Rotem; Rosental, Benyamin; Porgador, Angel

2015-01-01

Capsule: We observed that first trimester pregnancy loss is associated with an altered expression profile of the three isoforms of the NK receptor NKp30 expressed by NKs in PBMC and placental tissue. In this study, we aimed to investigate whether first trimester pregnancy loss is associated with differences in expression of NKp30 splice variants (isoforms) in maternal peripheral blood or placental tissue. We conducted a prospective case–control study; a total of 33 women undergoing dilation and curettage due to first trimester pregnancy loss were further subdivided into groups with sporadic or recurrent pregnancy loss. The control group comprises women undergoing elective termination of pregnancy. The qPCR approach was employed to assess the relative expression of NKp30 isoforms as well as the total expression of NKp30 and NKp46 receptors between the selected groups. Results show that in both PBMC and placental tissue, NKp46 and NKp30 expressions were mildly elevated in the pregnancy loss groups compared with the elective group. In particular, NKp46 elevation was significant. Moreover, expression analysis of NKp30 isoforms manifested a different profile between PBMC and the placenta. NKp30-a and NKp30-b isoforms in the placental tissue, but not in PBMC, showed a significant increase in the pregnancy loss groups compared with the elective group. Placental expression of NKp30 activating isoforms-a and -b in the pregnancy loss groups was negatively correlated with PLGF expression. By contrast, placental expression of these isoforms in the elective group was positively correlated with TNFα, IL-10, and VEGF-A expression. The altered expression of NKp30 activating isoforms in placental tissue from patients with pregnancy loss compared to the elective group and the different correlations with cytokine expression point to the involvement of NKp30-mediated function in pregnancy loss. PMID:26082773

Cadmium-induced ethylene production and responses in Arabidopsis thaliana rely on ACS2 and ACS6 gene expression

PubMed Central

2014-01-01

Background Anthropogenic activities cause metal pollution worldwide. Plants can absorb and accumulate these metals through their root system, inducing stress as a result of excess metal concentrations inside the plant. Ethylene is a regulator of multiple plant processes, and is affected by many biotic and abiotic stresses. Increased ethylene levels have been observed after exposure to excess metals but it remains unclear how the increased ethylene levels are achieved at the molecular level. In this study, the effects of cadmium (Cd) exposure on the production of ethylene and its precursor 1-aminocyclopropane-1-carboxylic acid (ACC), and on the expression of the ACC Synthase (ACS) and ACC Oxidase (ACO) multigene families were investigated in Arabidopsis thaliana. Results Increased ethylene release after Cd exposure was directly measurable in a system using rockwool-cultivated plants; enhanced levels of the ethylene precursor ACC together with higher mRNA levels of ethylene responsive genes: ACO2, ETR2 and ERF1 also indicated increased ethylene production in hydroponic culture. Regarding underlying mechanisms, it was found that the transcript levels of ACO2 and ACO4, the most abundantly expressed members of the ACO multigene family, were increased upon Cd exposure. ACC synthesis is the rate-limiting step in ethylene biosynthesis, and transcript levels of both ACS2 and ACS6 showed the highest increase and became the most abundant isoforms after Cd exposure, suggesting their importance in the Cd-induced increase of ethylene production. Conclusions Cadmium induced the biosynthesis of ACC and ethylene in Arabidopsis thaliana plants mainly via the increased expression of ACS2 and ACS6. This was confirmed in the acs2-1acs6-1 double knockout mutants, which showed a decreased ethylene production, positively affecting leaf biomass and resulting in a delayed induction of ethylene responsive gene expressions without significant differences in Cd contents between wild-type and mutant plants. PMID:25082369

Microenvironments in tuberculous granulomas are delineated by distinct populations of macrophage subsets and expression of nitric oxide synthase and arginase isoforms

PubMed Central

Mattila, Joshua T.; Ojo, Olabisi O.; Kepka-Lenhart, Diane; Marino, Simeone; Kim, Jin Hee; Eum, Seok Yong; Via, Laura E.; Barry, Clifton E.; Klein, Edwin; Kirschner, Denise E.; Morris, Sidney M.; Lin, Philana Ling; Flynn, JoAnne L.

2013-01-01

Macrophages in granulomas are both anti-mycobacterial effector and host cell for Mycobacterium tuberculosis(M.tb), yet basic aspects of macrophage diversity and function within the complex structures of granulomas remain poorly understood. To address this, we examined myeloid cell phenotypes and expression of enzymes correlated with host defense in macaque and human granulomas. Macaque granulomas had upregulated inducible and endothelial nitric oxide synthase (iNOS and eNOS) and arginase (Arg1 and Arg2) expression and enzyme activity compared to non-granulomatous tissue. Immunohistochemical analysis indicated macrophages adjacent to uninvolved normal tissue were more likely to express CD163, while epithelioid macrophages in regions where bacteria reside strongly expressed CD11c, CD68 and HAM56. Calprotectin-positive neutrophils were abundant in regions adjacent to caseum. iNOS, eNOS, Arg1 and Arg2 proteins were identified in macrophages and localized similarly in granulomas across species, with greater eNOS expression and ratio of iNOS:Arg1 expression in epithelioid macrophages, as compared to cells in the lymphocyte cuff. iNOS, Arg1 and Arg2 expression in neutrophils was also identified. The combination of phenotypic and functional markers support that macrophages with anti-inflammatory phenotypes localized to outer regions of granulomas while the inner regions were more likely to contain macrophages with pro-inflammatory, presumably bactericidal, phenotypes. Together these data support the concept that granulomas have organized microenvironments that balance anti-microbial anti-inflammatory responses to limit pathology in the lungs. PMID:23749634

DEIsoM: a hierarchical Bayesian model for identifying differentially expressed isoforms using biological replicates

PubMed Central

Peng, Hao; Yang, Yifan; Zhe, Shandian; Wang, Jian; Gribskov, Michael; Qi, Yuan

2017-01-01

Abstract Motivation High-throughput mRNA sequencing (RNA-Seq) is a powerful tool for quantifying gene expression. Identification of transcript isoforms that are differentially expressed in different conditions, such as in patients and healthy subjects, can provide insights into the molecular basis of diseases. Current transcript quantification approaches, however, do not take advantage of the shared information in the biological replicates, potentially decreasing sensitivity and accuracy. Results We present a novel hierarchical Bayesian model called Differentially Expressed Isoform detection from Multiple biological replicates (DEIsoM) for identifying differentially expressed (DE) isoforms from multiple biological replicates representing two conditions, e.g. multiple samples from healthy and diseased subjects. DEIsoM first estimates isoform expression within each condition by (1) capturing common patterns from sample replicates while allowing individual differences, and (2) modeling the uncertainty introduced by ambiguous read mapping in each replicate. Specifically, we introduce a Dirichlet prior distribution to capture the common expression pattern of replicates from the same condition, and treat the isoform expression of individual replicates as samples from this distribution. Ambiguous read mapping is modeled as a multinomial distribution, and ambiguous reads are assigned to the most probable isoform in each replicate. Additionally, DEIsoM couples an efficient variational inference and a post-analysis method to improve the accuracy and speed of identification of DE isoforms over alternative methods. Application of DEIsoM to an hepatocellular carcinoma (HCC) dataset identifies biologically relevant DE isoforms. The relevance of these genes/isoforms to HCC are supported by principal component analysis (PCA), read coverage visualization, and the biological literature. Availability and implementation The software is available at https://github.com/hao-peng/DEIsoM Contact pengh@alumni.purdue.edu Supplementary information Supplementary data are available at Bioinformatics online. PMID:28595376

Tumor associated CD70 expression is involved in promoting tumor migration and macrophage infiltration in GBM.

PubMed

Ge, Haitao; Mu, Luyan; Jin, Linchun; Yang, Changlin; Chang, Yifan Emily; Long, Yu; DeLeon, Gabriel; Deleyrolle, Loic; Mitchell, Duane A; Kubilis, Paul S; Lu, Dunyue; Qi, Jiping; Gu, Yunhe; Lin, Zhiguo; Huang, Jianping

2017-10-01

Tumor migration/metastasis and immunosuppression are major obstacles in effective cancer therapy. Incidentally, these 2 hurdles usually coexist inside tumors, therefore making therapy significantly more complicated, as both oncogenic mechanisms must be addressed for successful therapeutic intervention. Our recent report highlights that the tumor expression of a TNF family member, CD70, is correlated with poor survival for primary gliomas. In this study, we investigated how CD70 expression by GBM affects the characteristics of tumor cells and the tumor microenvironment. We found that the ablation of CD70 in primary GBM decreased CD44 and SOX2 gene expression, and inhibited tumor migration, growth and the ability to attract monocyte-derived M2 macrophages in vitro. In the tumor microenvironment, CD70 was associated with immune cell infiltrates, such as T cells; myeloid-derived suppressor cells; and monocytes/macrophages based on the RNA-sequencing profile. The CD163+ macrophages were far more abundant than T cells were. This overwhelming level of macrophages was identified only in GBM and not in low-grade gliomas and normal brain specimens, implying their tumor association. CD70 was detected only on tumor cells, not on macrophages, and was highly correlated with CD163 gene expression in primary GBM. Additionally, the co-expression of the CD70 and CD163 genes was found to correlate with decreased survival for patients with primary GBM. Together, these data suggest that CD70 expression is involved in promoting tumor aggressiveness and immunosuppression via tumor-associated macrophage recruitment/activation. Our current efforts to target this molecule using chimeric antigen receptor T cells hold great potential for treating patients with GBM. © 2017 UICC.

Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS

PubMed Central

Doublier, Sophie; Zennaro, Cristina; Musante, Luca; Spatola, Tiziana; Candiano, Giovanni; Bruschi, Maurizio; Besso, Luca; Cedrino, Massimo; Carraro, Michele; Ghiggeri, Gian Marco; Camussi, Giovanni

2017-01-01

CD40/CD40 ligand (CD40L) dyad, a co-stimulatory bi-molecular complex involved in the adaptive immune response, has also potent pro-inflammatory actions in haematopoietic and non-haematopoietic cells. We describe here a novel role for soluble CD40L (sCD40L) as modifier of glomerular permselectivity directly acting on glomerular epithelial cells (GECs). We found that stimulation of CD40, constitutively expressed on GEC cell membrane, by the sCD40L rapidly induced redistribution and loss of nephrin in GECs, and increased albumin permeability in isolated rat glomeruli. Pre-treatment with inhibitors of CD40-CD40L interaction completely prevented these effects. Furthermore, in vivo injection of sCD40L induced a significant reduction of nephrin and podocin expression in mouse glomeruli, although no significant increase of urine protein/creatinine ratio was observed after in vivo injection. The same effects were induced by plasma factors partially purified from post-transplant plasma exchange eluates of patients with focal segmental glomerulosclerosis (FSGS), and were blocked by CD40-CD40L inhibitors. Moreover, 17 and 34 kDa sCD40L isoforms were detected in the same plasmapheresis eluates by Western blotting. Finally, the levels of sCD40Lwere significantly increased in serum of children both with steroid-sensitive and steroid-resistant nephrotic syndrome (NS), and in adult patients with biopsy-proven FSGS, compared to healthy subjects, but neither in children with congenital NS nor in patients with membranous nephropathy. Our results demonstrate that sCD40L directly modifies nephrin and podocin distribution in GECs. Moreover, they suggest that sCD40L contained in plasmapheresis eluates from FSGS patients with post-transplant recurrence may contribute, presumably cooperating with other mediators, to FSGS pathogenesis by modulating glomerular permeability. PMID:29155846

Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS.

PubMed

Doublier, Sophie; Zennaro, Cristina; Musante, Luca; Spatola, Tiziana; Candiano, Giovanni; Bruschi, Maurizio; Besso, Luca; Cedrino, Massimo; Carraro, Michele; Ghiggeri, Gian Marco; Camussi, Giovanni; Lupia, Enrico

2017-01-01

CD40/CD40 ligand (CD40L) dyad, a co-stimulatory bi-molecular complex involved in the adaptive immune response, has also potent pro-inflammatory actions in haematopoietic and non-haematopoietic cells. We describe here a novel role for soluble CD40L (sCD40L) as modifier of glomerular permselectivity directly acting on glomerular epithelial cells (GECs). We found that stimulation of CD40, constitutively expressed on GEC cell membrane, by the sCD40L rapidly induced redistribution and loss of nephrin in GECs, and increased albumin permeability in isolated rat glomeruli. Pre-treatment with inhibitors of CD40-CD40L interaction completely prevented these effects. Furthermore, in vivo injection of sCD40L induced a significant reduction of nephrin and podocin expression in mouse glomeruli, although no significant increase of urine protein/creatinine ratio was observed after in vivo injection. The same effects were induced by plasma factors partially purified from post-transplant plasma exchange eluates of patients with focal segmental glomerulosclerosis (FSGS), and were blocked by CD40-CD40L inhibitors. Moreover, 17 and 34 kDa sCD40L isoforms were detected in the same plasmapheresis eluates by Western blotting. Finally, the levels of sCD40Lwere significantly increased in serum of children both with steroid-sensitive and steroid-resistant nephrotic syndrome (NS), and in adult patients with biopsy-proven FSGS, compared to healthy subjects, but neither in children with congenital NS nor in patients with membranous nephropathy. Our results demonstrate that sCD40L directly modifies nephrin and podocin distribution in GECs. Moreover, they suggest that sCD40L contained in plasmapheresis eluates from FSGS patients with post-transplant recurrence may contribute, presumably cooperating with other mediators, to FSGS pathogenesis by modulating glomerular permeability.

Immunophenotypic characterization and tenogenic differentiation of mesenchymal stromal cells isolated from equine umbilical cord blood.

PubMed

Mohanty, Niharika; Gulati, Baldev R; Kumar, Rajesh; Gera, Sandeep; Kumar, Pawan; Somasundaram, Rajesh K; Kumar, Sandeep

2014-06-01

Mesenchymal stem cells (MSCs) isolated from umbilical cord blood (UCB) in equines have not been well characterized with respect to the expression of pluripotency and mesenchymal markers and for tenogenic differentiation potential in vitro. The plastic adherent fibroblast-like cells isolated from 13 out of 20 UCB samples could proliferate till passage 20. The cells expressed pluripotency markers (OCT4, NANOG, and SOX2) and MSC surface markers (CD90, CD73, and CD105) by RT-PCR, but did not express CD34, CD45, and CD14. On immunocytochemistry, the isolated cells showed expression of CD90 and CD73 proteins, but tested negative for CD34 and CD45. In flow cytometry, CD29, CD44, CD73, and CD90 were expressed by 96.36 ± 1.28%, 93.40 ± 0.70%, 73.23 ± 1.29% and 46.75 ± 3.95% cells, respectively. The UCB-MSCs could be differentiated to tenocytes by culturing in growth medium supplemented with 50 ng/ml of BMP-12 by day 10. The differentiated cells showed the expression of mohawk homeobox (Mkx), collagen type I alpha 1 (Col1α1), scleraxis (Scx), tenomodulin (Tnmd) and decorin (Dcn) by RT-PCR. In addition, flow cytometry detected tenomodulin and decorin protein in 95.65 ± 2.15% and 96.30 ± 1.00% of differentiated cells in comparison to 11.30 ± 0.10% and 19.45 ± 0.55% cells, respect vely in undifferentiated control cells. The findings support the observation that these cells may be suitable for therapeutic applications, including ruptured tendons in racehorses.

Tissue Non-specific Alkaline Phosphatase Expression is Needed for the Full Stimulation of T Cells and T Cell-Dependent Colitis.

PubMed

Hernández-Chirlaque, Cristina; Gámez-Belmonte, Reyes; Ocón, Borja; Martínez-Moya, Patricia; Wirtz, Stefan; Sánchez de Medina, Fermín; Martínez-Augustin, Olga

2017-07-01

Two alkaline phosphatase isoforms, intestinal [IAP] and tissue non-specific alkaline phosphatase [TNAP], are coexpressed in mouse colon, with the latter predominating in colitis. We aimed to examine the role of TNAP in T lymphocytes, using heterozygous TNAP+/- mice [as TNAP-/- mice are non-viable]. In vitro primary cultures and in vivo T cell models using TNAP+/- mice were used. Stimulated splenocytes [lipopolysaccharide and concanavalin A] and T lymphocytes [concanavalin A and a-CD3/a-CD28] showed a decreased cytokine production and expression when compared with wild-type [WT] cells. Decreased T cell activation was reproduced by the TNAP inhibitors levamisole, theophylline, and phenylalanine in WT cells. Intraperitoneal administration of anti-CD3 in vivo resulted in reduced plasma cytokine levels, and decreased activation of splenocytes and T cells ex vivo in TNAP+/- mice. We further tested the hypothesis that TNAP expressed in T lymphocytes is involved in T cell activation and inflammation, using the lymphocyte transfer model of colitis. Rag1-/- mice were transferred with T naïve cells [CD4+ CD62L+] from TNAP+/- or WT mice and developed colitis, which was attenuated in the group receiving TNAP+/- cells. Compared with WT, T cells from TNAP+/- mice showed a decreased capacity for proliferation, with no change in differentiation. Our results offer clear evidence that TNAP modulates T lymphocyte function and specifically T cell-dependent colitis. This was associated with distinct changes in the type of TNAP expressed, probably because of changes in glycosylation. Copyright © 2016 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com

Comparison of differentiation potential of male mouse adipose tissue and bone marrow derived-mesenchymal stem cells into germ cells

PubMed Central

Hosseinzadeh Shirzeily, Maryam; Pasbakhsh, Parichehr; Amidi, Fardin; Mehrannia, Kobra; Sobhani, Aligholi

2013-01-01

Background: Recent publications about differentiation of stem cells to germ cells have motivated researchers to make new approaches to infertility. In vitro production of germ cells improves understanding differentiation process of male and female germ cells. Due to the problem of using embryonic stem cells (ESC), it’s necessary the mentioned cells be replaced with some adult multi-potent stem cells in laboratories. Objective: The aim of this study was to obtain germ cells from appropriate source beyond ESC and compare differential potentials of adipocytes derived stem cells (ADMSCs) with bone marrow derived stem cells (BMMSCs). Materials and Methods: To find multi-potential entity, after providing purified ADMSCs and BMMSCs, differentiation to osteoblast and adipocyte was confirmed by using appropriate culture medium. To confirm mesenchymal lineage production superficial markers (expression of CD90 and CD44 and non-expression of CD45 and CD31) were investigated by flowcytometry. Then the cells were differentiated to germ cells in inductive medium containing retinoic acid for 7days. To evaluate germ cells characteristic markers [Dazl (Deleted in azoospermia-like), Mvh (Mouse vasa homolog gene), Stra8 (Stimulated by retinoic acid) and Scp3 (Synaptonemal complex protein 3)] flowcytometry, imunoflorescence and real time PCR were used. Results: Both types of cells were able to differentiate into osteoblast and adipocyte cells and presentation of stem cell superficial markers (CD90, CD44) and absence of endothelial and blood cell markers (CD31, CD45) were confirmative The flowcytometry, imunoflorescence and real time PCR results showed remarkable expression of germ cells characteristic markers (Mvh, Dazl, Stra8, and Scp3). Conclusion: It was found that although ADMSCs were attained easier and also cultured and differentiated rapidly, germ cell markers were expressed in BMMSCs significantly more than ADMSCs. This article extracted from M.Sc. thesis. (Maryam Hosseinzadeh Shirzeily) PMID:24639722

Immunophenotyping and activation status of maternal peripheral blood leukocytes during pregnancy and labour, both term and preterm.

PubMed

Zhang, Jianhong; Shynlova, Oksana; Sabra, Sally; Bang, Annie; Briollais, Laurent; Lye, Stephen J

2017-10-01

The onset of labour in rodents and in humans is associated with physiological inflammation which is manifested by infiltration of activated maternal peripheral leukocytes (mPLs) into uterine tissues. Here, we used flow cytometry to immunophenotype mPLs throughout gestation and labour, both term and preterm. Peripheral blood was collected from non-pregnant women and pregnant women in the 1st, 2nd and 3rd trimesters. Samples were also collected from women in active labour at term (TL) or preterm (PTL) and compared with women term not-in-labour (TNIL) and preterm not-in-labour (PTNIL). Different leukocyte populations were identified by surface markers such as CD45, CD14, CD15, CD3, CD4, CD8, CD19 and CD56. Their activation status was measured by the expression levels of CD11b, CD44, CD55, CD181 and CD192 proteins. Of all circulating CD45+ leukocytes, we detected significant increases in CD15+ granulocytes (i) in pregnant women versus non-pregnant; (ii) in TL women versus TNIL and versus pregnant women in the 1st/2nd/3rd trimester; (iii) in PTL women versus PTNIL. TL was characterized by (iv) increased expressions of CD11b, CD55 and CD192 on granulocytes; (v) increased mean fluorescent intensity (MFI) of CD55 and CD192 on monocytes; (vi) increased CD44 MFI on CD3+ lymphocytes as compared to late gestation. In summary, we have identified sub-populations of mPLs that are specifically activated in association with gestation (granulocytes) or with the onset of labour (granulocytes, monocytes and lymphocytes). Additionally, beta regression analysis created a set of reference values to rank this association between immune markers of pregnancy and to identify activation status with potential prognostic and diagnostic capability. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

The lupus susceptibility gene Pbx1 regulates the balance between follicular helper T cell and regulatory T cell differentiation

PubMed Central

Choi, Seung-Chul; Hutchinson, Tarun E.; Titov, Anton A.; Seay, Howard R.; Li, Shiwu; Brusko, Todd M.; Croker, Byron P.; Salek-Ardakani, Shahram; Morel, Laurence

2016-01-01

Pbx1 controls chromatin accessibility to a large number of genes and is entirely conserved between mice and humans. The Pbx1-d dominant negative isoform is more frequent in the CD4+ T cells from lupus patients than from healthy controls. Pbx1-d is associated with the production of autoreactive T cells in mice carrying the Sle1a1 lupus susceptibility locus. Transgenic expression of Pbx1-d in CD4+ T cells reproduced the phenotypes of Sle1a1 mice, with increased inflammatory functions of CD4+ T cells and impaired regulatory T cell homeostasis. Pbx1-d Tg also expanded the number of follicular helper T cells in a cell-intrinsic and antigen-specific manner that was enhanced in recall responses, and resulted in TH1-biased antibodies. Moreover, Pbx1-d Tg CD4+ T cells upregulated the expression of miR-10a, miR-21 and miR-155, which have been implicated in Treg and TFH cell homeostasis. Our results suggest that Pbx1-d impacts lupus development by regulating effector T cell differentiation and promoting TFH cells at the expense of Treg cells. In addition, our results identify Pbx1 as a novel regulator of CD4+ T cell effector function. PMID:27296664

Comparison of DNA aneuploidy, chromosome 1 abnormalities, MYCN amplification and CD44 expression as prognostic factors in neuroblastoma.

PubMed

Christiansen, H; Sahin, K; Berthold, F; Hero, B; Terpe, H J; Lampert, F

1995-01-01

A comparison of the prognostic impact of five molecular variables in a large series was made, including tests of their nonrandom association and multivariate analysis. Molecular data were available for 377 patients and MYCN amplification, cytogenetic chromosome 1p deletion, loss of chromosome 1p heterozygosity, DNA ploidy and CD44 expression were investigated. Their interdependence and influence on event-free survival was tested uni- and multivariately using Pearson's chi 2-test, Kaplan-Meier estimates, log rank tests and the Cox's regression model. MYCN amplification was present in 18% (58/322) of cases and predicted poorer prognosis in localised (P < 0.001), metastatic (P = 0.002) and even 4S (P = 0.040) disease. CD44 expression was found in 86% (127/148) of cases, and was a marker for favourable outcome in patients with neuroblastoma stages 1-3 (P = 0.003) and 4 (P = 0.017). Chromosome 1p deletion was cytogenetically detected in 51% (28/55), and indicated reduced event-free survival in localised neuroblastoma (P = 0.020). DNA ploidy and loss of heterozygosity on chromosome 1p were of less prognostic value. Most factors of prognostic significance were associated with each other. By multivariate analysis, MYCN was selected as the only relevant factor. Risk estimation of high discriminating power is, therefore, possible for patients with localised and metastatic neuroblastoma using stage and MYCN.

Cyclophilin B induces integrin-mediated cell adhesion by a mechanism involving CD98-dependent activation of protein kinase C-delta and p44/42 mitogen-activated protein kinases.

PubMed

Melchior, Aurélie; Denys, Agnès; Deligny, Audrey; Mazurier, Joël; Allain, Fabrice

2008-02-01

Initially identified as a cyclosporin-A binding protein, cyclophilin B (CyPB) is an inflammatory mediator that induces adhesion of T lymphocytes to fibronectin, by a mechanism dependent on CD147 and alpha 4 beta 1 integrins. Recent findings have suggested that another cell membrane protein, CD98, may cooperate with CD147 to regulate beta1 integrin functions. Based on these functional relationships, we examined the contribution of CD98 in the pro-adhesive activity of CyPB, by utilizing the responsive promonocyte cell line THP-1. We demonstrated that cross-linking CD98 with CD98-AHN-18 antibody mimicked the responses induced by CyPB, i.e. homotypic aggregation, integrin-mediated adhesion to fibronectin and activation of p44/42 MAPK. Consistent with previous data, immunoprecipitation confirmed the existence of a heterocomplex wherein CD147, CD98 and beta1 integrins were associated. We then demonstrated that CyPB-induced cell adhesion and p44/42 MAPK activation were dependent on the participation of phosphoinositide 3-kinase and subsequent activation of protein kinase C-delta. Finally, silencing the expression of CD98 by RNA interference potently reduced CyPB-induced cell responses, thus confirming the role of CD98 in the pro-adhesive activity of CyPB. Altogether, our results support a model whereby CyPB induces integrin-mediated adhesion via interaction with a multimolecular unit formed by the association between CD147, CD98 and beta1 integrins.

Mammary stem cell and macrophage markers are enriched in normal tissue adjacent to inflammatory breast cancer.

PubMed

Reddy, Jay P; Atkinson, Rachel L; Larson, Richard; Burks, Jared K; Smith, Daniel; Debeb, Bisrat G; Ruffell, Brian; Creighton, Chad J; Bambhroliya, Arvind; Reuben, James M; Van Laere, Steven J; Krishnamurthy, Savitri; Symmans, William F; Brewster, Abenaa M; Woodward, Wendy A

2018-06-01

We hypothesized that breast tissue not involved by tumor in inflammatory breast cancer (IBC) patients contains intrinsic differences, including increased mammary stem cells and macrophage infiltration, which may promote the IBC phenotype. Normal breast parenchyma ≥ 5 cm away from primary tumors was obtained from mastectomy specimens. This included an initial cohort of 8 IBC patients and 60 non-IBC patients followed by a validation cohort of 19 IBC patients and 25 non-IBC patients. Samples were immunostained for either CD44 + CD49f + CD133/2 + mammary stem cell markers or the CD68 macrophage marker and correlated with IBC status. Quantitation of positive cells was determined using inForm software from PerkinElmer. We also examined the association between IBC status and previously published tumorigenic stem cell and IBC tumor signatures in the validation cohort samples. 8 of 8 IBC samples expressed isolated CD44 + CD49f + CD133/2 + stem cell marked cells in the initial cohort as opposed to 0/60 non-IBC samples (p = 0.001). Similarly, the median number of CD44 + CD49f + CD133/2 + cells was significantly higher in the IBC validation cohort as opposed to the non-IBC validation cohort (25.7 vs. 14.2, p = 0.007). 7 of 8 IBC samples expressed CD68 + histologically confirmed macrophages in initial cohort as opposed to 12/48 non-IBC samples (p = 0.001). In the validation cohort, the median number of CD68 + cells in IBC was 3.7 versus 1.0 in the non-IBC cohort (p = 0.06). IBC normal tissue was positively associated with a tumorigenic stem cell signature (p = 0.02) and with a 79-gene IBC signature (p < 0.001). Normal tissue from IBC patients is enriched for both mammary stem cells and macrophages and has higher association with both a tumorigenic stem cell signature and IBC-specific tumor signature. Collectively, these data suggest that IBC normal tissue differs from non-IBC tissue. Whether these changes occur before the tumor develops or is induced by tumor warrants further investigation.

Regulated Expression of a Calmodulin Isoform Alters Growth and Development in Potato

NASA Technical Reports Server (NTRS)

Poovaiah, B. W.; Takezawa, D.; An, G.; Han, T.-J.

1996-01-01

A transgene approach was taken to study the consequences of altered expression of a calmodutin iso-form on plant growth and development. Eight genomic clones of potato calmodulin (PCM 1 to 8) have been isolated and characterized. Among the potato calmodulin isoforms studied, PCM 1 differs from the other isoforms because of its unique amino acid substitutions. Transgenic potato plants were produced carrying sense construct of PCM 1 fused to the CAMV 35S promoter. Transgenic plants showing a moderate increase in PCM 1 MRNA exhibited strong apical dominance, produced elongated tubers, and were taller than the controls. Interestingly, the plants expressing the highest level of PCM 1 MRNA did not form underground tubers. Instead, these transgenic plants produced aerial tubers when allowed to grow for longer periods. The expression of different calmodulin isoforms (PCM 1, 5, 6, and 8) was studied in transgenic plants. Among the four potato calmodulin isoforms, only the expression of PCM 1 MRNA was altered in transgenic plants, while the expression of other isoforms was not significantly altered. Western analysis revealed increased PCM 1 protein in transgenic plants, indicating that the expression of both MRNA and protein are altered in transgenic plants. These results suggest that increasing the expression of PCM 1 alters growth and development in potato plants.

Binding and internalization of NGR-peptide-targeted liposomal doxorubicin (TVT-DOX) in CD13-expressing cells and its antitumor effects.

PubMed

Garde, Seema V; Forté, André J; Ge, Michael; Lepekhin, Eugene A; Panchal, Chandra J; Rabbani, Shafaat A; Wu, Jinzi J

2007-11-01

In an effort to develop new agents and molecular targets for the treatment of cancer, aspargine-glycine-arginine (NGR)-targeted liposomal doxorubicin (TVT-DOX) is being studied. The NGR peptide on the surface of liposomal doxorubicin (DOX) targets an aminopeptidase N (CD13) isoform, specific to the tumor neovasculature, making it a promising strategy. To further understand the molecular mechanisms of action, we investigated cell binding, kinetics of internalization as well as cytotoxicity of TVT-DOX in vitro. We demonstrate the specific binding of TVT-DOX to CD13-expressing endothelial [human umbilical vein endothelial cells (HUVEC) and Kaposi sarcoma-derived endothelial cells (SLK)] and tumor (fibrosarcoma, HT-1080) cells in vitro. Following binding, the drug was shown to internalize through the endosomal pathway, eventually leading to the localization of doxorubicin in cell nuclei. TVT-DOX showed selective toxicity toward CD13-expressing HUVEC, sparing the CD13-negative colon-cancer cells, HT-29. Additionally, the nontargeted counterpart of TVT-DOX, Caelyx, was less cytotoxic to the CD13-positive HUVECs demonstrating the advantages of NGR targeting in vitro. The antitumor activity of TVT-DOX was tested in nude mice bearing human prostate-cancer xenografts (PC3). A significant growth inhibition (up to 60%) of PC3 tumors in vivo was observed. Reduction of tumor vasculature following treatment with TVT-DOX was also apparent. We further compared the efficacies of TVT-DOX and free doxorubicin in the DOX-resistant colon-cancer model, HCT-116, and observed the more pronounced antitumor effects of the TVT-DOX formulation over free DOX. The potential utility of TVT-DOX in a variety of vascularized solid tumors is promising.

Phosphatidylinositol 4,5-Bisphosphate Clusters the Cell Adhesion Molecule CD44 and Assembles a Specific CD44-Ezrin Heterocomplex, as Revealed by Small Angle Neutron Scattering

DOE PAGES

Khajeh, Jahan Ali; Ju, Jeong Ho; Gupta, Yogesh K.; ...

2015-01-08

The cell adhesion molecule CD44 regulates diverse cellular functions, including cell-cell and cell-matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co-localizes with the membrane-cytoskeleton adapter protein Ezrin, which links the CD44 assembled receptor signaling complexes to the cytoskeletal actin and organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered and adopts an autoinhibited conformation, which prevents CD44ct from binding directly to activated Ezrin in solution. Binding to the signaling lipid phosphatidylinositol 4,5-biphosphlate (PIP2) disrupts autoinhibition in CD44ct, and activates CD44ct to associate with Ezrin.more » Further, using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific hetero-tetramer complex of CD44ct with Ezrin. This study reveals a novel autoregulation mechanism in the cytoplasmic tail of CD44 and the role of PIP2 in mediating the assembly of multimeric CD44ct-Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of multimeric PIP2-CD44-Ezrin complexes.« less

Isoforms, structures, and functions of versatile spectraplakin MACF1

PubMed Central

Hu, Lifang; Su, Peihong; Li, Runzhi; Yin, Chong; Zhang, Yan; Shang, Peng; Yang, Tuanmin; Qian, Airong

2016-01-01

Spectraplakins are crucially important communicators, linking cytoskeletal components to each other and cellular junctions. Microtubule actin crosslinking factor 1 (MACF1), also known as actin crosslinking family 7 (ACF7), is a member of the spectraplakin family. It is expressed in numerous tissues and cells as one extensively studied spectraplakin. MACF1 has several isoforms with unique structures and well-known function to be able to crosslink F-actin and microtubules. MACF1 is one versatile spectraplakin with various functions in cell processes, embryo development, tissue-specific functions, and human diseases. The importance of MACF1 has become more apparent in recent years. Here, we summarize the current knowledge on the presence and function of MACF1 and provide perspectives on future research of MACF1 based on our studies and others. [BMB Reports 2016; 49(1): 37-44] PMID:26521939

miR-375 induces human decidua basalis-derived stromal cells to become insulin-producing cells.

PubMed

Shaer, Anahita; Azarpira, Negar; Vahdati, Akbar; Karimi, Mohammad Hosein; Shariati, Mehrdad

2014-09-01

This paper focuses on the development of renewable sources of isletreplacement tissue for the treatment of type I diabetes mellitus. Placental tissue-derived mesenchymal stem cells (MSCs) are a promising source for regenerative medicine due to their plasticity and easy availability. They have the potential to differentiate into insulin-producing cells. miR-375 is a micro RNA that is expressed in the pancreas and involved in islet development. Human placental decidua basalis MSCs (PDB-MSCs) were cultured from full-term human placenta. The immunophenotype of the isolated cells was checked for CD90, CD105, CD44, CD133 and CD34 markers. The MSCs (P3) were chemically transfected with hsa-miR-375. Total RNA was extracted 4 and 6 days after transfection. The expressions of insulin, NGN3, GLUT2, PAX4, PAX6, KIR6.2, NKX6.1, PDX1, and glucagon genes were evaluated using real-time qPCR. On day 6, we tested the potency of the clusters in response to the high glucose challenge and assessed the presence of insulin and NGN3 proteins via immunocytochemistry. Flow cytometry analysis confirmed that more than 90% of the cells were positive for CD90, CD105 and CD44 and negative for CD133 and CD34. Morphological changes were followed from day 2. Cell clusters formed during day 6. Insulin-producing clusters showed a deep red color with DTZ. The expression of pancreatic-specific transcription factors increased remarkably during the four days after transfection and significantly increased on day 7. The clusters were positive for insulin and NGN3 proteins, and C-peptide and insulin secretion increased in response to changes in the glucose concentration (2.8 mM and 16.7 mM). In conclusion, the MSCs could be programmed into functional insulin-producing cells by transfection of miR-375.

Boronic acid-tethered amphiphilic hyaluronic acid derivative-based nanoassemblies for tumor targeting and penetration.

PubMed

Jeong, Jae Young; Hong, Eun-Hye; Lee, Song Yi; Lee, Jae-Young; Song, Jae-Hyoung; Ko, Seung-Hak; Shim, Jae-Seong; Choe, Sunghwa; Kim, Dae-Duk; Ko, Hyun-Jeong; Cho, Hyun-Jong

2017-04-15

(3-Aminomethylphenyl)boronic acid (AMPB)-installed hyaluronic acid-ceramide (HACE)-based nanoparticles (NPs), including manassantin B (MB), were fabricated for tumor-targeted delivery. The amine group of AMPB was conjugated to the carboxylic acid group of hyaluronic acid (HA) via amide bond formation, and synthesis was confirmed by spectroscopic methods. HACE-AMPB/MB NPs with a 239-nm mean diameter, narrow size distribution, negative zeta potential, and >90% drug encapsulation efficiency were fabricated. Exposed AMPB in the outer surface of HACE-AMPB NPs (in the aqueous environment) may react with sialic acid of cancer cells. The improved cellular accumulation efficiency, in vitro antitumor efficacy, and tumor penetration efficiency of HACE-AMPB/MB NPs, compared with HACE/MB NPs, in MDA-MB-231 cells (CD44 receptor-positive human breast adenocarcinoma cells) may be based on the CD44 receptor-mediated endocytosis and phenylboronic acid-sialic acid interaction. Enhanced in vivo tumor targetability, infiltration efficiency, and antitumor efficacies of HACE-AMPB NPs, compared with HACE NPs, were observed in a MDA-MB-231 tumor-xenografted mouse model. In addition to passive tumor targeting (based on an enhanced permeability and retention effect) and active tumor targeting (interaction between HA and CD44 receptor), the phenylboronic acid-sialic acid interaction can play important roles in augmented tumor targeting and penetration of HACE-AMPB NPs. STATEMENT OF SIGNIFICANCE: (3-Aminomethylphenyl)boronic acid (AMPB)-tethered hyaluronic acid-ceramide (HACE)-based nanoparticles (NPs), including manassantin B (MB), were fabricated and their tumor targeting and penetration efficiencies were assessed in MDA-MB-231 (CD44 receptor-positive human adenocarcinoma) tumor models. MB, which exhibited antitumor efficacies via the inhibition of angiogenesis and hypoxia inducible factor (HIF)-1, was entrapped in HACE-AMPB NPs in this study. Phenylboronic acid located in the outer surface of HACE-AMPB/MB NPs (in the aqueous milieu) may react with the sialic acid over-expressed in cancer cells and intramolecular B‒O bond can be formed. This phenylboronic acid-sialic acid interaction may provide additional tumor targeting and penetration potentials together with an enhanced permeability and retention (EPR) effect (passive tumor targeting) and HA-CD44 receptor interaction (active tumor targeting). Developed HACE-AMPB NP may be one of promising nanocarriers for the imaging and therapy of CD44 receptor-expressed cancers. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

A structured sparse regression method for estimating isoform expression level from multi-sample RNA-seq data.

PubMed

Zhang, L; Liu, X J

2016-06-03

With the rapid development of next-generation high-throughput sequencing technology, RNA-seq has become a standard and important technique for transcriptome analysis. For multi-sample RNA-seq data, the existing expression estimation methods usually deal with each single-RNA-seq sample, and ignore that the read distributions are consistent across multiple samples. In the current study, we propose a structured sparse regression method, SSRSeq, to estimate isoform expression using multi-sample RNA-seq data. SSRSeq uses a non-parameter model to capture the general tendency of non-uniformity read distribution for all genes across multiple samples. Additionally, our method adds a structured sparse regularization, which not only incorporates the sparse specificity between a gene and its corresponding isoform expression levels, but also reduces the effects of noisy reads, especially for lowly expressed genes and isoforms. Four real datasets were used to evaluate our method on isoform expression estimation. Compared with other popular methods, SSRSeq reduced the variance between multiple samples, and produced more accurate isoform expression estimations, and thus more meaningful biological interpretations.

A single amino-acid change in a highly conserved motif of gp41 elicits HIV-1 neutralization and protects against CD4 depletion.

PubMed

Petitdemange, Caroline; Achour, Abla; Dispinseri, Stefania; Malet, Isabelle; Sennepin, Alexis; Ho Tsong Fang, Raphaël; Crouzet, Joël; Marcelin, Anne-Geneviève; Calvez, Vincent; Scarlatti, Gabriella; Debré, Patrice; Vieillard, Vincent

2013-09-01

The induction of neutralizing antibodies against conserved regions of the human immunodeficiency virus type 1 (HIV-1) envelope protein is a major goal of vaccine strategies. We previously identified 3S, a critical conserved motif of gp41 that induces the NKp44L ligand of an activating NK receptor. In vivo, anti-3S antibodies protect against the natural killer (NK) cell-mediated CD4 depletion that occurs without efficient viral neutralization. Specific substitutions within the 3S peptide motif were prepared by directed mutagenesis. Virus production was monitored by measuring the p24 production. Neutralization assays were performed with immune-purified antibodies from immunized mice and a cohort of HIV-infected patients. Expression of NKp44L on CD4(+) T cells and degranulation assay on activating NK cells were both performed by flow cytometry. Here, we show that specific substitutions in the 3S motif reduce viral infection without affecting gp41 production, while decreasing both its capacity to induce NKp44L expression on CD4(+) T cells and its sensitivity to autologous NK cells. Generation of antibodies in mice against the W614 specific position in the 3S motif elicited a capacity to neutralize cross-clade viruses, notable in its magnitude, breadth, and durability. Antibodies against this 3S variant were also detected in sera from some HIV-1-infected patients, demonstrating both neutralization activity and protection against CD4 depletion. These findings suggest that a specific substitution in a 3S-based immunogen might allow the generation of specific antibodies, providing a foundation for a rational vaccine that combine a capacity to neutralize HIV-1 and to protect CD4(+) T cells.

Nanotechniques Inactivate Cancer Stem Cells

NASA Astrophysics Data System (ADS)

Goltsev, Anatoliy N.; Babenko, Natalya N.; Gaevskaya, Yulia A.; Bondarovich, Nikolay A.; Dubrava, Tatiana G.; Ostankov, Maksim V.; Chelombitko, Olga V.; Malyukin, Yuriy V.; Klochkov, Vladimir K.; Kavok, Nataliya S.

2017-06-01

One of the tasks of current oncology is identification of cancer stem cells and search of therapeutic means capable of their specific inhibition. The paper presents the data on phenotype characteristics of Ehrlich carcinoma cells as convenient and easy-to-follow model of tumor growth. The evidence of cancer stem cells as a part of Ehrlich carcinoma and significance of CD44+ and CD44- subpopulations in maintaining the growth of this type of tumor were demonstrated. A high (tenfold) tumorigenic activity of the Ehrlich carcinoma CD44+ cells if compared to CD44- cells was proven. In this pair of comparison, the CD44+ cells had a higher potential of generating in peritoneal cavity of CD44high, CD44+CD24-, CD44+CD24+ cell subpopulations, highlighting the presence of cancer stem cells in a pool of CD44+ cells.

Expression and cytokine regulation of immune recognition elements by normal human biliary epithelial and established liver cell lines in vitro.

PubMed

Cruickshank, S M; Southgate, J; Selby, P J; Trejdosiewicz, L K

1998-10-01

Biliary epithelial cells are targets of immune-mediated attack in conditions such as primary biliary cirrhosis and allograft rejection. This has been attributed to the ability of biliary epithelial cells to express ligands for T cell receptors. We aimed to investigate the expression of immune recognition elements and the effects of pro-inflammatory and anti-inflammatory cytokines on cell surface phenotypes of normal human biliary epithelial cells and established human liver-derived (PLC/PRF/5, HepG2, Hep3B and CC-SW) lines. Cells were cultured in the presence or absence of cytokines for 72 h, and expression of cell surface molecules was assessed by flow cytometry and immunofluorescence. All cell lines expressed MHC class I, ICAM-1 (CD54), LFA-3 (CD58) and EGF receptor, and all but Hep3B expressed Fas/Apo-1 (CD95). Unlike hepatocyte-derived cell lines, biliary epithelial cells and CC-SW expressed CD40 and CD44. As expected, IFNgamma and TNFalpha upregulated expression of ICAM-1, MHC class I and MHC class II, particularly in biliary epithelial cells. TGFbeta downregulated these molecules and downregulated CD95 on biliary epithelial cells, but upregulated LFA-3. The Th2 cytokines had little effect, although IL-4 upregulated CD95 expression on biliary epithelial cells. IFNgamma upregulated CD40 expression on biliary epithelial cells, CC-SW and HepG2. These findings imply that biliary epithelial cells may be capable of interacting with activated T lymphocytes via CD40 and LFA-3, which are thought to be important T cell accessory ligands for T cell activation in a B7-independent manner. Sensitivity to pro-inflammatory cytokines and expression of CD95 may explain why biliary epithelial cells are primary targets for autoimmune attack.

Differential Pre-mRNA Splicing Regulates Nnat Isoforms in the Hypothalamus after Gastric Bypass Surgery in Mice

PubMed Central

Scott, William R.; Gelegen, Cigdem; Chandarana, Keval; Karra, Efthimia; Yousseif, Ahmed; Amouyal, Chloé; Choudhury, Agharul I.; Andreelli, Fabrizio; Withers, Dominic J.; Batterham, Rachel L.

2013-01-01

Background Neuronatin (NNAT) is an endoplasmic reticulum proteolipid implicated in intracellular signalling. Nnat is highly-expressed in the hypothalamus, where it is acutely regulated by nutrients and leptin. Nnat pre-mRNA is differentially spliced to create Nnat-α and -β isoforms. Genetic variation of NNAT is associated with severe obesity. Currently, little is known about the long-term regulation of Nnat. Methods Expression of Nnat isoforms were examined in the hypothalamus of mice in response to acute fast/feed, chronic caloric restriction, diet-induced obesity and modified gastric bypass surgery. Nnat expression was assessed in the central nervous system and gastrointestinal tissues. RTqPCR was used to determine isoform-specific expression of Nnat mRNA. Results Hypothalamic expression of both Nnat isoforms was comparably decreased by overnight and 24-h fasting. Nnat expression was unaltered in diet-induced obesity, or subsequent switch to a calorie restricted diet. Nnat isoforms showed differential expression in the hypothalamus but not brainstem after bypass surgery. Hypothalamic Nnat-β expression was significantly reduced after bypass compared with sham surgery (P = 0.003), and was positively correlated with post-operative weight-loss (R2 = 0.38, P = 0.01). In contrast, Nnat-α expression was not suppressed after bypass surgery (P = 0.19), and expression did not correlate with reduction in weight after surgery (R2 = 0.06, P = 0.34). Hypothalamic expression of Nnat-β correlated weakly with circulating leptin, but neither isoform correlated with fasting gut hormone levels post- surgery. Nnat expression was detected in brainstem, brown-adipose tissue, stomach and small intestine. Conclusions Nnat expression in hypothalamus is regulated by short-term nutrient availability, but unaltered by diet-induced obesity or calorie restriction. While Nnat isoforms in the hypothalamus are co-ordinately regulated by acute nutrient supply, after modified gastric bypass surgery Nnat isoforms show differential expression. These results raise the possibility that in the radically altered nutrient and hormonal milieu created by bypass surgery, resultant differential splicing of Nnat pre-mRNA may contribute to weight-loss. PMID:23527188

Overexpressed long noncoding RNA CRNDE with distinct alternatively spliced isoforms in multiple cancers.

PubMed

Ma, Xuefei; Zhang, Wei; Zhang, Rong; Li, Jingming; Li, Shufen; Ma, Yunlin; Jin, Wen; Wang, Kankan

2018-05-26

Alternative splicing is a tightly regulated process that contributes to cancer development. CRNDE is a long noncoding RNA with alternative splicing and is implicated in the pathogenesis of several cancers. However, whether deregulated expression of CRNDE is common and which isoforms are mainly involved in cancers remain unclear. In this study, we report that CRNDE is aberrantly expressed in the majority of solid and hematopoietic malignancies. The investigation of CRNDE expression in normal samples revealed that CRNDE was expressed in a tissue- and cell-specific manner. Further comparison of CRNDE expression in 2938 patient samples from 15 solid and hematopoietic tumors showed that CRNDE was significantly overexpressed in 11 malignancies, including 3 reported and 8 unreported, and also implicated that the overexpressed isoforms differed in various cancer types. Furthermore, anti-cancer drugs could efficiently repress CRNDE overexpression in cancer cell lines and primary samples, and even had different impacts on the expression of CRNDE isoforms. Finally, experimental profiles of 12 alternatively spliced isoforms demonstrated that the spliced variant CRNDE-g was the most highly expressed isoform in multiple cancer types. Collectively, our results emphasize the cancer-associated feature of CRNDE and its spliced isoforms, and may provide promising targets for cancer diagnosis and therapy.

Syndecan-1/CD147 association is essential for cyclophilin B-induced activation of p44/42 mitogen-activated protein kinases and promotion of cell adhesion and chemotaxis.

PubMed

Pakula, Rachel; Melchior, Aurélie; Denys, Agnès; Vanpouille, Christophe; Mazurier, Joël; Allain, Fabrice

2007-05-01

Many of the biological functions attributed to cell surface proteoglycans are dependent on the interaction with extracellular mediators through their heparan sulphate (HS) moieties and the participation of their core proteins in signaling events. A class of recently identified inflammatory mediators is secreted cyclophilins, which are mostly known as cyclosporin A-binding proteins. We previously demonstrated that cyclophilin B (CyPB) triggers chemotaxis and integrin-mediated adhesion of T lymphocytes mainly of the CD4+/CD45RO+ phenotype. These activities are related to interactions with two types of binding sites, CD147 and cell surface HS. Here, we demonstrate that CyPB-mediated adhesion of CD4+/CD45RO+ T cells is related to p44/42 mitogen-activated protein kinase (MAPK) activation by a mechanism involving CD147 and HS proteoglycans (HSPG). Although HSPG core proteins are represented by syndecan-1, -2, -4, CD44v3 and betaglycan in CD4+/CD45RO+ T cells, we found that only syndecan-1 is physically associated with CD147. The intensity of the heterocomplex increased in response to CyPB, suggesting a transient enhancement and/or stabilization in the association of CD147 to syndecan-1. Pretreatment with anti-syndecan-1 antibodies or knockdown of syndecan-1 expression by RNA interference dramatically reduced CyPB-induced p44/p42 MAPK activation and consequent migration and adhesion, supporting the model in which syndecan-1 serves as a binding subunit to form the fully active receptor of CyPB. Altogether, our findings provide a novel example of a soluble mediator in which a member of the syndecan family plays a critical role in efficient interaction with signaling receptors and initiation of cellular responses.

Activation of bone marrow-derived mesenchymal stromal cells-a new mechanism of defocused low-energy shock wave in regenerative medicine.

PubMed

Zhao, Yong; Wang, Jinbang; Wang, Muwen; Sun, Peng; Chen, Ji; Jin, Xunbo; Zhang, Haiyang

2013-12-01

Defocused low-energy shock wave (DLSW) therapy has shown effectiveness in regenerative medicine. The mechanism of action was mainly focused on the pathophysiological improvement at the wound tissues. In this study, the activation of stem cells treated by DLSW was first examined as an important pathway during the healing process. Cultured rat bone marrow-derived mesenchymal stromal cells (BMSC) were treated by DLSW before each passage. The untreated BMSC served as a control. The secretions of vascular endothelial growth factor (VEGF) and CXC ligand 5 (CXCL5) were tested by means of enzyme-linked immunoassay. Flow cytometry was performed to analyze the BMSC (passage 4) surface antigen expressions (CD166, CD44 and CD34). The expressions of proliferating cell nuclear antigen and Ki67 were analyzed by means of Western blot. The healing abilities of conditioned media of shocked and unshocked BMSC were examined by Matrigel-based capillary-like tube formation assay and rat major pelvic ganglia culture test. The shocked BMSC secreted more VEGF and CXCL5 than did those of unshocked BMSC. The expressions of CD166, CD44 and CD34 showed no significant differences (P > 0.05) between the shocked and unshocked BMSC. The shocked BMSC demonstrated higher expressions of proliferating cell nuclear antigen (P < 0.01) and Ki67 (P < 0.01) than did those of unshocked BMSC. The shocked BMSC conditioned medium showed higher ability to enhance the growth of major pelvic ganglia neurites (P < 0.05) and Matrigel-based endothelial tube-like formation (P < 0.05). DLSW did not interfere with the expressions of cell surface markers. DLSW enhanced the secretion and proliferation of BMSC and promoted angiogenesis and nerve regeneration in vitro. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Phosphatidylinositol 4,5-Bisphosphate Clusters the Cell Adhesion Molecule CD44 and Assembles a Specific CD44-Ezrin Heterocomplex, as Revealed by Small Angle Neutron Scattering*

PubMed Central

Chen, Xiaodong; Khajeh, Jahan Ali; Ju, Jeong Ho; Gupta, Yogesh K.; Stanley, Christopher B.; Do, Changwoo; Heller, William T.; Aggarwal, Aneel K.; Callaway, David J. E.; Bu, Zimei

2015-01-01

The cell adhesion molecule CD44 regulates diverse cellular functions, including cell-cell and cell-matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co-localizes with the membrane-cytoskeleton adapter protein Ezrin that links the CD44 assembled receptor signaling complexes to the cytoskeletal actin network, which organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered. Upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2), CD44ct clusters into aggregates. Further, contrary to the generally accepted model, CD44ct does not bind directly to the FERM domain of Ezrin or to the full-length Ezrin but only forms a complex with FERM or with the full-length Ezrin in the presence of PIP2. Using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific heterotetramer complex of CD44ct with Ezrin. This study reveals the role of PIP2 in clustering CD44 and in assembling multimeric CD44-Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of CD44 clusters and the multimeric PIP2-CD44-Ezrin complexes. PMID:25572402

Phosphatidylinositol 4,5-bisphosphate clusters the cell adhesion molecule CD44 and assembles a specific CD44-Ezrin heterocomplex, as revealed by small angle neutron scattering.

PubMed

Chen, Xiaodong; Khajeh, Jahan Ali; Ju, Jeong Ho; Gupta, Yogesh K; Stanley, Christopher B; Do, Changwoo; Heller, William T; Aggarwal, Aneel K; Callaway, David J E; Bu, Zimei

2015-03-06

The cell adhesion molecule CD44 regulates diverse cellular functions, including cell-cell and cell-matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co-localizes with the membrane-cytoskeleton adapter protein Ezrin that links the CD44 assembled receptor signaling complexes to the cytoskeletal actin network, which organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered. Upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2), CD44ct clusters into aggregates. Further, contrary to the generally accepted model, CD44ct does not bind directly to the FERM domain of Ezrin or to the full-length Ezrin but only forms a complex with FERM or with the full-length Ezrin in the presence of PIP2. Using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific heterotetramer complex of CD44ct with Ezrin. This study reveals the role of PIP2 in clustering CD44 and in assembling multimeric CD44-Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of CD44 clusters and the multimeric PIP2-CD44-Ezrin complexes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Verification of ALDH Activity as a Biomarker in Colon Cancer Stem Cells-Derived HT-29 Cell Line.

PubMed

Khorrami, Samaneh; Zavaran Hosseini, Ahmad; Mowla, Seyed Javad; Malekzadeh, Reza

2015-10-01

Recent evidence has suggested that epithelial cancers including colorectal cancer (CRC) have driven by a small population of self-renewing, multi-potent cells termed cancer stem cells (CSCs) which could be responsible for recurrence of cancer. Aldehyde dehydrogenase 1 (ALDH1) activity has used as a functional stem cell biomarker to isolate CSCs in different cancers such as colorectal cancer. The main aim of this research was to determine the utility of ALDH1 activity along with CD44 and EPCAM in identifying stem cell-like cells in human HT-29 colonic adenocarcinoma cell line. In this experimental study, colon CSCs biomarkers including CD44, EPCAM and ALDH1 in colonospheres and parent cells have analyzed by flow cytometry. The expression levels of stemness genes in spheroid and parental cells have investigated using SYBR Green real-time PCR. In addition, in vivo xenografts assay has performed to determine tumorigenic potential of tumor spheroid cells in nude mice. According to results, over 92% of spheroids were CD44+/EpCAM+, while parent cells only have expressed 38% of CD44/EpCAM biomarkers (P < 0.001). Controversially, ALDH activity was about 2-fold higher in the parent cells than spheroid cells (P < 0.05). In comparison with the parental cells, expression levels of ''stemness'' genes, like Sox2, Oct4, Nanog, C-myc, and Klf4 have significantly increased in colonosphere cells (P < 0.05). Further, administration of 2500 spheroids could be sufficient to initiate tumor growth in nude mice, while 1x106 of parental cells has needed to form tumor. For the first time, we have shown that colonospheres with low ALDH1 activity has indicated increased tumorigenic potential and stemness properties. So, it hasn't seemed that ALDH1 could become a useful biomarker to identify CSCs population in HT-29 cell line.

The natural flavonoid apigenin sensitizes human CD44+ prostate cancer stem cells to cisplatin therapy.

PubMed

Erdogan, Suat; Turkekul, Kader; Serttas, Rıza; Erdogan, Zeynep

2017-04-01

Prostate cancer (PCa) is the second most common type of cancer and the fifth leading cause of cancer-related death among men. Development of chemoresistance, tumor relapse and metastasis remain major barriers to effective treatment and all been identified to be associated with cancer stem cells (CSCs). Natural flavonoids such as apigenin have been shown to have the ability to improve the therapeutic efficacy of common chemotherapy agents through CSCs sensitization. Thus, the aim of this study was to evaluate the combination of apigenin with cisplatin on CD44 + PCa stem cell growth and migration. Platinum-based anti-neoplastic drugs have been used to treat a number of malignancies including PCa. However, acquired resistance and side effects unfortunately have limited cisplatin's use. A CD44 + subpopulation was isolated from human androgen-independent PC3 PCa cells by using human CD44-PE antibody. IC 50 values were determined by MTT test. RT-qPCR, Western blot analyses and image-based cytometer were used to investigate apoptosis, cell cycle and their underlying molecular mechanisms. Cell migration was evaluated by wound healing test. The combination of the IC 50 doses of apigenin (15μM) and cisplatin (7.5μM) for 48h significantly enhanced cisplatin's cytotoxic and apoptotic effects through downregulation of Bcl-2, sharpin and survivin; and upregulation of caspase-8, Apaf-1 and p53 mRNA expression. The combined therapy suppressed the phosphorylation of p-PI3K and p-Akt, inhibited the protein expression of NF-κB, and downregulated the cell cycle by upregulating p21, as well as cyclin dependent kinases CDK-2, -4, and -6. Apigenin significantly increased the inhibitory effects of cisplatin on cell migration via downregulation of Snail expression. In conclusion, our study showed the possible therapeutic approach of using apigenin to potentially increase the effects of cisplatin by targeting CSCs subset in prostate cancer. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Targeting CD44 with nanoparticles in head and neck squamous cell carcinoma: A novel therapeutic strategy against cancer stem cells

NASA Astrophysics Data System (ADS)

Thapa, Ranjeeta

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide and is associated with significant morbidity and mortality. Advances in multi-modality treatments have only minimally improved survival rates in the past several years. Recent attention has been focused on the hypothesis that cancer stem cells (CSCs) may be responsible for the failure of current treatments. In HNSCC, a CSC population is contained within the cell fraction that expresses high levels of CD44. CD44 is a cell surface glycoprotein and was the first CSC marker to be described in solid malignancies. in this study, hyaluronan conjugated, dextran-coated super paramagnetic iron-oxide nanoparticles (HA-DESPIONs) were used to target the CD44 population in CD44-overexpressed HNSCC cell lines for treatment by establishing the interaction of HA-DESPIONs with radiation and hyperthermia therapy. The first part of this dissertation studied the cytotoxic, radiosensitizing, and hyperthermic properties of the HA-DESPIONs using cell proliferation and clonogenic survival assays. Cells were grown, plated, treated with HA-DESPIONs, irradiated/exposed to local hyperthermia, and then analyzed for apoptosis. HA-DESPIONs proved to be relatively non-toxic and nonradiosensitizing. However, temperature-dependent cell survival reduction upon incubation with HA-DESPIONs was observed with evidence of apoptotic cell death. These results supported further development of an alternating magnetic field (AMF) approach to activate the HADESPIONs attached to CSCs. In the second part of the dissertation, an AMF generator was constructed and its heat generating effect was tested via kinetic and dose-dependent bulk heating experiments by exposing magnetic nanoparticles to AMF. For elimination of the CD44 population, cells were treated with HA-DESPIONs/DESPIONs, exposed to AMF, and processed for flow cytometrybased apoptosis analysis. Magnetic nanoparticles caused concentration-dependent bulk heating in response to AMF resulting in a significant temperature rise. Following exposure to AMF, DESPIONs were unable to induce targeted hyperthermia and hence had no effect on CD44 cell death in HNSCC cells. However, there was significant cell death in the CD44 population treated with HA-DESPIONs and exposed to AMF. This effect was observed only when the AMF was turned on. These results demonstrated that HA-DESPIONs caused targeted cell-death in CD44overexpressing cells. This may be a promising strategy to specifically target CSCs for the treatment of HNSCC.

Characterization of Amniotic Stem Cells

PubMed Central

Koike, Chika; Zhou, Kaixuan; Takeda, Yuji; Fathy, Moustafa; Okabe, Motonori; Yoshida, Toshiko; Nakamura, Yukio; Kato, Yukio

2014-01-01

Abstract The amnion membrane is developed from embryo-derived cells, and amniotic cells have been shown to exhibit multidifferentiation potential. These cells represent a desirable source for stem cells for a variety of reasons. However, to date very few molecular analyses of amnion-derived cells have been reported, and efficient markers for isolating the stem cells remain unclear. This paper assesses the characterization of amnion-derived cells as stem cells by examining stemness marker expressions for amnion-derived epithelial cells and mesenchymal cells by flow cytometry, immunocytochemistry, and quantitative PCR. Flow cytometry revealed that amnion epithelial cells expressed CD133, CD 271, and TRA-1-60, whereas mecenchymal cells expressed CD44, CD73, CD90, and CD105. Immunohistochemistry showed that both cells expressed the stemness markers Oct3/4, Sox2, Klf4, and SSEA4. Stemness genes' expression in amnion epithelial cells, mesenchymal cells, fibroblast, bone marrow–derived mesenchymal stem cells (MSCs), and induced pluripotent stem cells (iPSCs) was compared by quantitative reverse-transcription polymerase chain reaction (RT-PCR). Amnion-derived epithelial cells and mesenchymal cells expressed Oct3/4, Nanog, and Klf4 more than bone marrow–derived MSCs. The sorted TRA1-60–positive cells expressed Oct3/4, Nanog, and Klf4 more than unsorted cells or TRA1-60–negative cells. TRA1-60 can be a marker for isolating amnion epithelial stem cells. PMID:25068631

Doxorubicin attached to HPMA copolymer via amide bond modifies the glycosylation pattern of EL4 cells.

PubMed

Kovar, Lubomir; Etrych, Tomas; Kabesova, Martina; Subr, Vladimir; Vetvicka, David; Hovorka, Ondrej; Strohalm, Jiri; Sklenar, Jan; Chytil, Petr; Ulbrich, Karel; Rihova, Blanka

2010-08-01

To avoid the side effects of the anti-cancer drug doxorubicin (Dox), we conjugated this drug to a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer backbone. Dox was conjugated via an amide bond (Dox-HPMA(AM), PK1) or a hydrazone pH-sensitive bond (Dox-HPMA(HYD)). In contrast to Dox and Dox-HPMA(HYD), Dox-HPMA(AM) accumulates within the cell's intracellular membranes, including those of the Golgi complex and endoplasmic reticulum, both involved in protein glycosylation. Flow cytometry was used to determine lectin binding and cell death, immunoblot to characterize the presence of CD7, CD43, CD44, and CD45, and high-performance anion exchange chromatography with pulsed amperometric detector analysis for characterization of plasma membrane saccharide composition. Incubation of EL4 cells with Dox-HPMA(AM) conjugate, in contrast to Dox or Dox-HPMA(HYD), increased the amounts of membrane surface-associated glycoproteins, as well as saccharide moieties recognized by peanut agglutinin, Erythrina cristagalli, or galectin-1 lectins. Only Dox-HPMA(AM) increased expression of the highly glycosylated membrane glycoprotein CD43, while expression of others (CD7, CD44, and CD45) was unaffected. The binding sites for galectin-1 are present on CD43 molecule. Furthermore, we present that EL4 treated with Dox-HPMA(AM) possesses increased sensitivity to galectin-1-induced apoptosis. In this study, we demonstrate that Dox-HPMA(AM) treatment changes glycosylation of the EL4 T cell lymphoma surface and sensitizes the cells to galectin-1-induced apoptosis.

Regulated expression of a calmodulin isoform alters growth and development in potato.

PubMed

Poovaiah, B W; Takezawa, D; An, G; Han, T J

1996-01-01

A transgene approach was taken to study the consequences of altered expression of a calmodulin isoform on plant growth and development. Eight genomic clones of potato calmodulin (PCM1 to 8) have been isolated and characterized (Takezawa et al., 1995). Among the potato calmodulin isoforms studied, PCM1 differs from the other isoforms because of its unique amino acid substitutions. Transgenic potato plants were produced carrying sense construct of PCM1 fused to the CaMV 35S promoter. Transgenic plants showing a moderate increase in PCM1 mRNA exhibited strong apical dominance, produced elongated tubers, and were taller than the controls. Interestingly, the plants expressing the highest level of PCM1 mRNA did not form underground tubers. Instead, these transgenic plants produced aerial tubers when allowed to grow for longer periods. The expression of different calmodulin isoforms (PCM1, 5, 6, and 8) was studied in transgenic plants. Among the four potato calmodulin isoforms, only the expression of PCM1 mRNA was altered in transgenic plants, while the expression of other isoforms was not significantly altered. Western analysis revealed increased PCM1 protein in transgenic plants, indicating that the expression of both mRNA and protein are altered in transgenic plants. These results suggest that increasing the expression of PCM1 alters growth and development in potato plants.

Human umbilical cord mesenchymal stromal cells suppress MHC class II expression on rat vascular endothelium and prolong survival time of cardiac allograft

PubMed Central

Qiu, Ying; Yun, Mark M; Han, Xia; Zhao, Ruidong; Zhou, Erxia; Yun, Sheng

2014-01-01

Background: Human umbilical cord mesenchymal stromal cells (UC-MSCs) have low immunogenicity and immune regulation. To investigate immunomodulatory effects of human UC-MSCs on MHC class II expression and allograft, we transplanted heart of transgenic rats with MHC class II expression on vascular endothelium. Methods: UC-MSCs were obtained from human umbilical cords and confirmed with flow cytometry analysis. Transgenic rat line was established using the construct of human MHC class II transactivator gene (CIITA) under mouse ICAM-2 promoter control. The induced MHC class II expression on transgenic rat vascular endothelial cells (VECs) was assessed with immunohistological staining. And the survival time of cardiac allograft was compared between the recipients with and without UC-MSC transfusion. Results: Flow cytometry confirmed that the human UC-MSCs were positive for CD29, CD44, CD73, CD90, CD105, CD271, and negative for CD34 and HLA-DR. Repeated infusion of human UC-MSCs reduced MHC class II expression on vascular endothelia of transplanted hearts, and increased survival time of allograft. The UC-MSCs increased regulatory cytokines IL10, transforming growth factor (TGF)-β1 and suppressed proinflammatory cytokines IL2 and IFN-γ in vivo. The UC-MSC culture supernatant had similar effects on cytokine expression, and decreased lymphocyte proliferation in vitro. Conclusions: Repeated transfusion of the human UC-MSCs reduced MHC class II expression on vascular endothelia and prolonged the survival time of rat cardiac allograft. PMID:25126177

Increased Intraepithelial Vα24 Invariant NKT Cells in the Celiac Duodenum

PubMed Central

Montalvillo, Enrique; Bernardo, David; Martínez-Abad, Beatriz; Allegretti, Yessica; Fernández-Salazar, Luis; Calvo, Carmen; Chirdo, Fernando G.; Garrote, José A.; Arranz, Eduardo

2015-01-01

Celiac Disease (CD) is an interferon (IFN)γ-mediated duodenal hypersensitivity to wheat gluten occurring in genetically predisposed individuals. Gluten-free diet (GFD) leads to a complete remission of the disease. Vα24-restricted invariant NKT (iNKT) cells are important to maintain immune homeostasis in the gut mucosa because of their unique capacity to rapidly produce large quantities of both T-helper (Th)1 and Th2 cytokines upon stimulation. We studied the presence of these cells in the CD duodenum. Duodenal biopsies were obtained from 45 untreated-CD patients (uCD), 15 Gluten Free Diet-CD patients (GFD-CD), 44 non-inflamed non-CD controls (C-controls) and 15 inflamed non-CD controls (I-controls). Two populations from Spain and Argentina were recruited. Messenger RNA (mRNA) expression of Vα24-Jα18 (invariant TCRα chain of human iNKT cells), IFNγ and intracellular transcription factor Forkhead Box P3 (Foxp3), and flow cytometry intraepithelial lymphocyte (IEL) profile were determined. Both uCD and GFD-CD patients had higher Vα24-Jα18 mRNA levels than non-CD controls (I and C-controls). The expression of Vα24-Jα18 correlated with Marsh score for the severity of mucosal lesion and also with increased mRNA IFNγ levels. uCD and GFD-CD patients had decreased mRNA expression of FoxP3 but increased expression of Vα24-Jα18, which revealed a CD-like molecular profile. Increased numbers of iNKT cells were confirmed by flow cytometry within the intraepithelial lymphocyte compartment of uCD and GFD-CD patients and correlated with Vα24-Jα18 mRNA expression. In conclusion, we have found an increased number of iNKT cells in the duodenum from both uCD and GFD-CD patients, irrespective of the mucosal status. A CD-like molecular profile, defined by an increased mRNA expression of Vα24-Jα18 together with a decreased expression of FoxP3, may represent a pro-inflammatory signature of the CD duodenum. PMID:26529008

Increased Intraepithelial Vα24 Invariant NKT Cells in the Celiac Duodenum.

PubMed

Montalvillo, Enrique; Bernardo, David; Martínez-Abad, Beatriz; Allegretti, Yessica; Fernández-Salazar, Luis; Calvo, Carmen; Chirdo, Fernando G; Garrote, José A; Arranz, Eduardo

2015-10-30

Celiac Disease (CD) is an interferon (IFN)γ-mediated duodenal hypersensitivity to wheat gluten occurring in genetically predisposed individuals. Gluten-free diet (GFD) leads to a complete remission of the disease. Vα24-restricted invariant NKT (iNKT) cells are important to maintain immune homeostasis in the gut mucosa because of their unique capacity to rapidly produce large quantities of both T-helper (Th)1 and Th2 cytokines upon stimulation. We studied the presence of these cells in the CD duodenum. Duodenal biopsies were obtained from 45 untreated-CD patients (uCD), 15 Gluten Free Diet-CD patients (GFD-CD), 44 non-inflamed non-CD controls (C-controls) and 15 inflamed non-CD controls (I-controls). Two populations from Spain and Argentina were recruited. Messenger RNA (mRNA) expression of Vα24-Jα18 (invariant TCRα chain of human iNKT cells), IFNγ and intracellular transcription factor Forkhead Box P3 (Foxp3), and flow cytometry intraepithelial lymphocyte (IEL) profile were determined. Both uCD and GFD-CD patients had higher Vα24-Jα18 mRNA levels than non-CD controls (I and C-controls). The expression of Vα24-Jα18 correlated with Marsh score for the severity of mucosal lesion and also with increased mRNA IFNγ levels. uCD and GFD-CD patients had decreased mRNA expression of FoxP3 but increased expression of Vα24-Jα18, which revealed a CD-like molecular profile. Increased numbers of iNKT cells were confirmed by flow cytometry within the intraepithelial lymphocyte compartment of uCD and GFD-CD patients and correlated with Vα24-Jα18 mRNA expression. In conclusion, we have found an increased number of iNKT cells in the duodenum from both uCD and GFD-CD patients, irrespective of the mucosal status. A CD-like molecular profile, defined by an increased mRNA expression of Vα24-Jα18 together with a decreased expression of FoxP3, may represent a pro-inflammatory signature of the CD duodenum.

Lipid Raft Association Restricts CD44-Ezrin Interaction and Promotion of Breast Cancer Cell Migration

PubMed Central

Donatello, Simona; Babina, Irina S.; Hazelwood, Lee D.; Hill, Arnold D.K.; Nabi, Ivan R.; Hopkins, Ann M.

2012-01-01

Cancer cell migration is an early event in metastasis, the main cause of breast cancer-related deaths. Cholesterol-enriched membrane domains called lipid rafts influence the function of many molecules, including the raft-associated protein CD44. We describe a novel mechanism whereby rafts regulate interactions between CD44 and its binding partner ezrin in migrating breast cancer cells. Specifically, in nonmigrating cells, CD44 and ezrin localized to different membranous compartments: CD44 predominantly in rafts, and ezrin in nonraft compartments. After the induction of migration (either nonspecific or CD44-driven), CD44 affiliation with lipid rafts was decreased. This was accompanied by increased coprecipitation of CD44 and active (threonine-phosphorylated) ezrin-radixin-moesin (ERM) proteins in nonraft compartments and increased colocalization of CD44 with the nonraft protein, transferrin receptor. Pharmacological raft disruption using methyl-β-cyclodextrin also increased CD44-ezrin coprecipitation and colocalization, further suggesting that CD44 interacts with ezrin outside rafts during migration. Conversely, promoting CD44 retention inside lipid rafts by pharmacological inhibition of depalmitoylation virtually abolished CD44-ezrin interactions. However, transient single or double knockdown of flotillin-1 or caveolin-1 was not sufficient to increase cell migration over a short time course, suggesting complex crosstalk mechanisms. We propose a new model for CD44-dependent breast cancer cell migration, where CD44 must relocalize outside lipid rafts to drive cell migration. This could have implications for rafts as pharmacological targets to down-regulate cancer cell migration. PMID:23031255

CDKL2 promotes epithelial-mesenchymal transition and breast cancer progression

PubMed Central

Li, Linna; Liu, Chunping; Amato, Robert J.; Chang, Jeffrey T.; Du, Guangwei; Li, Wenliang

2014-01-01

The epithelial–mesenchymal transition (EMT) confers mesenchymal properties on epithelial cells and has been closely associated with the acquisition of aggressive traits by epithelial cancer cells. To identify novel regulators of EMT, we carried out cDNA screens that covered 500 human kinases. Subsequent characterization of candidate kinases led us to uncover cyclin-dependent kinase-like 2 (CDKL2) as a novel potent promoter for EMT and breast cancer progression. CDKL2-expressing human mammary gland epithelial cells displayed enhanced mesenchymal traits and stem cell-like phenotypes, which was acquired through activating a ZEB1/E-cadherin/β-catenin positive feedback loop and regulating CD44 mRNA alternative splicing to promote conversion of CD24high cells to CD44high cells. Furthermore, CDKL2 enhanced primary tumor formation and metastasis in a breast cancer xenograft model. Notably, CDKL2 is expressed significantly higher in mesenchymal human breast cancer cell lines than in epithelial lines, and its over-expression/amplification in human breast cancers is associated with shorter disease-free survival. Taken together, our study uncovered a major role for CDKL2 in promoting EMT and breast cancer progression. PMID:25333262

CD44v6: A metastasis-associated biomarker in patients with gastric cancer?

PubMed Central

Lu, Li; Huang, Fei; Zhao, Zhicheng; Li, Chuan; Liu, Tong; Li, Weidong; Fu, Weihua

2016-01-01

Abstract Background: The diagnostic and prognostic value of CD44v6 in patients with gastric cancer remains unclear. Therefore, a quantitative meta-analysis was conducted to determine the clinical value of CD44v6 in patients with gastric cancer. Methods: Sixteen studies with 2177 patients were included. Pooled odds ratios (ORs) and hazard ratio (HR) with 95% confidence intervals (CIs) were calculated to estimate the impact of CD44v6 in patients with gastric cancer on clinicopathological features and 5-year overall survival (OS). Sensitivity analysis, subgroup analysis, and regression analysis were introduced to evaluate the heterogeneity across the studies. Publication bias was also explored among the studies. Results: The meta-analysis showed that the upregulated CD44v6 was associated with lymph node metastasis (OR 1.91, 95% CI 1.19–3.08; P = 0.007), distant metastasis (OR 3.41, 95% CI 2.01–5.78; P = 0.000), high TNM stage (OR 2.29, 95% CI 1.10–4.75; P = 0.026), lymphatic vessel invasion (OR 1.59, 95% CI 1.21–2.09; P = 0.001), and vascular invasion (OR 1.57, 95% CI 1.19–2.07; P = 0.001). When excluded 1 study based on sensitivity analysis, pooled HR indicated that CD44v6 positive expression was correlated poor 5-year OS (OR 1.76, 95% CI 1.30–2.39; P = 0.000), meanwhile, heterogeneity was eliminated. The heterogeneity of Lauren type mainly existed in the big sample size subgroup. Different region and publication year might contribute to the heterogeneity of differentiation type. While the heterogeneity of lymph node mainly existed in Asian and big sample size group. Publication bias was observed among 12 studies on lymph node metastasis (Ppublication bias = 0.041), and 5 studies on TNM stage (Ppublication bias = 0.026). Conclusion: Taken together, CD44v6 overexpression might be correlated to the characteristics of tumor metastasis in gastric cancer, consisting with many mechanism studies. Therefore, CD44v6 might present a metastasis-associated biomarker in patients with gastric cancer. PMID:27977599

Improving RNA-Seq expression estimation by modeling isoform- and exon-specific read sequencing rate.

PubMed

Liu, Xuejun; Shi, Xinxin; Chen, Chunlin; Zhang, Li

2015-10-16

The high-throughput sequencing technology, RNA-Seq, has been widely used to quantify gene and isoform expression in the study of transcriptome in recent years. Accurate expression measurement from the millions or billions of short generated reads is obstructed by difficulties. One is ambiguous mapping of reads to reference transcriptome caused by alternative splicing. This increases the uncertainty in estimating isoform expression. The other is non-uniformity of read distribution along the reference transcriptome due to positional, sequencing, mappability and other undiscovered sources of biases. This violates the uniform assumption of read distribution for many expression calculation approaches, such as the direct RPKM calculation and Poisson-based models. Many methods have been proposed to address these difficulties. Some approaches employ latent variable models to discover the underlying pattern of read sequencing. However, most of these methods make bias correction based on surrounding sequence contents and share the bias models by all genes. They therefore cannot estimate gene- and isoform-specific biases as revealed by recent studies. We propose a latent variable model, NLDMseq, to estimate gene and isoform expression. Our method adopts latent variables to model the unknown isoforms, from which reads originate, and the underlying percentage of multiple spliced variants. The isoform- and exon-specific read sequencing biases are modeled to account for the non-uniformity of read distribution, and are identified by utilizing the replicate information of multiple lanes of a single library run. We employ simulation and real data to verify the performance of our method in terms of accuracy in the calculation of gene and isoform expression. Results show that NLDMseq obtains competitive gene and isoform expression compared to popular alternatives. Finally, the proposed method is applied to the detection of differential expression (DE) to show its usefulness in the downstream analysis. The proposed NLDMseq method provides an approach to accurately estimate gene and isoform expression from RNA-Seq data by modeling the isoform- and exon-specific read sequencing biases. It makes use of a latent variable model to discover the hidden pattern of read sequencing. We have shown that it works well in both simulations and real datasets, and has competitive performance compared to popular methods. The method has been implemented as a freely available software which can be found at https://github.com/PUGEA/NLDMseq.

Progesterone receptor isoforms expression pattern in the rat brain during the estrous cycle.

PubMed

Guerra-Araiza, C; Cerbón, M A; Morimoto, S; Camacho-Arroyo, I

2000-03-24

Progesterone receptor (PR) isoforms expression was determined in the hypothalamus, the preoptic area, the hippocampus and the frontal cerebral cortex of the rat at 12:00 h on each day of the estrous cycle by using reverse transcription coupled to polymerase chain reaction. Rats under a 14:10 h light-dark cycle, with lights on at 06:00 h were used. We found that PR-B isoform was predominant in the hypothalamus, the preoptic area and the frontal cerebral cortex. Both PR isoforms were similarly expressed in the hippocampus. The highest PR-B expression was found on proestrus day in the hypothalamus; on metestrus in the preoptic area; and on diestrus in the frontal cortex. We observed no changes in PR isoforms expression in the hippocampus during the estrous cycle. These results indicate that PR isoforms expression is differentially regulated during the estrous cycle in distinct brain regions and that PR-B may be involved in progesterone actions upon the hypothalamus, the preoptic area and the frontal cortex of the rat.

Expression of cytoprotective proteins, heat shock protein 70and metallothioneins, in tissues ofOstrea edulis exposed to heat andheavy metals

PubMed Central

Piano, Annamaria; Valbonesi, Paola; Fabbri, Elena

2004-01-01

Heat shock proteins (Hsps) are constitutively expressed in cells and involved in protein folding, assembly, degradation, intracellular localization, etc, acting as molecular chaperones. However, their overexpression represents a ubiquitous molecular mechanism to cope with stress. Hsps are classified into families, and among them the Hsp70 family appears to be the most evolutionary preserved and distributed in animals. In this study, the expression of Hsp70 and the related messenger ribonucleic acid (mRNA) has been studied in Ostrea edulis after exposure to heat and heavy metals; moreover, levels of metallothioneins (MTs), another class of stress-induced proteins, have contemporaneously been assessed in the same animals. Thermal stress caused the expression of a 69-kDa inducible isoform in gills of O edulis but not in the digestive gland. Northern dot blot analysis confirmed that the transcription of Hsp69-mRNA occurs within 3 hours of stress recovery after oyster exposure at 32 and 35°C. Hsp69-mRNA transcripts were not present in the gills of animals exposed to 38°C after 3 hours of poststress recovery, but they were detected after 24 hours. The expression of the 69-kDa protein in O edulis exposed to 38°C was rather low or totally absent, suggesting that the biochemical machinery at the base of the heat shock response is compromised. Together with the expected increase in MT content, the oysters exposed to Cd showed a significant enhancement of Hsp70, although there was no clear appearance of Hsp69. Interestingly, the levels of MT were significantly increased in the tissues of individuals exposed to thermal stress. Unlike oysters, heat did not provoke the expression of inducible Hsp isoforms in Mytilus galloprovincialis, Tapes philippinarum, and Scapharca inaequivalvis, although it significantly enhanced the expression of constitutive proteins of the 70-kDa family. The expression of newly synthesized Hsp70 isoforms does not seem therefore a common feature in bivalves exposed to thermal stress. PMID:15497500

Radiobiological characteristics of cancer stem cells from esophageal cancer cell lines

PubMed Central

Wang, Jian-Lin; Yu, Jing-Ping; Sun, Zhi-Qiang; Sun, Su-Ping

2014-01-01

AIM: To study the cancer stem cell population in esophageal cancer cell lines KYSE-150 and TE-1 and identify whether the resulting stem-like spheroid cells display cancer stem cells and radiation resistance characteristics. METHODS: A serum-free medium (SFM) suspension was used to culture esophageal cancer stem cell lines and enrich the esophageal stem-like spheres. A reverse transcription polymerase chain reaction assay was used to detect stem cell gene expression in the spheroid cells. Radiosensitivity of stem-like spheres and parental cells were evaluated by clonogenic assays. Furthermore, different cells after different doses of irradiation were tested to evaluate the change in sphere formation, cell cycle and CD44+CD271+ expression of tumor stem-like spheroid cells using flow cytometry before and after irradiation. RESULTS: The cells were observed to generate an increased number of spheres in SFM with increasing cell passage. Radiation increased the rate of generation of stem-like spheres in both types of cells. The average survival fraction (SF2) of the cultured KYSE-150 compared with TE-1 stem-like spheres after 2 Gy of radiation was 0.81 ± 0.03 vs 0.87 ± 0.01 (P < 0.05), while the average SF2 of KYSE-150 compared with TE-1 parental cells was 0.69 ± 0.04 vs 0.80 ± 0.03, P < 0.05. In the esophageal parental cells, irradiation dose-dependently induced G2 arrest. Stem-like esophageal spheres were resistant to irradiation-induced G2 arrest without significant changes in the percentage population of irradiated stem-like cells. Under irradiation at 0, 4, and 8 Gy, the CD44+CD271+ cell percentage for KYSE150 parental cells was 1.08% ± 0.03% vs 1.29% ± 0.07% vs 1.11% ± 0.09%, respectively; the CD44+CD271+ cell percentage for TE1 parental cells was 1.16% ± 0.11% vs 0.97% ± 0.08% vs 1.45% ± 0.35%, respectively. The differences were not statistically significant. Under irradiation at 0, 4, and 8 Gy, the CD44+CD271+ cell percentage for KYSE-150 stem-like spheres was 35.83% ± 1.23% vs 44.9% ± 1.67% vs 57.77% ± 1.88%, respectively; the CD44+CD271+ cell percentage for TE1 stem-like spheres was 16.07% ± 0.91% vs 22.67% ± 1.12%, 16.07% ± 0.91% vs 33.27% ± 1.07%, respectively. The 4 and 8 Gy irradiated KYSE-150 and TE-1 stem-like spheres were compared with the 0 Gy irradiated group, and the differences were statistically significant (P < 0.05). CONCLUSION: The KYSE-150 and TE-1 stem-like spheres are more radioresistant than their parental cells which may suggest that cancer stem cells are related to radioresistance. PMID:25561796

Radiobiological characteristics of cancer stem cells from esophageal cancer cell lines.

PubMed

Wang, Jian-Lin; Yu, Jing-Ping; Sun, Zhi-Qiang; Sun, Su-Ping

2014-12-28

To study the cancer stem cell population in esophageal cancer cell lines KYSE-150 and TE-1 and identify whether the resulting stem-like spheroid cells display cancer stem cells and radiation resistance characteristics. A serum-free medium (SFM) suspension was used to culture esophageal cancer stem cell lines and enrich the esophageal stem-like spheres. A reverse transcription polymerase chain reaction assay was used to detect stem cell gene expression in the spheroid cells. Radiosensitivity of stem-like spheres and parental cells were evaluated by clonogenic assays. Furthermore, different cells after different doses of irradiation were tested to evaluate the change in sphere formation, cell cycle and CD44(+)CD271(+) expression of tumor stem-like spheroid cells using flow cytometry before and after irradiation. The cells were observed to generate an increased number of spheres in SFM with increasing cell passage. Radiation increased the rate of generation of stem-like spheres in both types of cells. The average survival fraction (SF2) of the cultured KYSE-150 compared with TE-1 stem-like spheres after 2 Gy of radiation was 0.81 ± 0.03 vs 0.87 ± 0.01 (P < 0.05), while the average SF2 of KYSE-150 compared with TE-1 parental cells was 0.69 ± 0.04 vs 0.80 ± 0.03, P < 0.05. In the esophageal parental cells, irradiation dose-dependently induced G2 arrest. Stem-like esophageal spheres were resistant to irradiation-induced G2 arrest without significant changes in the percentage population of irradiated stem-like cells. Under irradiation at 0, 4, and 8 Gy, the CD44(+)CD271(+) cell percentage for KYSE150 parental cells was 1.08% ± 0.03% vs 1.29% ± 0.07% vs 1.11% ± 0.09%, respectively; the CD44(+)CD271(+) cell percentage for TE1 parental cells was 1.16% ± 0.11% vs 0.97% ± 0.08% vs 1.45% ± 0.35%, respectively. The differences were not statistically significant. Under irradiation at 0, 4, and 8 Gy, the CD44(+)CD271(+) cell percentage for KYSE-150 stem-like spheres was 35.83% ± 1.23% vs 44.9% ± 1.67% vs 57.77% ± 1.88%, respectively; the CD44(+)CD271(+) cell percentage for TE1 stem-like spheres was 16.07% ± 0.91% vs 22.67% ± 1.12%, 16.07% ± 0.91% vs 33.27% ± 1.07%, respectively. The 4 and 8 Gy irradiated KYSE-150 and TE-1 stem-like spheres were compared with the 0 Gy irradiated group, and the differences were statistically significant (P < 0.05). The KYSE-150 and TE-1 stem-like spheres are more radioresistant than their parental cells which may suggest that cancer stem cells are related to radioresistance.

Absence of Metallothionein 3 Expression in Breast Cancer is a Rare, But Favorable Marker of Outcome that is Under Epigenetic Control

PubMed Central

Somji, Seema; Garrett, Scott H.; Zhou, Xu Dong; Zheng, Yun; Sens, Donald A.; Sens, Mary Ann

2010-01-01

Cadmium (Cd+2), a known carcinogen mimics the effects of estrogen in the uterus and mammary gland suggesting its possible involvement in the development and progression of breast cancer. This lab showed through analysis of a small set of archival human diagnostic specimens that the third isoform of the classic Cd+2 binding protein metallothionein (MT-3), is not expressed in normal breast tissue, but is expressed in some breast cancers and that expression tends to correlate with a poor disease outcome. The goals of the present study were to verify that overexpression of MT-3 in a large set of archival human diagnostic specimens tends to correlate with poor disease outcome and define the mechanism of MT-3 gene regulation in the normal breast epithelial cell. The results showed that MT-3 was expressed in approximately 90% of all breast cancers and was absent in normal breast epithelium. The lack of MT-3 staining in some cancers correlated with a favorable patient outcome. High frequency of MT-3 staining was also found for in situ breast cancer suggesting that MT-3 might be an early biomarker for breast cancer. The study also demonstrated that the MCF-10A cell line, an immortalized, non-tumorigenic model of human breast epithelial cells, displayed no basal expression of MT-3, nor was it induced by Cd+2. Treatment of the MCF-10A cells with the demethylation agent, 5-Aza-2′-deoxycytidine, or the histone deacetylase inhibitor, MS-275, restored MT-3 mRNA expression. It was also shown that the MT-3 metal regulatory elements are potentially active binders of protein factors following treatment with these inhibitors suggesting that MT-3 expression may be subject to epigenetic regulation. PMID:21170156

An enteric pathogen Salmonella enterica serovar Typhimurium suppresses tumor growth by downregulating CD44high and CD4T regulatory (Treg) cell expression in mice: the critical role of lipopolysaccharide and Braun lipoprotein in modulating tumor growth.

PubMed

Liu, T; Chopra, A K

2010-02-01

An antitumor activity associated with several bacterial pathogens, including Salmonella enterica serovar Typhimurium, has been reported; however, the underlying immunological mechanism(s) that lead to an antitumor effect are currently unclear. Furthermore, such pathogens cannot be used to suppress tumor growth because of their potential for causing sepsis. Recently, we reported the characterization of S. Typhimurium isogenic mutants from which Braun lipoprotein genes (lppA and B) and the multicopy repressor of high temperature requirement (msbB) gene were deleted. In a mouse infection model, two mutants, namely, lppB/msbB and lppAB/msbB, minimally induced proinflammatory cytokine production at high doses and were nonlethal to animals. We showed that immunization of mice with these mutants, followed by challenge with the wild-type S. Typhimurium, could significantly suppress tumor growth, as evidenced by an 88% regression in tumor size in lppB/msbB mutant-immunized animals over a 24-day period. However, the lppAB/msbB mutant alone was not effective in modulating tumor growth in mice, although the lppB/msbB mutant alone caused marginal regression in tumor size. Importantly, we showed that CD44(+) cells grew much faster than CD44(-) cells from human liver tumors in mice, leading us to examine the possibility that S. Typhimurium might downregulate CD44 in tumors and splenocytes of mice. Consequently, we found in S. Typhimurium-infected mice that tumor size regression could indeed be related to the downregulation of CD44(high) and CD4(+)CD25(+) T(reg) cells. Importantly, the role of lipopolysaccharide and Braun lipoprotein was critical in S. Typhimurium-induced antitumor immune responses. Taken together, we have defined new immune mechanisms leading to tumor suppression in mice by S. Typhimurium.

Cannabidiol Modulates the Immunophenotype and Inhibits the Activation of the Inflammasome in Human Gingival Mesenchymal Stem Cells

PubMed Central

Libro, Rosaliana; Scionti, Domenico; Diomede, Francesca; Marchisio, Marco; Grassi, Gianpaolo; Pollastro, Federica; Piattelli, Adriano; Bramanti, Placido; Mazzon, Emanuela; Trubiani, Oriana

2016-01-01

Human Gingival Mesenchymal Stem Cells (hGMSCs) are multipotential cells that can expand and differentiate in culture under specific and standardized conditions. In the present study, we have investigated whether in vitro pre-treatment of hGMSCs with Cannabidiol (CBD) can influence their expression profile, improving the therapeutic potential of this cell culture. Following CBD treatment (5 μM) for 24 h, gene expression analysis through Next Generation Sequencing (NGS) has revealed several genes differentially expressed between CBD-treated hGMSCs (CBD-hGMSCs) and control cells (CTR-hGMSCs) that were linked to inflammation and apoptosis. In particular, we have demonstrated that CBD treatment in hGMSCs prevented the activation of the NALP3-inflammasome pathway by suppressing the levels of NALP3, CASP1, and IL18, and in parallel, inhibited apoptosis, as demonstrated by the suppression of Bax. CBD treatment was also able to modulate the expression of the well-known mesenchymal stem cell markers (CD13, CD29, CD73, CD44, CD90, and CD166), and other surface antigens. Specifically, CBD led to the downregulation of genes codifying for antigens involved in the activation of the immune system (CD109, CD151, CD40, CD46, CD59, CD68, CD81, CD82, CD99), while it led to the upregulation of those implicated in the inhibition of the immune responses (CD47, CD55, CD276). In conclusion, the present study will provide a new simple and reproducible method for preconditioning hGMSCs with CBD, before transplantation, as an interesting strategy for improving the hGMSCs molecular phenotype, reducing the risk of immune or inflammatory reactions in the host, and in parallel, for increasing their survival and thus, their long-term therapeutic efficacy. PMID:27932991

FAM13A is associated with non-small cell lung cancer (NSCLC) progression and controls tumor cell proliferation and survival

PubMed Central

Heim, Lisanne; Trump, Sonja; Mittler, Susanne; Sopel, Nina; Andreev, Katerina; Ferrazzi, Fulvia; Ekici, Arif B.; Rieker, Ralf; Springel, Rebekka; Assmann, Vera L.; Lechmann, Matthias; Koch, Sonja; Engelhardt, Marina; Trufa, Denis I.; Sirbu, Horia; Hartmann, Arndt; Finotto, Susetta

2017-01-01

ABSTRACT Genome-wide association studies (GWAS) associated Family with sequence similarity 13, member A (FAM13A) with non-small cell lung cancer (NSCLC) occurrence. Here, we found increased numbers of FAM13A protein expressing cells in the tumoral region of lung tissues from a cohort of patients with NSCLC. Moreover, FAM13A inversely correlated with CTLA4 but directly correlated with HIF1α levels in the control region of these patients. Consistently, FAM13A RhoGAP was found to be associated with T cell effector molecules like HIF1α and Tbet and was downregulated in immunosuppressive CD4+CD25+Foxp3+CTLA4+ T cells. TGFβ, a tumor suppressor factor, as well as siRNA to FAM13A, suppressed both isoforms of FAM13A and inhibited tumor cell proliferation. RNA-Seq analysis confirmed this finding. Moreover, siRNA to FAM13A induced TGFβ levels. Finally, in experimental tumor cell migration, FAM13A was induced and TGFβ accelerated this process by inducing cell migration, HIF1α, and the FAM13A RhoGAP isoform. Furthermore, siRNA to FAM13A inhibited tumor cell proliferation and induced cell migration without affecting HIF1α. In conclusion, FAM13A is involved in tumor cell proliferation and downstream of TGFβ and HIF1α, FAM13A RhoGAP is associated with Th1 gene expression and lung tumor cell migration. These findings identify FAM13A as key regulator of NSCLC growth and progression. PMID:28197372

Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord.

PubMed

Wang, Hwai-Shi; Hung, Shih-Chieh; Peng, Shu-Tine; Huang, Chun-Chieh; Wei, Hung-Mu; Guo, Yi-Jhih; Fu, Yu-Show; Lai, Mei-Chun; Chen, Chin-Chang

2004-01-01

The Wharton's jelly of the umbilical cord contains mucoid connective tissue and fibroblast-like cells. Using flow cytometric analysis, we found that mesenchymal cells isolated from the umbilical cord express matrix receptors (CD44, CD105) and integrin markers (CD29, CD51) but not hematopoietic lineage markers (CD34, CD45). Interestingly, these cells also express significant amounts of mesenchymal stem cell markers (SH2, SH3). We therefore investigated the potential of these cells to differentiate into cardiomyocytes by treating them with 5-azacytidine or by culturing them in cardiomyocyte-conditioned medium and found that both sets of conditions resulted in the expression of cardiomyocyte markers, namely N-cadherin and cardiac troponin I. We also showed that these cells have multilineage potential and that, under suitable culture conditions, are able to differentiate into cells of the adipogenic and osteogenic lineages. These findings may have a significant impact on studies of early human cardiac differentiation, functional genomics, pharmacological testing, cell therapy, and tissue engineering by helping to eliminate worrying ethical and technical issues.

Distinct Transcript Isoforms of the Atypical Chemokine Receptor 1 (ACKR1) / Duffy Antigen Receptor for Chemokines (DARC) Gene Are Expressed in Lymphoblasts and Altered Isoform Levels Are Associated with Genetic Ancestry and the Duffy-Null Allele

PubMed Central

Davis, Melissa B.; Walens, Andrea; Hire, Rupali; Mumin, Kauthar; Brown, Andrea M.; Ford, DeJuana; Howerth, Elizabeth W.; Monteil, Michele

2015-01-01

The Atypical ChemoKine Receptor 1 (ACKR1) gene, better known as Duffy Antigen Receptor for Chemokines (DARC or Duffy), is responsible for the Duffy Blood Group and plays a major role in regulating the circulating homeostatic levels of pro-inflammatory chemokines. Previous studies have shown that one common variant, the Duffy Null (Fy-) allele that is specific to African Ancestry groups, completely removes expression of the gene on erythrocytes; however, these individuals retain endothelial expression. Additional alleles are associated with a myriad of clinical outcomes related to immune responses and inflammation. In addition to allele variants, there are two distinct transcript isoforms of DARC which are expressed from separate promoters, and very little is known about the distinct transcriptional regulation or the distinct functionality of these protein isoforms. Our objective was to determine if the African specific Fy- allele alters the expression pattern of DARC isoforms and therefore could potentially result in a unique signature of the gene products, commonly referred to as antigens. Our work is the first to establish that there is expression of DARC on lymphoblasts. Our data indicates that people of African ancestry have distinct relative levels of DARC isoforms expressed in these cells. We conclude that the expression of both isoforms in combination with alternate alleles yields multiple Duffy antigens in ancestry groups, depending upon the haplotypes across the gene. Importantly, we hypothesize that DARC isoform expression patterns will translate into ancestry-specific inflammatory responses that are correlated with the axis of pro-inflammatory chemokine levels and distinct isoform-specific interactions with these chemokines. Ultimately, this work will increase knowledge of biological mechanisms underlying disparate clinical outcomes of inflammatory-related diseases among ethnic and geographic ancestry groups. PMID:26473357

BCc1, the novel antineoplastic nanocomplex, showed potent anticancer effects in vitro and in vivo.

PubMed

Kalanaky, Somayeh; Hafizi, Maryam; Fakharzadeh, Saideh; Vasei, Mohammad; Langroudi, Ladan; Janzamin, Ehsan; Hashemi, Seyed Mahmoud; Khayamzadeh, Maryam; Soleimani, Masoud; Akbari, Mohammad Esmaeil; Nazaran, Mohammad Hassan

2016-01-01

In spite of all the efforts and researches on anticancer therapeutics, an absolute treatment is still a myth. Therefore, it is necessary to utilize novel technologies in order to synthesize smart multifunctional structures. In this study, for the first time, we have evaluated the anticancer effects of BCc1 nanocomplex by vitro and in vivo studies, which is designed based on the novel nanochelating technology. Human breast adenocarcinoma cell line (MCF-7) and mouse embryonic fibroblasts were used for the in vitro study. Antioxidant potential, cell toxicity, apoptosis induction, and CD44 and CD24 protein expression were evaluated after treatment of cells with different concentrations of BCc1 nanocomplex. For the in vivo study, mammary tumor-bearing female Balb/c mice were treated with different doses of BCc1 and their effects on tumor growth rate and survival were evaluated. BCc1 decreased CD44 protein expression and increased CD24 protein expression. It induced MCF-7 cell apoptosis but at the same concentrations did not have negative effects on mouse embryonic fibroblasts viability and protected them against oxidative stress. Treatment with nanocomplex increased survival and reduced the tumor size growth in breast cancer-bearing balb/c mice. These results demonstrate that BCc1 has the capacity to be assessed as a new anticancer agent in complementary studies.

Auraptene Attenuates Malignant Properties of Esophageal Stem-Like Cancer Cells.

PubMed

Saboor-Maleki, Saffiyeh; Rassouli, Fatemeh B; Matin, Maryam M; Iranshahi, Mehrdad

2017-08-01

The high incidence of esophageal squamous cell carcinoma has been reported in selected ethnic populations including North of Iran. Low survival rate of esophageal carcinoma is partially due to the presence of stem-like cancer cells with chemotherapy resistance. In the current study, we aimed to determine the effects of auraptene, an interesting dietary coumarin with various biological activities, on malignant properties of stem-like esophageal squamous cell carcinoma, in terms of sensitivity to anticancer drugs and expression of specific markers. To do so, the half maximal inhibitory concentration values of auraptene, cisplatin, paclitaxel, and 5-fluorouracil were determined on esophageal carcinoma cells (KYSE30 cell line). After administrating combinatorial treatments, including nontoxic concentrations of auraptene + cisplatin, paclitaxel, or 5-fluorouracil, sensitivity of cells to chemical drugs and also induced apoptosis were assessed. In addition, quantitative real-time polymerase chain reaction was used to study changes in the expression of tumor suppressor proteins 53 and 21 ( P53 and P21), cluster of differentiation 44 ( CD44), and B cell-specific Moloney murine leukemia virus integration site 1 ( BMI-1) upon treatments. Results of thiazolyl blue assay revealed that auraptene significantly ( P < .05) increased toxicity of cisplatin, paclitaxel, and 5-fluorouracil in KYSE30 cells, specifically 72 hours after treatment. Conducting an apoptosis assay using flow cytometry also confirmed the synergic effects of auraptene. Results of quantitative real-time polymerase chain reaction revealed significant ( P < .05) upregulation of P53 and P21 upon combinatorial treatments and also downregulation of CD44 and BMI-1 after auraptene administration. Current study provided evidence, for the first time, that auraptene attenuates the properties of esophageal stem-like cancer cells through enhancing sensitivity to chemical agents and reducing the expression of CD44 and BMI-1 markers.

Serglycin as a potential biomarker for glioma: association of serglycin expression, extent of mast cell recruitment and glioblastoma progression

PubMed Central

Roy, Ananya; Attarha, Sanaz; Weishaupt, Holger; Edqvist, Per-Henrik; Swartling, Fredrik J.; Bergqvist, Michael; Siebzehnrubl, Florian A.; Smits, Anja; Pontén, Fredrik; Tchougounova, Elena

2017-01-01

Serglycin is an intracellular proteoglycan with a unique ability to adopt highly divergent structures by glycosylation with variable types of glycosaminoglycans (GAGs) when expressed by different cell types. Serglycin is overexpressed in aggressive cancers suggesting its protumorigenic role. In this study, we explored the expression of serglycin in human glioma and its correlation with survival and immune cell infiltration. We demonstrate that serglycin is expressed in glioma and that increased expression predicts poor survival of patients. Analysis of serglycin expression in a large cohort of low- and high-grade human glioma samples reveals that its expression is grade dependent and is positively correlated with mast cell (MC) infiltration. Moreover, serglycin expression in patient-derived glioma cells is significantly increased upon MC co-culture. This is also accompanied by increased expression of CXCL12, CXCL10, as well as markers of cancer progression, including CD44, ZEB1 and vimentin. In conclusion, these findings indicate the importance of infiltrating MCs in glioma by modulating signaling cascades involving serglycin, CD44 and ZEB1. The present investigation reveals serglycin as a potential prognostic marker for glioma and demonstrates an association with the extent of MC recruitment and glioma progression, uncovering potential future therapeutic opportunities for patients. PMID:28445977

Overexpression of c-kit(CD117), relevant with microvessel density, is an independent survival prognostic factor for patients with HBV-related hepatocellular carcinoma.

PubMed

Yan, Weiwei; Zhu, Zhenyu; Pan, Fei; Huang, Ang; Dai, Guang-Hai

2018-01-01

To explore new biomarkers for indicating the recurrence and prognosis in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) patients after tumor resection, we investigated the expression and prognostic value of c-kit(CD117) in HBV-related HCC. Immunohistochemistry was used to estimate the expression of c-kit(CD117) and CD34 in the liver cancer tissues. The correlations between the expression of these biomarkers and the clinicopathologic characteristics were analyzed. The positive rate of c-kit(CD117) expression in 206 HCC cases was 48.1%, and c-kit expression was significantly related with CD34-positive microvessel density. CD34-microvessel density numbers were much higher in c-kit(+) HCC tissues than in c-kit(-) HCC tissues (44.13±17.01 vs 26.87±13.16, P =0.003). The expression of c-kit was significantly higher in patients with Edmondson grade III-IV ( P <0.001) and TNM stage III ( P <0.001). Moreover, Kaplan-Meier survival analysis showed that c-kit ( P <0.001) expression was correlated with reduced disease-free survival (DFS). Multivariate analysis identified c-kit as an independent poor prognostic factor of DFS in HCC patients ( P <0.001). Increased c-kit expression could be considered as an independent unfavorable prognostic factor for predicting DFS in HBV-related HCC patients after surgery. These results could be used to identify patients at a higher risk of early tumor recurrence and poor prognosis.

AF1q is a novel TCF7 co-factor which activates CD44 and promotes breast cancer metastasis.

PubMed

Park, Jino; Schlederer, Michaela; Schreiber, Martin; Ice, Ryan; Merkel, Olaf; Bilban, Martin; Hofbauer, Sebastian; Kim, Soojin; Addison, Joseph; Zou, Jie; Ji, Chunyan; Bunting, Silvia T; Wang, Zhengqi; Shoham, Menachem; Huang, Gang; Bago-Horvath, Zsuzsanna; Gibson, Laura F; Rojanasakul, Yon; Remick, Scot; Ivanov, Alexey; Pugacheva, Elena; Bunting, Kevin D; Moriggl, Richard; Kenner, Lukas; Tse, William

2015-08-21

AF1q is an MLL fusion partner that was identified from acute myeloid leukemia (AML) patients with t (1; 11) (q21; q23) chromosomal abnormality. The function of AF1q is not yet fully known, however, elevated AF1q expression is associated with poor clinical outcomes in various malignancies. Here, we show that AF1q specifically binds to T-cell-factor-7 (TCF7) in the Wnt signaling pathway and results in transcriptional activation of CD44 as well as multiple downstream targets of the TCF7/LEF1. In addition, enhanced AF1q expression promotes breast cancer cell proliferation, migration, mammosphere formation, and chemo-resistance. In xenograft models, enforced AF1q expression in breast cancer cells also promotes liver metastasis and lung colonization. In a cohort of 63 breast cancer patients, higher percentages of AF1q-positive cancer cells in primary sites were associated with significantly poorer overall survival (OS), disease-free survival (DFS), and brain metastasis-free survival (b-MFS). Using paired primary/metastatic samples from the same patients, we demonstrate that AF1q-positive breast cancer cells become dynamically dominant in the metastatic sites compared to the primary sites. Our findings indicate that breast cancer cells with a hyperactive AF1q/TCF7/CD44 regulatory axis in the primary sites may represent "metastatic founder cells" which have invasive properties.

Loss of the two major leaf isoforms of sucrose-phosphate synthase in Arabidopsis thaliana limits sucrose synthesis and nocturnal starch degradation but does not alter carbon partitioning during photosynthesis

PubMed Central

Volkert, Kathrin; Debast, Stefan; Voll, Lars M.; Voll, Hildegard; Schießl, Ingrid; Hofmann, Jörg; Schneider, Sabine; Börnke, Frederik

2014-01-01

Sucrose (Suc)-phosphate synthase (SPS) catalyses one of the rate-limiting steps in the synthesis of Suc in plants. The Arabidopsis genome contains four annotated SPS genes which can be grouped into three different families (SPSA1, SPSA2, SPSB, and SPSC). However, the functional significance of this multiplicity of SPS genes is as yet only poorly understood. All four SPS isoforms show enzymatic activity when expressed in yeast although there is variation in sensitivity towards allosteric effectors. Promoter–reporter gene analyses and quantitative real-time reverse transcription–PCR studies indicate that no two SPS genes have the same expression pattern and that AtSPSA1 and AtSPSC represent the major isoforms expressed in leaves. An spsa1 knock-out mutant showed a 44% decrease in leaf SPS activity and a slight increase in leaf starch content at the end of the light period as well as at the end of the dark period. The spsc null mutant displayed reduced Suc contents towards the end of the photoperiod and a concomitant 25% reduction in SPS activity. In contrast, an spsa1/spsc double mutant was strongly impaired in growth and accumulated high levels of starch. This increase in starch was probably not due to an increased partitioning of carbon into starch, but was rather caused by an impaired starch mobilization during the night. Suc export from excised petioles harvested from spsa1/spsc double mutant plants was significantly reduced under illumination as well as during the dark period. It is concluded that loss of the two major SPS isoforms in leaves limits Suc synthesis without grossly changing carbon partitioning in favour of starch during the light period but limits starch degradation during the dark period. PMID:24994761

Human breast adipose tissue: characterization of factors that change during tumor progression in human breast cancer.

PubMed

Fletcher, Sabrina Johanna; Sacca, Paula Alejandra; Pistone-Creydt, Mercedes; Coló, Federico Andrés; Serra, María Florencia; Santino, Flavia Eliana; Sasso, Corina Verónica; Lopez-Fontana, Constanza Matilde; Carón, Rubén Walter; Calvo, Juan Carlos; Pistone-Creydt, Virginia

2017-02-07

Adipose microenvironment is involved in signaling pathways that influence breast cancer. We aim to characterize factors that are modified: 1) in tumor and non tumor human breast epithelial cell lines when incubated with conditioned media (CMs) from human breast cancer adipose tissue explants (hATT) or normal breast adipose tissue explants (hATN); 2) in hATN-CMs vs hATT-CMs; 3) in the tumor associated adipocytes vs. non tumor associated adipocytes. We used hATN or hATT- CMs on tumor and non-tumor breast cancer cell lines. We evaluated changes in versican, CD44, ADAMTS1 and Adipo R1 expression on cell lines or in the different CMs. In addition we evaluated changes in the morphology and expression of these factors in slices of the different adipose tissues. The statistical significance between different experimental conditions was evaluated by one-way ANOVA. Tukey's post-hoc tests were performed within each individual treatment. hATT-CMs increase versican, CD44, ADAMTS1 and Adipo R1 expression in breast cancer epithelial cells. Furthermore, hATT-CMs present higher levels of versican expression compared to hATN-CMs. In addition, we observed a loss of effect in cellular migration when we pre-incubated hATT-CMs with chondroitinase ABC, which cleaves GAGs chains bound to the versican core protein, thus losing the ability to bind to CD44. Adipocytes associated with the invasive front are reduced in size compared to adipocytes that are farther away. Also, hATT adipocytes express significantly higher amounts of versican, CD44 and Adipo R1, and significantly lower amounts of adiponectin and perilipin, unlike hATN adipocytes. We conclude that hATT secrete a different set of proteins compared to hATN. Furthermore, versican, a proteoglycan that is overexpressed in hATT-CMs compared to hATN-CMs, might be involved in the tumorogenic behavior observed in both cell lines employed. In addition, we may conclude that adipocytes from the tumor microenvironment show a less differentiated state than adipocytes from normal microenvironment. This would indicate a loss of normal functions in mature adipocytes (such as energy storage), in support of others that might favor tumor growth.

Expression Profiles of Ligands for Activating Natural Killer Cell Receptors on HIV Infected and Uninfected CD4⁺ T Cells.

PubMed

Tremblay-McLean, Alexandra; Bruneau, Julie; Lebouché, Bertrand; Lisovsky, Irene; Song, Rujun; Bernard, Nicole F

2017-10-12

Natural Killer (NK) cell responses to HIV-infected CD4 T cells (iCD4) depend on the integration of signals received through inhibitory (iNKR) and activating NK receptors (aNKR). iCD4 activate NK cells to inhibit HIV replication. HIV infection-dependent changes in the human leukocyte antigen (HLA) ligands for iNKR on iCD4 are well documented. By contrast, less is known regarding the HIV infection related changes in ligands for aNKR on iCD4. We examined the aNKR ligand profiles HIV p24⁺ HIV iCD4s that maintained cell surface CD4 (iCD4⁺), did not maintain CD4 (iCD4 - ) and uninfected CD4 (unCD4) T cells for expression of unique long (UL)-16 binding proteins-1 (ULBP-1), ULBP-2/5/6, ULBP-3, major histocompatibility complex (MHC) class 1-related (MIC)-A, MIC-B, CD48, CD80, CD86, CD112, CD155, Intercellular adhesion molecule (ICAM)-1, ICAM-2, HLA-E, HLA-F, HLA-A2, HLA-C, and the ligands to NKp30, NKp44, NKp46, and killer immunoglobulin-like receptor 3DS1 (KIR3DS1) by flow cytometry on CD4 T cells from 17 HIV-1 seronegative donors activated and infected with HIV. iCD4⁺ cells had higher expression of aNKR ligands than did unCD4. However, the expression of aNKR ligands on iCD4 where CD4 was downregulated (iCD4 - ) was similar to (ULBP-1, ULBP-2/5/6, ULBP-3, MIC-A, CD48, CD80, CD86 and CD155) or significantly lower than (MIC-B, CD112 and ICAM-2) what was observed on unCD4. Thus, HIV infection can be associated with increased expression of aNKR ligands or either baseline or lower than baseline levels of aNKR ligands, concomitantly with the HIV-mediated downregulation of cell surface CD4 on infected cells.

1,25-DIHYDROXYVITAMIN D3 INDUCES MONOCYTIC DIFFERENTIATION OF HUMAN MYELOID LEUKEMIA CELLS BY REGULATING C/EBPβ EXPRESSION THROUGH MEF2C

PubMed Central

Zheng, Ruifang; Wang, Xuening; Studzinski, George P.

2015-01-01

Myogenic enhancer factor2 (Mef2) consists of a family of transcription factors involved in morphogenesis of skeletal, cardiac and smooth muscle cells. Among the four isoforms (Mef2A, 2B, 2C, and 2D), Mef2C was also found to play important roles in hematopoiesis. At myeloid progenitor level, Mef2C expression favors monocytic differentiation. Previous studies from our laboratory demonstrated that ERK5 was activated in 1,25-dihydroxyvitamin D3 (1,25D)-induced monocytic differentiation in AML cells and ERK5 activation was accompanied by increased Mef2C phosphorylation. We therefore examined the role of Mef2C in 1,25D-induced monocytic differentiation in AML cell lines (HL60, U937 and THP1) and found that knockdown of Mef2C with small interfering RNA (siRNA) significantly decreases the expression of the monocytic marker, CD14, without affecting the expression of the general myeloid marker, CD11b. CCAAT/Enhancer-binding protein (C/EBP) β, which can bind to CD14 promoter and increase its transcription, has been shown to be the downstream effector of 1,25D-induced monocytic differentiation in AML cells. When Mef2C was knocked down, expression of C/EBPβ was reduced at both mRNA and protein levels. The protein expression levels of cell cycle regulators, p27Kip1 and cyclin D1, were not affected by Mef2C knockdown, nor the monopoiesis related transcription factor, ATF2 (Activating Transcription Factor 2). Thus, we conclude that 1,25D-induced monocytic differentiation, and CD14 expression in particular, is mediated through activation of ERK5-Mef2C-C/EBPβ signaling pathway, and that Mef2C does not seem to modulate cell cycle progression. PMID:25448741

Reviewing and Updating the Major Molecular Markers for Stem Cells

PubMed Central

Calloni, Raquel; Cordero, Elvira Alicia Aparicio; Henriques, João Antonio Pêgas

2013-01-01

Stem cells (SC) are able to self-renew and to differentiate into many types of committed cells, making SCs interesting for cellular therapy. However, the pool of SCs in vivo and in vitro consists of a mix of cells at several stages of differentiation, making it difficult to obtain a homogeneous population of SCs for research. Therefore, it is important to isolate and characterize unambiguous molecular markers that can be applied to SCs. Here, we review classical and new candidate molecular markers that have been established to show a molecular profile for human embryonic stem cells (hESCs), mesenchymal stem cells (MSCs), and hematopoietic stem cells (HSCs). The commonly cited markers for embryonic ESCs are Nanog, Oct-4, Sox-2, Rex-1, Dnmt3b, Lin-28, Tdgf1, FoxD3, Tert, Utf-1, Gal, Cx43, Gdf3, Gtcm1, Terf1, Terf2, Lefty A, and Lefty B. MSCs are primarily identified by the expression of CD13, CD29, CD44, CD49e, CD54, CD71, CD73, CD90, CD105, CD106, CD166, and HLA-ABC and lack CD14, CD31, CD34, CD45, CD62E, CD62L, CD62P, and HLA-DR expression. HSCs are mainly isolated based on the expression of CD34, but the combination of this marker with CD133 and CD90, together with a lack of CD38 and other lineage markers, provides the most homogeneous pool of SCs. Here, we present new and alternative markers for SCs, along with microRNA profiles, for these cells. PMID:23336433

Stemness and angiogenic gene expression changes of serial-passage human amnion mesenchymal cells.

PubMed

Fatimah, Simat Siti; Tan, Geok Chin; Chua, Kienhui; Fariha, Mohd Manzor Nur; Tan, Ay Eeng; Hayati, Abdul Rahman

2013-03-01

Particular attention has been directed towards human amnion mesenchymal stem cells (HAMCs) due to their accessibility, availability and immunomodulatory properties. Therefore, the aim of the present study was to determine the temporal changes of stemness and angiogenic gene expressions of serial-passage HAMCs. HAMCs were isolated from human term placenta and cultured in serial passages in culture medium supplemented with 10% fetal bovine serum. Morphological analysis, growth kinetic and CFU-F assay of HAMCs were assessed. In vitro differentiation and the immunophenotype of HAMCs at P5 were also analyzed. Quantitative PCR was used to determine the stemness, angiogenic and endothelial gene expression of cultured HAMCs after serial passage. Cultured HAMCs displayed intermediate epitheloid-fibroblastoid morphology at an initial culture and the fibroblastoid features became more pronounced in later passages. They showed high clonogenic activity and faster proliferation at later passages with colony forming efficiency of 0.88%. HAMCs were successfully differentiated into adipocytes, osteocytes and neuron-like cells. Most HAMCs expressed CD9, CD44, CD73, CD90 and HLA-A,B,C but negligibly expressed CD31, CD34, CD45, CD117 and HLA-DR,DP,DQ. After serial passage, stemness genes Oct-3/4, Sox-2, Nanog3, Rex-1, FGF-4 and FZD-9 expressions significantly decreased. Of the angiogenic genes PECAM-1, bFGF, eNOS, VEGFR-2, VEGF, and vWF expressions also decreased significantly except angiopoietin-1 which significantly increased. No significant differences were observed in ABCG-2, BST-1, nestin, PGF and HGF expressions after serial passage. These results suggested that cultured HAMCs could be an alternative source of stem cells and may have the potential for angiogenesis and hence its use in stem-cell based therapy. Copyright © 2012 Elsevier Inc. All rights reserved.

Insulin Restores Gestational Diabetes Mellitus–Reduced Adenosine Transport Involving Differential Expression of Insulin Receptor Isoforms in Human Umbilical Vein Endothelium

PubMed Central

Westermeier, Francisco; Salomón, Carlos; González, Marcelo; Puebla, Carlos; Guzmán-Gutiérrez, Enrique; Cifuentes, Fredi; Leiva, Andrea; Casanello, Paola; Sobrevia, Luis

2011-01-01

OBJECTIVE To determine whether insulin reverses gestational diabetes mellitus (GDM)–reduced expression and activity of human equilibrative nucleoside transporters 1 (hENT1) in human umbilical vein endothelium cells (HUVECs). RESEARCH DESIGN AND METHODS Primary cultured HUVECs from full-term normal (n = 44) and diet-treated GDM (n = 44) pregnancies were used. Insulin effect was assayed on hENT1 expression (protein, mRNA, SLC29A1 promoter activity) and activity (initial rates of adenosine transport) as well as endothelial nitric oxide (NO) synthase activity (serine1177 phosphorylation, l-citrulline formation). Adenosine concentration in culture medium and umbilical vein blood (high-performance liquid chromatography) as well as insulin receptor A and B expression (quantitative PCR) were determined. Reactivity of umbilical vein rings to adenosine and insulin was assayed by wire myography. Experiments were in the absence or presence of l-NG-nitro-l-arginine methyl ester (l-NAME; NO synthase inhibitor) or ZM-241385 (an A2A-adenosine receptor antagonist). RESULTS Umbilical vein blood adenosine concentration was higher, and the adenosine- and insulin-induced NO/endothelium-dependent umbilical vein relaxation was lower in GDM. Cells from GDM exhibited increased insulin receptor A isoform expression in addition to the reported NO–dependent inhibition of hENT1-adenosine transport and SLC29A1 reporter repression, and increased extracellular concentration of adenosine and NO synthase activity. Insulin reversed all these parameters to values in normal pregnancies, an effect blocked by ZM-241385 and l-NAME. CONCLUSIONS GDM and normal pregnancy HUVEC phenotypes are differentially responsive to insulin, a phenomenon where insulin acts as protecting factor for endothelial dysfunction characteristic of this syndrome. Abnormal adenosine plasma levels, and potentially A2A-adenosine receptors and insulin receptor A, will play crucial roles in this phenomenon in GDM. PMID:21515851

CD44 Is a Negative Cell Surface Marker for Pluripotent Stem Cell Identification during Human Fibroblast Reprogramming

PubMed Central

Vaz, Candida; Tanavde, Vivek; Lakshmipathy, Uma

2014-01-01

Induced pluripotent stem cells (iPSCs) are promising tools for disease research and cell therapy. One of the critical steps in establishing iPSC lines is the early identification of fully reprogrammed colonies among unreprogrammed fibroblasts and partially reprogrammed intermediates. Currently, colony morphology and pluripotent stem cell surface markers are used to identify iPSC colonies. Through additional clonal characterization, we show that these tools fail to distinguish partially reprogrammed intermediates from fully reprogrammed iPSCs. Thus, they can lead to the selection of suboptimal clones for expansion. A subsequent global transcriptome analysis revealed that the cell adhesion protein CD44 is a marker that differentiates between partially and fully reprogrammed cells. Immunohistochemistry and flow cytometry confirmed that CD44 is highly expressed in the human parental fibroblasts used for the reprogramming experiments. It is gradually lost throughout the reprogramming process and is absent in fully established iPSCs. When used in conjunction with pluripotent cell markers, CD44 staining results in the clear identification of fully reprogrammed cells. This combination of positive and negative surface markers allows for easier and more accurate iPSC detection and selection, thus reducing the effort spent on suboptimal iPSC clones. PMID:24416407

MCT1, MCT4 and CD147 gene polymorphisms in healthy horses and horses with myopathy.

PubMed

Mykkänen, A K; Koho, N M; Reeben, M; McGowan, C M; Pösö, A R

2011-12-01

Polymorphisms in human lactate transporter proteins (monocarboxylate transporters; MCTs), especially the MCT1 isoform, can affect lactate transport activity and cause signs of exercise-induced myopathy. Muscles express MCT1, MCT4 and CD147, an ancillary protein, indispensable for the activity of MCT1 and MCT4. We sequenced the coding sequence (cDNA) of horse MCT4 for the first time and examined polymorphisms in the cDNA of MCT1, MCT4 and CD147 of 16 healthy horses. To study whether signs of myopathy are linked to the polymorphisms, biopsy samples were taken from 26 horses with exercise-induced recurrent myopathy. Two polymorphisms that cause a change in amino acid sequence were found in MCT1 (Val(432)Ile and Lys(457)Gln) and one in CD147 (Met(125)Val). All polymorphisms in MCT4 were silent. Mutations in MCT1 or CD147 in equine muscle were not associated with myopathy. In the future, a functional study design is needed to evaluate the physiological role of the polymorphisms found. Copyright © 2010 Elsevier Ltd. All rights reserved.

Regulation of Asymmetric Division and CD8+ T Lymphocyte Fate Specification by PKCζ and PKCλ/ι

PubMed Central

Metz, Patrick J.; Arsenio, Janilyn; Kakaradov, Boyko; Kim, Stephanie H.; Remedios, Kelly A.; Oakley, Katherine; Akimoto, Kazunori; Ohno, Shigeo; Yeo, Gene W.; Chang, John T.

2015-01-01

During an immune response against a microbial pathogen, activated naïve T lymphocytes give rise to effector cells that provide acute host defense and memory cells that provide long-lived immunity. It has been shown that T lymphocytes can undergo asymmetric division, enabling the daughter cells to inherit unequal amounts of fate-determining proteins and thereby acquire distinct fates from their inception. Here, we show that the absence of the atypical protein kinase C (aPKC) isoforms, PKCζ and PKCλ/ι, disrupts asymmetric CD8+ T lymphocyte division. These alterations were associated with aberrant acquisition of a ‘pre-effector’ transcriptional program, detected by single-cell gene expression analyses, in lymphocytes that had undergone their first division in vivo and enhanced differentiation toward effector fates at the expense of memory fates. Together, these results demonstrate a role for aPKC in regulating asymmetric division and the specification of divergent CD8+ T lymphocyte fates early during an immune response. PMID:25617472

MDR1 siRNA loaded hyaluronic acid-based CD44 targeted nanoparticle systems circumvent paclitaxel resistance in ovarian cancer

PubMed Central

Yang, Xiaoqian; lyer, Arun K.; Singh, Amit; Choy, Edwin; Hornicek, Francis J.; Amiji, Mansoor M.; Duan, Zhenfeng

2015-01-01

Development of multidrug resistance (MDR) is an almost universal phenomenon in patients with ovarian cancer, and this severely limits the ultimate success of chemotherapy in the clinic. Overexpression of the MDR1 gene and corresponding P-glycoprotein (Pgp) is one of the best known MDR mechanisms. MDR1 siRNA based strategies were proposed to circumvent MDR, however, systemic, safe, and effective targeted delivery is still a major challenge. Cluster of differentiation 44 (CD44) targeted hyaluronic acid (HA) based nanoparticle has been shown to successfully deliver chemotherapy agents or siRNAs into tumor cells. The goal of this study is to evaluate the ability of HA-PEI/HA-PEG to deliver MDR1 siRNA and the efficacy of the combination of HA-PEI/HA-PEG/MDR1 siRNA with paclitaxel to suppress growth of ovarian cancer. We observed that HA-PEI/HA-PEG nanoparticles can efficiently deliver MDR1 siRNA into MDR ovarian cancer cells, resulting in down-regulation of MDR1 and Pgp expression. Administration of HA-PEI/HA-PEG/MDR1 siRNA nanoparticles followed by paclitaxel treatment induced a significant inhibitory effect on the tumor growth, decreased Pgp expression and increased apoptosis in MDR ovarian cancer mice model. Our findings suggest that CD44 targeted HA-PEI/HA-PEG/MDR1 siRNA nanoparticles can serve as a therapeutic tool with great potentials to circumvent MDR in ovarian cancer. PMID:25687880

MDR1 siRNA loaded hyaluronic acid-based CD44 targeted nanoparticle systems circumvent paclitaxel resistance in ovarian cancer

NASA Astrophysics Data System (ADS)

Yang, Xiaoqian; Lyer, Arun K.; Singh, Amit; Choy, Edwin; Hornicek, Francis J.; Amiji, Mansoor M.; Duan, Zhenfeng

2015-02-01

Development of multidrug resistance (MDR) is an almost universal phenomenon in patients with ovarian cancer, and this severely limits the ultimate success of chemotherapy in the clinic. Overexpression of the MDR1 gene and corresponding P-glycoprotein (Pgp) is one of the best known MDR mechanisms. MDR1 siRNA based strategies were proposed to circumvent MDR, however, systemic, safe, and effective targeted delivery is still a major challenge. Cluster of differentiation 44 (CD44) targeted hyaluronic acid (HA) based nanoparticle has been shown to successfully deliver chemotherapy agents or siRNAs into tumor cells. The goal of this study is to evaluate the ability of HA-PEI/HA-PEG to deliver MDR1 siRNA and the efficacy of the combination of HA-PEI/HA-PEG/MDR1 siRNA with paclitaxel to suppress growth of ovarian cancer. We observed that HA-PEI/HA-PEG nanoparticles can efficiently deliver MDR1 siRNA into MDR ovarian cancer cells, resulting in down-regulation of MDR1 and Pgp expression. Administration of HA-PEI/HA-PEG/MDR1 siRNA nanoparticles followed by paclitaxel treatment induced a significant inhibitory effect on the tumor growth, decreased Pgp expression and increased apoptosis in MDR ovarian cancer mice model. Our findings suggest that CD44 targeted HA-PEI/HA-PEG/MDR1 siRNA nanoparticles can serve as a therapeutic tool with great potentials to circumvent MDR in ovarian cancer.

Characterization of the expression of the pro-metastatic Mena(INV) isoform during breast tumor progression.

PubMed

Oudin, Madeleine J; Hughes, Shannon K; Rohani, Nazanin; Moufarrej, Mira N; Jones, Joan G; Condeelis, John S; Lauffenburger, Douglas A; Gertler, Frank B

2016-03-01

Several functionally distinct isoforms of the actin regulatory Mena are produced by alternative splicing during tumor progression. Forced expression of the Mena(INV) isoform drives invasion, intravasation and metastasis. However, the abundance and distribution of endogenously expressed Mena(INV) within primary tumors during progression remain unknown, as most studies to date have only assessed relative mRNA levels from dissociated tumor samples. We have developed a Mena(INV) isoform-specific monoclonal antibody and used it to examine Mena(INV) expression patterns in mouse mammary and human breast tumors. Mena(INV) expression increases during tumor progression and to examine the relationship between Mena(INV) expression and markers for epithelial or mesenchymal status, stemness, stromal cell types and hypoxic regions. Further, while Mena(INV) robustly expressed in vascularized areas of the tumor, it is not confined to cells adjacent to blood vessels. Altogether, these data demonstrate the specificity and utility of the anti-Mena(INV)-isoform specific antibody, and provide the first description of endogenous Mena(INV) protein expression in mouse and human tumors.

Expression of Metallothionein and Vascular Endothelial Growth Factor Isoforms in Breast Cancer Cells.

PubMed

Wierzowiecka, Barbara; Gomulkiewicz, Agnieszka; Cwynar-Zajac, Lucja; Olbromski, Mateusz; Grzegrzolka, Jedrzej; Kobierzycki, Christopher; Podhorska-Okolow, Marzenna; Dziegiel, Piotr

2016-01-01

Metallothioneins (MTs) are low-molecular-weight and cysteine-rich proteins that bind heavy metal ions and oxygen-free radicals. MTs are commonly expressed in various tissues of mammals and are involved in regulation of cell proliferation and differentiation, and may be engaged in angiogenesis. Expression of MTs has been studied in many cancer types, especially breast cancer. The research results indicate that MTs may play important, although not yet fully known, roles in cancer angiogenesis. The aim of this study was to analyze the level of gene expression of selected MT isoforms induced with zinc ions in correlation with vascular endothelial growth factor (VEGF) isoforms in in vitro models of breast cancer. The studies were carried out in three breast cancer cell lines (MCF-7, SK-BR-3, MDA-MB-231). An epithelial cell line derived from normal breast tissue (Me16c) was used as a control. The levels of expression of selected MT isoforms and selected genes involved in angiogenesis were studied with real-time PCR. Expression of different MT isoforms was induced by zinc ions to differing degrees in individual breast cancer cell lines. An increase in the expression of some MT isoforms was associated with a slight increase in the level of expression of VEGFA. The research results may indicate certain correlation between an increased expression of selected MT isoforms and a pro-angiogenic factor VEGF in specific types of breast cancer cells. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Characterisation of monoclonal antibodies specific for hamster leukocyte differentiation molecules.

PubMed

Rees, Jennifer; Haig, David; Mack, Victoria; Davis, William C

2017-01-01

Flow cytometry was used to identify mAbs that recognize conserved epitopes on hamster leukocyte differentiation molecules (hLDM) and also to characterize mAbs developed against hLDM. Initial screening of mAbs developed against LDMs in other species yielded mAbs specific for the major histocompatibility (MHC) II molecule, CD4 and CD18. Screening of sets of mAbs developed against hLDM yielded 22 new mAbs, including additional mAbs to MHC II molecules and mAbs that recognize LDMs expressed on all leukocytes, granulocytes, all lymphocytes, all T cells, a subset of T cells, or on all B cells. Based on comparison of the pattern of expression of LDMs expressed on all hamster leukocytes with the patterns of expression of known LDMs in other species, as detected by flow cytometry (FC), four mAbs are predicted to recognize CD11a, CD44, and CD45. Cross comparison of mAbs specific for a subset of hamster T cells with a cross reactive mAb known to recognize CD4 in mice and one recognising CD8 revealed they recognize CD4. The characterization of these mAbs expands opportunities to use hamsters as an additional model species to investigate the mechanisms of immunopathogenesis of infectious diseases. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

The ThPOK transcription factor differentially affects the development and function of self-specific CD8(+) T cells and regulatory CD4(+) T cells.

PubMed

Twu, Yuh-Ching; Teh, Hung-Sia

2014-03-01

The zinc finger transcription factor ThPOK plays a crucial role in CD4 T-cell development and CD4/CD8 lineage decision. In ThPOK-deficient mice, developing T cells expressing MHC class II-restricted T-cell receptors are redirected into the CD8 T-cell lineage. In this study, we investigated whether the ThPOK transgene affected the development and function of two additional types of T cells, namely self-specific CD8 T cells and CD4(+) FoxP3(+) T regulatory cells. Self-specific CD8 T cells are characterized by high expression of CD44, CD122, Ly6C, 1B11 and proliferation in response to either IL-2 or IL-15. The ThPOK transgene converted these self-specific CD8 T cells into CD4 T cells. The converted CD4(+) T cells are no longer self-reactive, lose the characteristics of self-specific CD8 T cells, acquire the properties of conventional CD4 T cells and survive poorly in peripheral lymphoid organs. By contrast, the ThPOK transgene promoted the development of CD4(+) FoxP3(+) regulatory T cells resulting in an increased recovery of CD4(+) FoxP3(+) regulatory T cells that expressed higher transforming growth factor-β-dependent suppressor activity. These studies indicate that the ThPOK transcription factor differentially affects the development and function of self-specific CD8 T cells and CD4(+) FoxP3(+) regulatory T cells. © 2013 John Wiley & Sons Ltd.

Recruited brain tumor-derived mesenchymal stem cells contribute to brain tumor progression.

PubMed

Behnan, Jinan; Isakson, Pauline; Joel, Mrinal; Cilio, Corrado; Langmoen, Iver A; Vik-Mo, Einar O; Badn, Wiaam

2014-05-01

The identity of the cells that contribute to brain tumor structure and progression remains unclear. Mesenchymal stem cells (MSCs) have recently been isolated from normal mouse brain. Here, we report the infiltration of MSC-like cells into the GL261 murine glioma model. These brain tumor-derived mesenchymal stem cells (BT-MSCs) are defined with the phenotype (Lin-Sca-1+CD9+CD44+CD166+/-) and have multipotent differentiation capacity. We show that the infiltration of BT-MSCs correlates to tumor progression; furthermore, BT-MSCs increased the proliferation rate of GL261 cells in vitro. For the first time, we report that the majority of GL261 cells expressed mesenchymal phenotype under both adherent and sphere culture conditions in vitro and that the non-MSC population is nontumorigenic in vivo. Although the GL261 cell line expressed mesenchymal phenotype markers in vitro, most BT-MSCs are recruited cells from host origin in both wild-type GL261 inoculated into green fluorescent protein (GFP)-transgenic mice and GL261-GFP cells inoculated into wild-type mice. We show the expression of chemokine receptors CXCR4 and CXCR6 on different recruited cell populations. In vivo, the GL261 cells change marker profile and acquire a phenotype that is more similar to cells growing in sphere culture conditions. Finally, we identify a BT-MSC population in human glioblastoma that is CD44+CD9+CD166+ both in freshly isolated and culture-expanded cells. Our data indicate that cells with MSC-like phenotype infiltrate into the tumor stroma and play an important role in tumor cell growth in vitro and in vivo. Thus, we suggest that targeting BT-MSCs could be a possible strategy for treating glioblastoma patients. © 2013 AlphaMed Press.

Effects of low-doses of Bacillus spp. from permafrost on differentiation of bone marrow cells.

PubMed

Kalyonova, L F; Novikova, M A; Kostolomova, E G

2015-01-01

The effects of a new microorganism species (Bacillus spp., strain M3) isolated from permafrost specimens from Central Yakutia (Mamontova Mountain) on the bone marrow hemopoiesis were studied on laboratory mice. Analysis of the count and immunophenotype of bone marrow cells indicated that even in low doses (1000-5000 microbial cells) these microorganisms modulated hemopoiesis and lymphopoiesis activity. The percentage of early hemopoietic precursors (CD117(+)CD34(-)) increased, intensity of lymphocyte precursor proliferation and differentiation (CD25(+)CD44(-)) decreased, and the percentage of lymphocytes released from the bone marrow (CD25(+)CD44(+)) increased on day 21 after injection of the bacteria. These changes in activity of hemopoiesis were associated with changes in the level of regulatory T lymphocytes (reduced expression of TCRαβ) and were most likely compensatory. The possibility of modulating hemopoiesis activity in the bone marrow by low doses of one microorganism strain isolated from the permafrost could be useful for evaluating the effects of other low dose bacteria on the bone marrow hemopoiesis.

Ethylene Receptor 1 (ETR1) Is Sufficient and Has the Predominant Role in Mediating Inhibition of Ethylene Responses by Silver in Arabidopsis thaliana*

PubMed Central

McDaniel, Brittany K.; Binder, Brad M.

2012-01-01

Ethylene influences many processes in Arabidopsis thaliana through the action of five receptor isoforms. All five isoforms use copper as a cofactor for binding ethylene. Previous research showed that silver can substitute for copper as a cofactor for ethylene binding activity in the ETR1 ethylene receptor yet also inhibit ethylene responses in plants. End-point and rapid kinetic analyses of dark-grown seedling growth revealed that the effects of silver are mostly dependent upon ETR1, and ETR1 alone is sufficient for the effects of silver. Ethylene responses in etr1-6 etr2-3 ein4-4 triple mutants were not blocked by silver. Transformation of these triple mutants with cDNA for each receptor isoform under the promoter control of ETR1 revealed that the cETR1 transgene completely rescued responses to silver while the cETR2 transgene failed to rescue these responses. The other three isoforms partially rescued responses to silver. Ethylene binding assays on the binding domains of the five receptor isoforms expressed in yeast showed that silver supports ethylene binding to ETR1 and ERS1 but not the other isoforms. Thus, silver may have an effect on ethylene signaling outside of the ethylene binding pocket of the receptors. Ethylene binding to ETR1 with silver was ∼30% of binding with copper. However, alterations in the Kd for ethylene binding to ETR1 and the half-time of ethylene dissociation from ETR1 do not underlie this lower binding. Thus, it is likely that the lower ethylene binding activity of ETR1 with silver is due to fewer ethylene binding sites generated with silver versus copper. PMID:22692214

Identification of Insulin Receptor Splice Variant B in Neurons by in situ Detection in Human Brain Samples.

PubMed

Spencer, Brian; Rank, Logan; Metcalf, Jeff; Desplats, Paula

2018-03-06

Insulin and its receptor are widely expressed in a variety of tissues throughout the body including liver, adipose tissue, liver and brain. The insulin receptor is expressed as two functionally distinct isoforms, differentiated by a single 12 amino acid exon. The two receptor isoforms, designated IR/A and IR/B, are expressed in a highly tissue and cell specific manner and relative proportions of the different isoforms vary during development, aging and disease states. The high degree of similarity between the two isoforms has prevented detailed studies as differentiation of the two isoforms by traditional immunological methods cannot be achieved. We describe here a new in situ RT-PCR/ FISH assay that allows for the visualization of IR/A and IR/B in tissue along with tissue specific markers. We used this new method to show for the first time that IR/A and IR/B are both expressed in neurons in the adult human brain. Thus, we present a method that enables the investigation of IR/A and IR/B insulin receptor isoform expression in situ in various tissues.

Expansion of PD-1-positive effector CD4 T cells in an experimental model of SLE: contribution to the self-organized criticality theory.

PubMed

Miyazaki, Yumi; Tsumiyama, Ken; Yamane, Takashi; Ito, Mitsuhiro; Shiozawa, Shunichi

2013-04-18

We have developed a systems biology concept to explain the origin of systemic autoimmunity. From our studies of systemic lupus erythematosus (SLE) we have concluded that this disease is the inevitable consequence of over-stimulating the host's immune system by repeated exposure to antigen to levels that surpass a critical threshold, which we term the system's "self-organized criticality". We observed that overstimulation of CD4 T cells in mice led to the development of autoantibody-inducing CD4 T cells (aiCD4 T) capable of generating various autoantibodies and pathological lesions identical to those observed in SLE. We show here that this is accompanied by the significant expansion of a novel population of effector T cells characterized by expression of programmed death-1 (PD-1)-positive, CD27(low), CD127(low), CCR7(low) and CD44(high)CD62L(low) markers, as well as increased production of IL-2 and IL-6. In addition, repeated immunization caused the expansion of CD8 T cells into fully-matured cytotoxic T lymphocytes (CTL) that express Ly6C(high)CD122(high) effector and memory markers. Thus, overstimulation with antigen leads to the expansion of a novel effector CD4 T cell population that expresses an unusual memory marker, PD-1, and that may contribute to the pathogenesis of SLE.

Triton-polyacrylamide gel electrophoresis and leucine aminopeptidase activity staining detect Triton-slowed bands including high-molecular-mass aminopeptidase N (CD13) isoform in cholestatic patient sera.

PubMed

Kawai, Makoto; Hara, Yukichi

2006-02-01

Western blotting of aminopeptidase N (APN) detects a high-molecular-mass isoform (260 kDa) [M. Kawai, Y. Otake, Y. Hara High-molecular-mass isoform of aminopeptidase N/CD13 in serum from cholestatic patients. Clin Chim Acta 330 (2003) 141-149] in cholestatic patient serum but is time-consuming. Human sera were electrophoresed on polyacrylamide gel containing Triton-X100 (Triton-PAGE) and stained with leucine-B-naphthylamide (LAP-staining). The stained bands were eluted from the gel, treated with N- and O-glycosidase if necessary, and analyzed by Western blotting [M. Kawai, Y. Otake, Y. Hara High-molecular-mass isoform of aminopeptidase N/CD13 in serum from cholestatic patients. Clin Chim Acta 330 (2003) 141-149]. Triton-PAGE and LAP-staining clearly detected fast bands in all the sera examined. Almost parallel with leucine aminopeptidase activity, slow bands were strongly stained in all 11 cholestatic patients but clearly stained in 3 out of 14 patients with hepatobiliary diseases other than cholestasis. PAGE with various concentrations of Triton showed that Triton slows down slow bands but not fast bands. Western blotting showed that Triton-PAGE-slow bands of cholestasis contained 140 and 260-kDa APN and that fast bands were slightly smaller than monomer-size slow bands after glycosidase treatment. Less time-consuming than Western blotting, Triton-PAGE and LAP-staining detect novel APN bands slowed by Triton and partly composed of the high-molecular-mass isoform in cholestasis. The slow bands seem to be homodimers of APN with transmembrane anchors. The polypeptide of the fast band seems to be processed differently from that of the slow band.

Pixel based SHG probes of extracellular matrix (ECM) alterations in ovarian cancer (Conference Presentation)

NASA Astrophysics Data System (ADS)

Campbell, Kirby R.; Chaudhary, Rajeev; Handel, Julia; Campagnola, Paul J.

2017-02-01

Remodeling of the extracellular matrix in human ovarian cancer, can be reflected in increased collagen concentration, changes in alignment and/or up-regulation of different collagen isoforms, including Col III. Using fibrillar gel models, we demonstrate that Col I and Col III can be quantitatively distinguished by 3 distinct SHG polarization specific metrics: i) determination of helical pitch angle via the single axis molecular model, ii) dipole alignment via anisotropy, and iii) chirality via SHG circular dichroism (SHG-CD). These sub-resolution differentiations are possible due to differences in the α helix angles of the two isoforms, which co-mingle in the same fibrils. We also investigated the mechanism of the SHG-CD response and show that unlike conventional CD, it is dominated by electric dipole interactions and is consistent with the two state SHG model. We further applied these 3 polarization resolved analyses to human normal, high risk, benign tumors, and malignant human ovarian tissues. We found that these tissues could all be differentiated by these metrics, where high grade tissues had analogous α-helical pitch angles to the in the Col I/Col III gel model. This confirms literature suggestions based on immunofluorescence and gene expression that Col III is up-regulated in high grade ovarian cancers. The different tissues also displayed differing anisotropies, indicating the fibril assemblies are distinct and likely do not result from remodeling of existing collagen but synthesis of new collagen. Importantly, these SHG polarization methods provide structural information not otherwise possible and can serve as label-free biomarkers for ovarian and other cancers.

Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets

PubMed Central

Möhle, Robert; Green, David; Moore, Malcolm A. S.; Nachman, Ralph L.; Rafii, Shahin

1997-01-01

We have shown that coculture of bone marrow microvascular endothelial cells with hematopoietic progenitor cells results in proliferation and differentiation of megakaryocytes. In these long-term cultures, bone marrow microvascular endothelial cell monolayers maintain their cellular integrity in the absence of exogenous endothelial growth factors. Because this interaction may involve paracrine secretion of cytokines, we evaluated megakaryocytic cells for secretion of vascular endothelial growth factor (VEGF). Megakaryocytes (CD41a+) were generated by ex vivo expansion of hematopoietic progenitor cells with kit-ligand and thrombopoietin for 10 days and further purified with immunomagnetic microbeads. Using reverse transcription–PCR, we showed that megakaryocytic cell lines (Dami, HEL) and purified megakaryocytes expressed mRNA of the three VEGF isoforms (121, 165, and 189 amino acids). Large quantities of VEGF (>1 ng/106 cells/3 days) were detected in the supernatant of Dami cells, ex vivo-generated megakaryocytes, and CD41a+ cells isolated from bone marrow. The constitutive secretion of VEGF by CD41a+ cells was stimulated by growth factors of the megakaryocytic lineage (interleukin 3, thrombopoietin). Western blotting of heparin–Sepharose-enriched supernatant mainly detected the isoform VEGF165. In addition, immunohistochemistry showed intracytoplasmic VEGF in polyploid megakaryocytes. Thrombin stimulation of megakaryocytes and platelets resulted in rapid release of VEGF within 30 min. We conclude that human megakaryocytes produce and secrete VEGF in an inducible manner. Within the bone marrow microenvironment, VEGF secreted by megakaryocytes may contribute to the proliferation of endothelial cells. VEGF delivered to sites of vascular injury by activated platelets may initiate angiogenesis. PMID:9012841

CD45RO enriches for activated, highly mutated human germinal center B cells

PubMed Central

Jackson, Stephen M.; Harp, Natessa; Patel, Darshna; Zhang, Jeffrey; Willson, Savannah; Kim, Yoon J.; Clanton, Christian

2007-01-01

To date, there is no consensus regarding the influence of different CD45 isoforms during peripheral B-cell development. Examining correlations between surface CD45RO expression and various physiologic processes ongoing during the germinal center (GC) reaction, we hypothesized that GC B cells, like T cells, that up-regulate surface RO should progressively acquire phenotypes commonly associated with activated, differentiating lymphocytes. GC B cells (IgD−CD38+) were subdivided into 3 surface CD45RO fractions: RO−, RO+/−, and RO+. We show here that the average number of mutations per IgVH transcript increased in direct correlation with surface RO levels. Conjunctional use of RO and CD69 further delineated low/moderately and highly mutated fractions. Activation-induced cytidine deaminase (AID) mRNA was slightly reduced among RO+ GC B cells, suggesting that higher mutation averages are unlikely due to elevated somatic mutation activity. Instead, RO+ GC B cells were negative for Annexin V, comprised mostly (93%) of CD77− centrocytes, and were enriched for CD69+ cells. Collectively, RO+ GC B cells occupy what seems to be a specialized niche comprised mostly of centrocytes that may be in transition between activation states. These findings are among the first to sort GC B cells into populations enriched for live mutated cells solely using a single extracellular marker. PMID:17644737

Altered expression of hyaluronan, HAS1-2, and HYAL1-2 in oral lichen planus.

PubMed

Siponen, Maria; Kullaa, Arja; Nieminen, Pentti; Salo, Tuula; Pasonen-Seppänen, Sanna

2015-07-01

Oral lichen planus (OLP) is an immune-mediated mucosal disease of unclear etiology and of unresolved pathogenesis. Hyaluronan (HA) is an extracellular matrix glycosaminoglycan involved in inflammation and tumor progression. However, its presence in OLP has not been reported. We therefore aimed to study the immunohistochemical expression of HA, its receptor CD44, hyaluronan synthases (HAS1-3), and hyaluronidases (HYAL1-2) in OLP. The presence of HA, CD44, HAS1-3, and HYAL1-2 was studied by immunohistochemical methods in 55 OLP and 23 control oral mucosal specimens (CTR). The localization, intensity, and differences of the epithelial expression between OLP and CTRs were analyzed. HA and CD44 were found on cell membranes in the epithelial basal and intermediate layers in CTR and OLP specimens. The HA staining intensity was stronger in the basal layer of the epithelium in OLP than in CTRs (P < 0.001). HAS1 (P = 0.001) and HAS2 (P < 0.001) showed stronger staining in the basal and weaker staining in the superficial (P < 0.001) epithelial layers in OLP than in CTRs. The immunostaining of HAS3 was low in both OLP and CTRs. Positive HYAL1 and HYAL2 staining were mainly found in the basal and intermediate epithelial layers, and their intensities were significantly increased in OLP, except HYAL 2 in the intermediate epithelial layer. HA, HAS1-2, and HYAL1-2 have altered expression in OLP compared to CTRs and may therefore have a role in OLP pathogenesis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Level of PAX5 in differential diagnosis of non-Hodgkin's lymphoma

PubMed Central

Bharti, Brij; Shukla, Sachin; Tripathi, Ratnakar; Mishra, Suman; Kumar, Mohan; Pandey, Manoj; Mishra, Rajnikant

2016-01-01

Background & objectives: The PAX5, a paired box transcription factor and B-cell activator protein (BSAP), activates B-cell commitment genes and represses non-B-cell lineage genes. About 14 transcript variants of PAX5 have been observed in human. Any alteration in its expression pattern leads to lymphogenesis or associated diseases and carcinogenesis in non-lymphoid tissues. Its mechanisms of function in pathophysiology of non-Hodgkin's lymphoma (NHL) are unclear. This study was intended to explore influence of PAX5 in cascade of NHL pathogenesis and diagnosis. Methods: Samples of 65 patients were evaluated by immunohistochemical staining for cellular localization of PAX5, CD19, CD3, cABL, p53, Ras and Raf and by TUNEL assay, RNA-isolation and reverse transcriptase (RT)-PCR, Western blot analysis, and lactate dehydrogenase (LDH) specific staining. Results: B-cell type NHL patients were positive for PAX5, p53, Ras, CD19, Raf and CD3. All of them showed TUNEL-positive cells. The differential expression pattern of PAX5, CD19, p53, CD3, ZAP70, HIF1α, Ras, Raf and MAPK (mitogen-activated protein kinase) at the levels of transcripts and proteins was observed. The LDH assay showed modulation of LDH4 and LDH5 isoforms in the lymph nodes of NHL patients. Interpretation & conclusions: The histological observations suggested that the patients represent diverse cases of NHL like mature B-cell type, mature T-cell type and high grade diffuse B-cell type NHL. The findings indicate that patients with NHL may also be analyzed for status of PAX5, CD19 and ZAP70, and their transcriptional and post-translational variants for the differential diagnosis of NHL and therapy. PMID:27748274

In vitro Catecholamine Exposure Produces Variable Effects on the B7 Costimulatory Pathway in Human Monocytic Cells

NASA Technical Reports Server (NTRS)

Salicru, A. N.; Crucian, B.; Sams, Clarence; Actor, J. K.; Marshall, G. D., Jr.

2006-01-01

Catecholamines have been associated with immunomodulation of the adaptive immune system towards a Th2 response in vitro. We therefore examined the role of in vitro epinephrine (EPI) and norepinephrine (NE) exposure on the B7 costimulatory expression of antigen presenting cells (APC) from human monocytic cell lines and human peripheral blood mononuclear cells (PBMC). THP1 monocytic cells and CD14+ cells from normal human PBMC were stimulated with lipopolysaccharide (LPS) and incubated with physiologic stress levels (10(exp -6) - 10(exp -8)M) of EPI or NE for 24 hours. Cells were subsequently stained with CD80 FITC, CD86 PE, and CD14 PC5 antibodies and analyzed by flow cytometry for changes in fluorescence and mean fluorescence intensity (MFI). Exposure of THP1 to EPI in vitro at concentrations of 10(exp -6), 10(exp -7) and 10(exp -8)M significantly decreased mean CD80 from 42 plus or minus 0.7% to 11 plus or minus 0.44%, 19.1 plus or minus 2.0%, and 30.7 plus or minus 2.1% expression, respectively (p less than 0.01). In addition, CD86 expression increased with EPI at 10(exp -6), 10(exp -7) and 10(exp -8) M from 9.2 plus or minus 0.52% to 41 plus or minus 3.8%, 26.4 plus or minus 1.9%, and 15.74 plus or minus 1.8% expression, respectively (p less than 0.01). Similar results for mean CD80 and CD86 percent expression were observed for CD14+ cells from PBMC with a sample size of N = 6 and for NE when substituted for EPI. The data show that in vitro exposure to catecholamines significantly decreases %CD86 expression and significantly increases %CD86 expression in THP1 cells and human CD14+ APC. Previous studies have suggested an association between increased CD86 expression and TH2 activity. Thus, these data suggest that immunomodulation by catecholamines results in part by the variable effects of the B7 costimulatory pathway in APC.

Universal monitoring of minimal residual disease in acute myeloid leukemia.

PubMed

Coustan-Smith, Elaine; Song, Guangchun; Shurtleff, Sheila; Yeoh, Allen Eng-Juh; Chng, Wee Joo; Chen, Siew Peng; Rubnitz, Jeffrey E; Pui, Ching-Hon; Downing, James R; Campana, Dario

2018-05-03

Optimal management of acute myeloid leukemia (AML) requires monitoring of treatment response, but minimal residual disease (MRD) may escape detection. We sought to identify distinctive features of AML cells for universal MRD monitoring. We compared genome-wide gene expression of AML cells from 157 patients with that of normal myeloblasts. Markers encoded by aberrantly expressed genes, including some previously associated with leukemia stem cells, were studied by flow cytometry in 240 patients with AML and in nonleukemic myeloblasts from 63 bone marrow samples. Twenty-two (CD9, CD18, CD25, CD32, CD44, CD47, CD52, CD54, CD59, CD64, CD68, CD86, CD93, CD96, CD97, CD99, CD123, CD200, CD300a/c, CD366, CD371, and CX3CR1) markers were aberrantly expressed in AML. Leukemia-associated profiles defined by these markers extended to immature CD34+CD38- AML cells; expression remained stable during treatment. The markers yielded MRD measurements matching those of standard methods in 208 samples from 52 patients undergoing chemotherapy and revealed otherwise undetectable MRD. They allowed MRD monitoring in 129 consecutive patients, yielding prognostically significant results. Using a machine-learning algorithm to reduce high-dimensional data sets to 2-dimensional data, the markers allowed a clear visualization of MRD and could detect 1 leukemic cell among more than 100,000 normal cells. The markers uncovered in this study allow universal and sensitive monitoring of MRD in AML. In combination with contemporary analytical tools, the markers improve the discrimination between leukemic and normal cells, thus facilitating data interpretation and, hence, the reliability of MRD results. National Cancer Institute (CA60419 and CA21765); American Lebanese Syrian Associated Charities; National Medical Research Council of Singapore (1299/2011); Viva Foundation for Children with Cancer, Children's Cancer Foundation, Tote Board & Turf Club, and Lee Foundation of Singapore.

Differential Properties of Cytomegalovirus pUL97 Kinase Isoforms Affect Viral Replication and Maribavir Susceptibility

PubMed Central

Webel, Rike; Hakki, Morgan; Prichard, Mark N.; Rawlinson, William D.; Marschall, Manfred

2014-01-01

ABSTRACT The human cytomegalovirus (HCMV)-encoded kinase pUL97 is required for efficient viral replication. Previous studies described two isoforms of pUL97, the full-length isoform (M1) and a smaller isoform likely resulting from translation initiation at codon 74 (M74). Here, we report the detection of a third pUL97 isoform during viral infection resulting from translation initiation at codon 157 (isoform M157). The consistent expression of isoform M157 as a minor component of pUL97 during infection with clinical and laboratory-adapted HCMV strains was suppressed when codon 157 was mutagenized. Viral mutants expressing specific isoforms were generated to compare their growth and drug susceptibility phenotypes, as well as pUL97 intracellular localization patterns and kinase activities. The exclusive expression of isoform M157 resulted in substantially reduced viral growth and resistance to the pUL97 inhibitor maribavir while retaining susceptibility to ganciclovir. Confocal imaging demonstrated reduced nuclear import of amino-terminal deletion isoforms compared to isoform M1. Isoform M157 showed reduced efficiency of various substrate protein interactions and autophosphorylation, whereas Rb phosphorylation was preserved. These results reveal differential properties of pUL97 isoforms that affect viral replication, with implications for the antiviral efficacy of maribavir. IMPORTANCE The HCMV UL97 kinase performs important functions in viral replication that are targeted by the antiviral drug maribavir. Here, we describe a naturally occurring short isoform of the kinase that when expressed by itself in a recombinant virus results in altered intracellular localization, impaired growth, and high-level resistance to maribavir compared to those of the predominant full-length counterpart. This is another factor to consider in explaining why maribavir appears to have variable antiviral activity in cell culture and in vivo. PMID:24522923

BORIS/CTCFL mRNA isoform expression and epigenetic regulation in epithelial ovarian cancer

PubMed Central

Link, Petra A.; Zhang, Wa; Odunsi, Kunle; Karpf, Adam R.

2013-01-01

Cancer germline (CG) genes are normally expressed in germ cells and aberrantly expressed in a variety of cancers; their immunogenicity has led to the widespread development of cancer vaccines targeting these antigens. BORIS/CTCFL is an autosomal CG antigen and promising cancer vaccine target. BORIS is the only known paralog of CTCF, a gene intimately involved in genomic imprinting, chromatin insulation, and nuclear regulation. We have previously shown that BORIS is expressed in epithelial ovarian cancer (EOC) and that its expression coincides with promoter and global DNA hypomethylation. Recently, 23 different BORIS mRNA variants have been described, and have been functionally grouped into six BORIS isoform families (sf1–sf6). In the present study, we have characterized the expression of BORIS isoform families in normal ovary (NO) and EOC, the latter of which were selected to include two groups with widely varying global DNA methylation status. We find selective expression of BORIS isoform families in NO, which becomes altered in EOC, primarily by the activation of BORIS sf1 in EOC. When comparing EOC samples based on methylation status, we find that BORIS sf1 and sf2 isoform families are selectively activated in globally hypomethylated tumors. In contrast, CTCF is downregulated in EOC, and the ratio of BORIS sf1, sf2, and sf6 isoform families as a function of CTCF is elevated in hypomethylated tumors. Finally, the expression of all BORIS isoform families was induced to varying extents by epigenetic modulatory drugs in EOC cell lines, particularly when DNMT and HDAC inhibitors were used in combination. PMID:23390377

Isoform-level gene expression patterns in single-cell RNA-sequencing data.

PubMed

Vu, Trung Nghia; Wills, Quin F; Kalari, Krishna R; Niu, Nifang; Wang, Liewei; Pawitan, Yudi; Rantalainen, Mattias

2018-02-27

RNA sequencing of single cells enables characterization of transcriptional heterogeneity in seemingly homogeneous cell populations. Single-cell sequencing has been applied in a wide range of researches fields. However, few studies have focus on characterization of isoform-level expression patterns at the single-cell level. In this study we propose and apply a novel method, ISOform-Patterns (ISOP), based on mixture modeling, to characterize the expression patterns of isoform pairs from the same gene in single-cell isoform-level expression data. We define six principal patterns of isoform expression relationships and describe a method for differential-pattern analysis. We demonstrate ISOP through analysis of single-cell RNA-sequencing data from a breast cancer cell line, with replication in three independent datasets. We assigned the pattern types to each of 16,562 isoform-pairs from 4,929 genes. Among those, 26% of the discovered patterns were significant (p<0.05), while remaining patterns are possibly effects of transcriptional bursting, drop-out and stochastic biological heterogeneity. Furthermore, 32% of genes discovered through differential-pattern analysis were not detected by differential-expression analysis. The effect of drop-out events, mean expression level, and properties of the expression distribution on the performances of ISOP were also investigated through simulated datasets. To conclude, ISOP provides a novel approach for characterization of isoformlevel preference, commitment and heterogeneity in single-cell RNA-sequencing data. The ISOP method has been implemented as a R package and is available at https://github.com/nghiavtr/ISOP under a GPL-3 license. mattias.rantalainen@ki.se. Supplementary data are available at Bioinformatics online.

CD45RA, a specific marker for leukaemia stem cell sub-populations in acute myeloid leukaemia.

PubMed

Kersten, Bas; Valkering, Matthijs; Wouters, Rolf; van Amerongen, Rosa; Hanekamp, Diana; Kwidama, Zinia; Valk, Peter; Ossenkoppele, Gert; Zeijlemaker, Wendelien; Kaspers, Gertjan; Cloos, Jacqueline; Schuurhuis, Gerrit J

2016-04-01

Chemotherapy resistant leukaemic stem cells (LSC) are thought to be responsible for relapses after therapy in acute myeloid leukaemia (AML). Flow cytometry can discriminate CD34(+) CD38(-) LSC and normal haematopoietic stem cells (HSC) by using aberrant expression of markers and scatter properties. However, not all LSC can be identified using currently available markers, so new markers are needed. CD45RA is expressed on leukaemic cells in the majority of AML patients. We investigated the potency of CD45RA to specifically identify LSC and HSC and improve LSC quantification. Compared to our best other markers (CLL-1, also termed CLEC12A, CD33 and CD123), CD45RA was the most reliable marker. Patients with high percentages (>90%) of CD45RA on CD34(+) CD38(-) LSC have 1·69-fold higher scatter values compared to HSC (P < 0·001), indicating a more mature CD34(+) CD38(-) phenotype. Patients with low (<10%) or intermediate (10-90%) CD45RA expression on LSC showed no significant differences to HSC (1·12- and 1·15-fold higher, P = 0·31 and P = 0·44, respectively). CD45RA-positive LSC tended to represent more favourable cytogenetic/molecular markers. In conclusion, CD45RA contributes to more accurate LSC detection and is recommended for inclusion in stem cell tracking panels. CD45RA may contribute to define new LSC-specific therapies and to monitor effects of anti-LSC treatment. © 2016 John Wiley & Sons Ltd.

Salivary proline-rich proteins and gluten: Do structural similarities suggest a role in celiac disease?

PubMed

Tian, Na; Messana, Irene; Leffler, Daniel A; Kelly, Ciaran P; Hansen, Joshua; Cabras, Tiziana; D'Alessandro, Alfredo; Schuppan, Detlef; Castagnola, Massimo; Helmerhorst, Eva J

2015-10-01

Gluten proteins, the culprits in celiac disease (CD), show striking similarities in primary structure with human salivary proline-rich proteins (PRPs). Both are enriched in proline and glutamine residues that often occur consecutively in their sequences. We investigated potential differences in the spectrum of salivary PRPs in health and CD. Stimulated salivary secretions were collected from CD patients, patients with refractory CD, patients with gastrointestinal complaints but no CD, and healthy controls. PRP isoforms/peptides were characterized by anionic and SDS-PAGE, PCR, and LC-ESI-MS. The gene frequencies of the acidic PRP isoforms PIF, Db, Pa, PRP1, and PRP2 did not differ between groups. At the protein level, PRPs peptides showed minor group differences, but these could not differentiate the CD and/or refractory CDs groups from the controls. This extensive study established that salivary PRPs, despite similarity to gluten proteins, show no apparent correlation with CD and thus will not serve as diagnostic markers for the disease. The structural basis for the tolerance to the gluten-like PRP proteins in CD is worthy of further exploration and may lead to the development of gluten-like analogs lacking immunogenicity that could be used therapeutically. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Estrogen receptor mRNA expression patterns in the liver and ovary of female rainbow trout over a complete reproductive cycle

PubMed Central

Nagler, James J.; Cavileer, Timothy D.; Verducci, Joseph S.; Schultz, Irvin R.; Hook, Sharon E.; Hayton, William L.

2012-01-01

Estrogens are critical hormones involved in reproduction and need to bind to estrogen receptors in target organs for biological activity. Fishes have two distinct estrogen receptor subtypes, alpha (α) and beta (β), with variable combinations of additional isoforms of each subtype dependent on the history of genome duplication within a taxon. The comparative expression patterns of estrogen receptor isoforms during the female reproductive cycle will provide important insights into the unique function and importance of each. The purpose of this study was to measure the mRNAs for the four estrogen receptor isoforms (erα1, erα2, erβ1, erβ2) in the liver and ovary of adult, female rainbow trout over the course of an annual reproductive cycle. The expression of estrogen receptor mRNA isoforms was measured by quantitative real-time RT-PCR. Several reproductive indices (gonadosomatic index, maximum oocyte diameter, plasma estradiol-17β, plasma vitellogenin, and ovulation) were also quantified for comparison and used in a correlation analysis to examine any inter-relationships. Of the four isoforms, the expression of erα1 was highest in the liver, and had a significant positive correlation with liver erβ1 expression. Liver expression of erα2 mRNA was the lowest, but showed a significant positive correlation with maximum oocyte diameter in the ovary. The pattern of the erβ isoforms in liver was one of initially elevated mRNA expression followed by a gradual decrease as reproductive development proceeded. In the ovary the erβ1 isoform had the highest mRNA expression of all estrogen receptor isoforms, at the beginning of the reproductive cycle, but then decreased afterward. Both ovarian erβ isoforms had a significant positive correlation with one another. In contrast, erα2 mRNA expression showed a high maximum level in the ovary near the end of the cycle along with a significant positive correlation with plasma estradiol-17β levels; the highest gonadosomatic indices, maximum oocyte diameter, and vitellogenin levels occurred then too. PMID:22732076

Ouabain interactions with the α4 isoform of the sodium pump trigger non-classical steroid hormone signaling and integrin expression in spermatogenic cells.

PubMed

Upmanyu, Neha; Dietze, Raimund; Kirch, Ulrike; Scheiner-Bobis, Georgios

2016-11-01

In addition to the ubiquitous α1 isoform of the sodium pump, sperm cells also express a male-specific α4 isoform whose function has been associated with sperm motility, fertility, and capacitation. Here we investigate in the murine spermatogenic cell line GC-2 interactions of the α4 isoform with the cardiotonic steroid ouabain in signaling cascades involved in the non-classical action of steroid hormones. Exposure of GC-2 cells to low concentrations of ouabain stimulates the phosphorylation of Erk1/2 and of the transcription factors CREB and ATF-1. As a consequence of this signaling cascade, ouabain stimulates on the mRNA level the expression of integrins αv, β3 and α5, whose expression is also modulated by the cAMP response element. Increased expression of integrins αv and β3 is also seen in cultures of seminiferous tubules exposed to 10nM ouabain. At the protein level we observed a significant stimulation of β3 integrin expression by ouabain. Abrogation of α4 isoform expression by siRNA leads to the complete suppression of all ouabain-induced signaling mentioned above, including its stimulatory effect on the expression of β3 integrin. The results presented here demonstrate for the first time the induction of signaling cascades through the interaction of ouabain with the α4 isoform in a germ-cell derived cell line. The novel finding that these interactions lead to increased expression of integrins in GC-2 cells and the confirmation of these results in the ex vivo experiments indicate that hormone/receptor-like interactions of ouabain with the α4 isoform might be of significance for male physiology. Copyright © 2016 Elsevier B.V. All rights reserved.

Isolation, expansion, and differentiation of goat adipose-derived stem cells.

PubMed

Ren, Yu; Wu, Haiqing; Zhou, Xueyuan; Wen, Jianxun; Jin, Muzi; Cang, Ming; Guo, Xudong; Wang, Qinglian; Liu, Dongjun; Ma, Yuzhen

2012-08-01

A goat adipose-derived stem cell (ADSC) line was established and compared to a rat line. Goat ADSC cells had normal diploidy after subculture. Proliferation of goat ADSCs was faster than rat cells in the same conditions. Both rat and goat ADSCs stained positively for vimentin, CD49d, CD44 and CD13, but stained negatively for CD34 and CD106. Bone nodules were apparent, and alizarin staining was positive after osteogenic induction. Cells expressing osteocalcin were positive by alkaline phosphatase (ALP) staining. After osteogenic induction, ossification nodules of goat ADSCs were larger than in rats, with dense ALP staining. Adipogenic induction resulting in lipid droplets and peroxisome proliferator-activated receptor (PPARγ2) expression were observed. Cartilage lacunae were formed and COL2A1 was expressed. More cartilage lacunae with better morphology were seen following differentiation of goat ADSC's using the hang-drop method. For goat ADSCs, results with both adherent-induced and hanging-drop induced cultures were better than for three-dimensional cultures. Copyright © 2012. Published by Elsevier India Pvt Ltd.

FcγRIIb expression in early stage chronic lymphocytic leukemia.

PubMed

Bosch, Rosa; Mora, Alba; Vicente, Eva Puy; Ferrer, Gerardo; Jansà, Sonia; Damle, Rajendra; Gorlatov, Sergey; Rai, Kanti; Montserrat, Emili; Nomdedeu, Josep; Pratcorona, Marta; Blanco, Laura; Saavedra, Silvana; Garrido, Ana; Esquirol, Albert; Garcia, Irene; Granell, Miquel; Martino, Rodrigo; Delgado, Julio; Sierra, Jorge; Chiorazzi, Nicholas; Moreno, Carol

2017-11-01

In normal B-cells, B-cell antigen receptor (BCR) signaling can be negatively regulated by the low-affinity receptor FcγRIIb (CD32b). To better understand the role of FcγRIIb in chronic lymphocytic leukemia (CLL), we correlated its expression on 155 samples from newly-diagnosed Binet A patients with clinical characteristics and outcome. FcγRIIb expression was similar in normal B-cells and leukemic cells, this being heterogenous among patients and within CLL clones. FcγRIIb expression did not correlate with well known prognostic markers [disease stage, serum beta-2 microglobulin (B2M), IGHV mutational status, expression of ZAP-70 and CD38, and cytogenetics] except for a weak concordance with CD49d. Moreover, patients with low FcγRIIb expression (69/155, 44.5%) required therapy earlier than those with high FcγRIIb expression (86/155, 55.5%) (median 151.4 months vs. not reached; p=.071). These results encourage further investigation on the role of FcγRIIb in CLL biology and prognostic significance in larger series of patients.

Skeletal myogenic differentiation of human urine-derived cells as a potential source for skeletal muscle regeneration.

PubMed

Chen, Wei; Xie, Minkai; Yang, Bin; Bharadwaj, Shantaram; Song, Lujie; Liu, Guihua; Yi, Shanhong; Ye, Gang; Atala, Anthony; Zhang, Yuanyuan

2017-02-01

Stem cells are regarded as possible cell therapy candidates for skeletal muscle regeneration. However, invasive harvesting of those cells can cause potential harvest-site morbidity. The goal of this study was to assess whether human urine-derived stem cells (USCs), obtained through non-invasive procedures, can differentiate into skeletal muscle linage cells (Sk-MCs) and potentially be used for skeletal muscle regeneration. In this study, USCs were harvested from six healthy individuals aged 25-55. Expression profiles of cell-surface markers were assessed by flow cytometry. To optimize the myogenic differentiation medium, we selected two from four different types of myogenic differentiation media to induce the USCs. Differentiated USCs were identified with myogenic markers by gene and protein expression. USCs were implanted into the tibialis anterior muscles of nude mice for 1 month. The results showed that USCs displayed surface markers with positive staining for CD24, CD29, CD44, CD73, CD90, CD105, CD117, CD133, CD146, SSEA-4 and STRO-1, and negative staining for CD14, CD31, CD34 and CD45. After myogenic differentiation, a change in morphology was observed from 'rice-grain'-like cells to spindle-shaped cells. The USCs expressed specific Sk-MC transcripts and protein markers (myf5, myoD, myosin, and desmin) after being induced with different myogenic culture media. Implanted cells expressed Sk-MC markers stably in vivo. Our findings suggest that USCs are able to differentiate into the Sk-MC lineage in vitro and after being implanted in vivo. Thus, they might be a potential source for cell injection therapy in the use of skeletal muscle regeneration. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

Lymphoid hyperplasia in transgenic mice over-expressing a secreted form of the human interleukin-1β gene product

PubMed Central

Björkdahl, O; Åkerblad, P; Gjörloff-wingren, A; Leanderson, T; Dohlsten, M

1999-01-01

To evaluate the biological effects of over-expression of interleukin-1β (IL-1β) on the immune system we have generated transgenic mice, expressing the IL-1β gene fused to a heterologous signal sequence under the control of the mouse immunoglobulin enhancer (Eμ). A prominent hyperplasia and a disturbed microarchitecture of lymphoid tissues were observed in the transgenic mice. The CD4+ T cells in the hyperplastic lymphoid organs seemed to invade the majority of the lymphoid organs including B-cell restricted areas. Analysis of lymph node cells revealed an increased frequency of CD4+ CD44high CD62L− T cells and local secretion of IL-2 and IL-4, compatible with an elevated number of activated T cells. Furthermore, significant levels of human IL-1β in sera and high concentrations of serum immunoglobulin G (IgG) were observed in the transgenic mice. The data suggest a role for IL-1β in controlling lymphoid microarchitecture and, when over-expressed, breaking the threshold in T-helper–B-cell interaction. PMID:10233687

Targeting cancer stem cells in breast cancer: potential anticancer properties of 6-shogaol and pterostilbene.

PubMed

Wu, Chi-Hao; Hong, Bo-Han; Ho, Chi-Tang; Yen, Gow-Chin

2015-03-11

Breast cancer stem cells (BCSCs) constitute a small fraction of the primary tumor that can self-renew and become a drug-resistant cell population, thus limiting the treatment effects of chemotherapeutic drugs. The present study evaluated the cytotoxic effects of five phytochemicals including 6-gingerol (6-G), 6-shogaol (6-S), 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5-HF), nobiletin (NOL), and pterostilbene (PTE) on MCF-7 breast cancer cells and BCSCs. The results showed that 6-G, 6-S, and PTE selectively killed BCSCs and had high sensitivity for BCSCs isolated from MCF-7 cells that expressed the surface antigen CD44(+)/CD24(-). 6-S and PTE induced cell necrosis phenomena such as membrane injury and bleb formation in BCSCs and inhibited mammosphere formation. In addition, 6-S and PTE increased the sensitivity of isolated BCSCs to chemotherapeutic drugs and significantly increased the anticancer activity of paclitaxel. Analysis of the underlying mechanism showed that 6-S and PTE decreased the expression of the surface antigen CD44 on BCSCs and promoted β-catenin phosphorylation through the inhibition of hedgehog/Akt/GSK3β signaling, thus decreasing the protein expression of downstream c-Myc and cyclin D1 and reducing BCSC stemness.

Expression of fibronectin ED-A+ and ED-B+ isoforms by human and experimental colorectal cancer. Contribution of cancer cells and tumor-associated myofibroblasts.

PubMed Central

Pujuguet, P.; Hammann, A.; Moutet, M.; Samuel, J. L.; Martin, F.; Martin, M.

1996-01-01

Alternative splicing of primary fibronectin (FN) mRNA results in the synthesis of different isoforms. ED-A+ and ED-B+ FN isoforms are absent from plasma FN and are representative of cellular FN. Their expression was studied in human and rat normal colon, in human colorectal carcinomas, and in transplanted tumors derived from a chemically-induced rat colon cancer. In normal colon, only the ED-A+ FN isoform was expressed as a thin deposit between crypt colonocytes and pericryptal myofibroblasts. Conversely, heavy ED-A+ FN deposits and lighter ED-B+ FN expression were found in the stroma of colorectal tumors in association with myofibroblasts surrounding tumor glands. Some colonic cancer cells also contained intracellular FN isoform granules and expressed FN mRNA. Tumor-associated myofibroblasts and some cancer cell lines were able to synthesize and deposit extracellular ED-A+ and ED-B+ FN in vitro. FN isoform deposition by tumor-associated myofibroblasts was not modulated by colon cancer cell-conditioned medium, but was strongly enhanced when myofibroblasts were cultured on colon cancer cell extracellular matrix or on laminin. These results show that the ED-A+ and ED-B+ FN isoforms were overexpressed in colorectal cancer. Cancer cells can deposit these FN isoforms directly and also stimulate their deposition by tumor-associated myofibroblasts. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 7 PMID:8579120

A new haemocyanin in cuttlefish (Sepia officinalis) eggs: sequence analysis and relevance during ontogeny

PubMed Central

2014-01-01

Background Haemocyanin is the respiratory protein of most of the Mollusca. In cephalopods and gastropods at least two distinct isoforms are differentially expressed. However, their physiological purpose is unknown. For the common cuttlefish Sepia officinalis, three isoforms are known so far, whereas for only two of them the complete mRNA sequences are available. In this study, we sequenced the complete mRNA of the third haemocyanin isoform and measured the relative expression of all three isoforms during embryogenesis to reveal a potential ontogenetic relevance. Results The cDNA of isoform 3 clearly correlates to the known Sepia officinalis haemocyanin subunits consisting of eight functional units and an internal duplicated functional unit d. Our molecular phylogenetic analyses reveal the third isoform representing a potentially ancestral haemocyanin isoform, and the analyses of the expression of haemocyanin type 3 reveal that haemocyanin type 3 only can be observed within eggs and during early development. Isoforms 1 and 2 are absent at these stages. After hatching, isoform 3 is downregulated, and isoform 1 and 2 are upregulated. Conclusions Our study clearly shows an embryonic relevance of the third isoform, which will be further discussed in the light of the changes in the physiological function of haemocyanin during ontogeny. Taken together with the fact that it could also be the isoform closest related to the common ancestor of cuttlefish haemocyanin, the phylogeny of cuttlefish haemocyanin may be recapitulated during its ontogeny. PMID:24499521

Resistance to age-dependent thymic atrophy in long-lived mice that are deficient in pregnancy-associated plasma protein A

PubMed Central

Vallejo, Abbe N.; Michel, Joshua J.; Bale, Laurie K.; Lemster, Bonnie H.; Borghesi, Lisa; Conover, Cheryl A.

2009-01-01

Pregnancy-associated plasma protein A (PAPPA) is a metalloproteinase that controls the tissue availability of insulin-like growth factor (IGF). Homozygous deletion of PAPPA in mice leads to lifespan extension. Since immune function is an important determinant of individual fitness, we examined the natural immune ecology of PAPPA−/− mice and their wild-type littermates reared under specific pathogen-free condition with aging. Whereas wild-type mice exhibit classic age-dependent thymic atrophy, 18-month-old PAPPA−/− mice maintain discrete thymic cortex and medulla densely populated by CD4+CD8+ thymocytes that are capable of differentiating into single-positive CD4 and CD8 T cells. Old PAPPA−/− mice have high levels of T cell receptor excision circles, and have bone marrows enriched for subsets of thymus-seeding progenitors. PAPPA−/− mice have an overall larger pool of naive T cells, and also exhibit an age-dependent accumulation of CD44+CD43+ memory T cells similar to wild-type mice. However, CD43+ T cell subsets of old PAPPA−/− mice have significantly lower prevalence of 1B11 and S7, glycosylation isoforms known to inhibit T cell activation with normal aging. In bioassays of cell activation, splenic T cells of old PAPPA−/− mice have high levels of activation antigens and cytokine production, and also elicit Ig production by autologous B cells at levels equivalent to young wild-type mice. These data suggest an IGF-immune axis of healthy longevity. Controlling the availability of IGF in the thymus by targeted manipulation of PAPPA could be a way to maintain immune homeostasis during postnatal development and aging. PMID:19549878

Reduced Expression of the Liver/Beta-Cell Glucose Transporter Isoform in Glucose-Insensitive Pancreatic Beta Cells of Diabetic Rats

NASA Astrophysics Data System (ADS)

Thorens, Bernard; Weir, Gordon C.; Leahy, John L.; Lodish, Harvey F.; Bonner-Weir, Susan

1990-09-01

Rats injected with a single dose of streptozocin at 2 days of age develop non-insulin-dependent diabetes 6 weeks later. The pancreatic beta islet cells of these diabetic rats display a loss of glucose-induced insulin secretion while maintaining sensitivity to other secretagogues such as arginine. We analyzed the level of expression of the liver/beta-cell glucose transporter isoform in diabetic islets by immunofluorescence staining of pancreas sections and by Western blotting of islet lysates. Islets from diabetic animals have a reduced expression of this beta-cell-specific glucose transporter isoform and the extent of reduction is correlated with the severity of hyperglycemia. In contrast, expression of this transporter isoform in liver is minimally modified by the diabetes. Thus a decreased expression of the liver/beta-cell glucose transporter isoform in beta cells is associated with the impaired glucose sensing characteristic of diabetic islets; our data suggest that this glucose transporter may be part of the beta-cell glucose sensor.

The Geonomic Organization of the CD28 Gene. Implications for the Regulation of CD28 mRNA Expression and Heterogeneity

DTIC Science & Technology

1990-07-01

doanrmsialecgtonptcelsvstsrtinrnsoa Partial primary structure of the alpha and beta chains of human tdomn ctmvity nat Nrento 320 ticl levsis.ti tasoa...L. Moretta. and C. MW. Croce. tlon and RNA splicing defects in five cloned j6- thalassaemia genes. 1987. Tp44 molecules Involved In antigen-independent T cell acti- Na t ure 302:59 1.

Delayed Expansion and Contraction of CD8+ T Cell Response during Infection with Virulent Salmonella typhimurium1

PubMed Central

Luu, Rachel A.; Gurnani, Komal; Dudani, Renu; Kammara, Rajagopal; van Faassen, Henk; Sirard, Jean-Claude; Krishnan, Lakshmi; Sad, Subash

2014-01-01

Ag presentation to CD8+ T cells often commences immediately after infection, which facilitates their rapid expansion and control of infection. Subsequently, the primed cells undergo rapid contraction. We report that this paradigm is not followed during infection with virulent Salmonella enterica, serovar Typhimurium (ST), an intracellular bacterium that replicates within phagosomes of infected cells. Although susceptible mice die rapidly (~7 days), resistant mice (129×1SvJ) harbor a chronic infection lasting ~60–90 days. Using rOVA-expressing ST (ST-OVA), we show that T cell priming is considerably delayed in the resistant mice. CD8+ T cells that are induced during ST-OVA infection undergo delayed expansion, which peaks around day 21, and is followed by protracted contraction. Initially, ST-OVA induces a small population of cycling central phenotype (CD62LhighIL-7RαhighCD44high) CD8+ T cells. However, by day 14–21, majority of the primed CD8+ T cells display an effector phenotype (CD62LlowIL-7RαlowCD44high). Subsequently, a progressive increase in the numbers of effector memory phenotype cells (CD62LlowIL-7RαhighCD44high) occurs. This differentiation program remained unchanged after accelerated removal of the pathogen with antibiotics, as majority of the primed cells displayed an effector memory phenotype even at 6 mo postinfection. Despite the chronic infection, CD8+ T cells induced by ST-OVA were functional as they exhibited killing ability and cytokine production. Importantly, even memory CD8+ T cells failed to undergo rapid expansion in response to ST-OVA infection, suggesting a delay in T cell priming during infection with virulent ST-OVA. Thus, phagosomal lifestyle may allow escape from host CD8+ T cell recognition, conferring a survival advantage to the pathogen. PMID:16849458

Human cytosolic glutathione-S-transferases: quantitative analysis of expression, comparative analysis of structures and inhibition strategies of isozymes involved in drug resistance.

PubMed

Mohana, Krishnamoorthy; Achary, Anant

2017-08-01

Glutathione-S-transferase (GST) inhibition is a strategy to overcome drug resistance. Several isoforms of human GSTs are present and they are expressed in almost all the organs. Specific expression levels of GSTs in various organs are collected from the human transcriptome data and analysis of the organ-specific expression of GST isoforms is carried out. The variations in the level of expressions of GST isoforms are statistically significant. The GST expression differs in diseased conditions as reported by many investigators and some of the isoforms of GSTs are disease markers or drug targets. Structure analysis of various isoforms is carried out and literature mining has been performed to identify the differences in the active sites of the GSTs. The xenobiotic binding H site is classified into H1, H2, and H3 and the differences in the amino acid composition, the hydrophobicity and other structural features of H site of GSTs are discussed. The existing inhibition strategies are compared. The advent of rational drug design, mechanism-based inhibition strategies, availability of high-throughput screening, target specific, and selective inhibition of GST isoforms involved in drug resistance could be achieved for the reversal of drug resistance and aid in the treatment of diseases.

Novel apigenin-loaded sodium hyaluronate nano-assemblies for targeting tumor cells.

PubMed

Zhao, Ting; He, Yue; Chen, Huali; Bai, Yan; Hu, Wenjing; Zhang, Liangke

2017-12-01

We aimed to construct a novel nano-assembly carrying apigenin (APG), a hydrophobic drug, and to evaluate its in vitro targeting ability for A549 cells overexpressing CD44 receptors. The apigenin-loaded sodium hyaluronate nano-assemblies (APG/SH-NAs) were assembled by multiple non-covalent interactions between sodium hyaluronate (SH) and APG. The prepared APG/SH-NAs exhibited a small average size and narrow particle size distribution. In addition, satisfactory encapsulation efficiency and drug loading were obtained. The drug release curves indicated that APG/SH-NAs achieved a sustainable drug-release effect due to the presence of hydrophilic materials. The in vitro cytotoxicity of APG/SH-NAs against A549 cells and HepG2 cells was evaluated, and the results indicated that the prepared APG/SH-NA showed higher cytotoxicity compared to apigenin suspensions. When CD44 receptors on the surface of A549 cells were blocked by the addition of excess SH, the cytotoxicity of APG/SH-NA was significantly reduced. However, similar phenomena were not observed in HepG2 cells with relatively low CD44 receptor expression. The resulting APG/SH-NAs could efficiently facilitate the internalization of APG into A549 cells, which might be due to their high affinity for CD44 receptors. Moreover, the apoptotic rate of APG/SH-NAs through receptor-mediated endocytosis mechanism was higher than that of the other groups in A549 cells. Thus, such nano-assemblies were considered to be an effective transport system with excellent affinity for CD44 receptors to allow the SH-mediated targeted delivery of APG. Copyright © 2017. Published by Elsevier Ltd.

Epithelial expression of extracellular matrix metalloproteinase inducer/CD147 and matrix metalloproteinase-2 in neoplasms and precursor lesions derived from cutaneous squamous cells: An immunohistochemical study.

PubMed

Ayva, Sebnem Kupana; Karabulut, Ayse Anil; Akatli, Ayşe Nur; Atasoy, Pinar; Bozdogan, Onder

2013-10-01

Extracellular matrix metalloproteinase inducer (CD147) is a transmembrane glycoprotein involved in the regulation of matrix metalloproteinases (MMPs). The study investigated CD147 and MMP-2 expression in epidermis of cutaneous squamous lesions. CD147 and MMP-2 expressions were evaluated immunohistochemically in 44 specimens: 18 actinic keratoses (AK), 6 squamous cell carcinomas in situ (SCCIS), 13 squamous cell carcinomas (SCC; peritumoral and invasive portions assessed), and 7 normal skins. Patterns of expression were assessed, with MMP-2 in nuclei (MMP-2n) and cytoplasm (MMP-2c) evaluated separately. The expression of each marker was quantified using a calculated immunohistochemical/histologic score (H-score). Correlations were analyzed for the marker H-scores in each study group. Associations between H-scores and histopathologic parameters were also evaluated. CD147 H-score was the highest in SCC (invasive islands), followed by AK, SCCIS, and control specimens, respectively. MMP-2n and MMP-2c H-scores were the highest in AK, followed by SCCIS, SCC, and control specimens, respectively. MMP-2c and MMP-2n H-scores were significantly higher in peritumoral epidermis than in invasive islands of SCC. MMP-2c and CD147 H-scores were positively correlated in the peritumoral SCCs. CD147 H-score was positively correlated with tumor differentiation in SCC. The findings suggest that overexpression of CD147 plays a role in the development of SCC. Copyright © 2013 Elsevier GmbH. All rights reserved.

Tissue specificity and regulation of the N-terminal diversity of reticulon 3

PubMed Central

Di Scala, Franck; Dupuis, Luc; Gaiddon, Christian; De Tapia, Marc; Jokic, Natasa; Gonzalez De Aguilar, Jose-Luis; Raul, Jean-Sébastien; Ludes, Bertrand; Loeffler, Jean-Philippe

2004-01-01

Over the last few years, the widely distributed family of reticulons (RTNs) is receiving renewed interest because of the implication of RTN4/Nogo in neurite regeneration. Four genes were identified in mammals and are referred to as RTN1, 2, 3 and the neurite outgrowth inhibitor RTN4/Nogo. In the present paper, we describe the existence of five new isoforms of RTN3 that differ in their N-termini, and analysed their tissue distribution and expression in neurons. We redefined the structure of human and murine rtn3 genes, and identified two supplementary exons that may generate up to seven putative isoforms arising by alternative splicing or differential promoter usage. We confirmed the presence of five of these isoforms at the mRNA and protein levels, and showed their preferential expression in the central nervous system. We analysed rtn3 expression in the cerebellum further, and observed increased levels of several of the RTN3 isoforms during cerebellum development and during in vitro maturation of cerebellar granule cells. This pattern of expression paralleled that shown by RTN4/Nogo isoforms. Specifically, RTN3A1 expression was down-regulated upon cell death of cerebellar granule neurons triggered by potassium deprivation. Altogether, our results demonstrate that the rtn3 gene generates multiple isoforms varying in their N-termini, and that their expression is tightly regulated in neurons. These findings suggest that RTN3 isoforms may contribute, by as yet unknown mechanisms, to neuronal survival and plasticity. PMID:15350194

Effects of Different Cell-Detaching Methods on the Viability and Cell Surface Antigen Expression of Synovial Mesenchymal Stem Cells.

PubMed

Tsuji, Kunikazu; Ojima, Miyoko; Otabe, Koji; Horie, Masafumi; Koga, Hideyuki; Sekiya, Ichiro; Muneta, Takeshi

2017-06-09

Flow cytometric analysis of cell surface antigens is a powerful tool for the isolation and characterization of stem cells residing in adult tissues. In contrast to the collection of hematopoietic stem cells, the process of enzymatic digestion is usually necessary to prepare mesenchymal stem cells (MSCs) suspensions, which can influence the expression of cell surface markers. In this study, we examined the effects of various cell-detaching reagents and digestion times on the expression of stem cell-related surface antigens and MSC functions. Human MSCs were detached from dishes using four different reagents: trypsin, TrypLE, collagenase, and a nonenzymatic cell dissociation reagent (C5789; Sigma-Aldrich). Following dissociation reagent incubations ranging from 5 to 120 min, cell surface markers were analyzed by flow cytometry. Trypsin and TrypLE quickly dissociated the cells within 5 min, while collagenase and C5789 required 60 min to obtain maximum cell yields. C5789 significantly decreased cell viability at 120 min. Trypsin treatment significantly reduced CD44+, CD55+, CD73+, CD105+, CD140a+, CD140b+, and CD201+ cell numbers within 30 min. Collagenase treatment reduced CD140a expression by 30 min. In contrast, TrypLE treatment did not affect the expression of any cell surface antigens tested by 30 min. Despite the significant loss of surface antigen expression after 60 min of treatment with trypsin, adverse effects of enzymatic digestion on multipotency of MSCs were limited. Overall, our data indicated that TrypLE is advantageous over other cell dissociation reagents tested for the rapid preparation of viable MSC suspensions.

Gene expression profiling in rat kidney after intratracheal exposure to cadmium-doped nanoparticles

NASA Astrophysics Data System (ADS)

Coccini, Teresa; Roda, Elisa; Fabbri, Marco; Sacco, Maria Grazia; Gribaldo, Laura; Manzo, Luigi

2012-08-01

While nephrotoxicity of cadmium is well documented, very limited information exists on renal effects of exposure to cadmium-containing nanomaterials. In this work, "omics" methodologies have been used to assess the action of cadmium-containing silica nanoparticles (Cd-SiNPs) in the kidney of Sprague-Dawley rats exposed intratracheally. Groups of animals received a single dose of Cd-SiNPs (1 mg/rat), CdCl2 (400 μg/rat) or 0.1 ml saline (control). Renal gene expression was evaluated 7 and 30 days post exposure by DNA microarray technology using the Agilent Whole Rat Genome Microarray 4x44K. Gene modulating effects were observed in kidney at both time periods after treatment with Cd-SiNPs. The number of differentially expressed genes being 139 and 153 at the post exposure days 7 and 30, respectively. Renal gene expression changes were also observed in the kidney of CdCl2-treated rats with a total of 253 and 70 probes modulated at 7 and 30 days, respectively. Analysis of renal gene expression profiles at day 7 indicated in both Cd-SiNP and CdCl2 groups downregulation of several cluster genes linked to immune function, oxidative stress, and inflammation processes. Differing from day 7, the majority of cluster gene categories modified by nanoparticles in kidney 30 days after dosing were genes implicated in cell regulation and apoptosis. Modest renal gene expression changes were observed at day 30 in rats treated with CdCl2. These results indicate that kidney may be a susceptible target for subtle long-lasting molecular alterations produced by cadmium nanoparticles locally instilled in the lung.

Inflammatory Mediators in Xanthelasma Palpebrarum: Histopathologic and Immunohistochemical Study.

PubMed

Govorkova, Maria S; Milman, Tatyana; Ying, Gui-Shuang; Pan, Wei; Silkiss, Rona Z

To evaluate the expression of inflammatory mediators in xanthelasma palpebrarum. In this retrospective histopathologic case-control study, xanthelasma specimens obtained from the private practice and pathology archives of 1 author (R.Z.S.) were analyzed and compared with the blepharoplasty tissues from age- and sex-matched controls. Paraffin-embedded tissue sections were stained with hematoxylin-eosin and CD3, CD20, CD163, cyclooxygenase-1, inducible nitric oxide synthase, matrix metallopeptidase-9, and myeloperoxidase antibodies. Immunostaining was quantified by light microscopy and with a computerized image analysis system of scanned images. Hematoxylin-eosin-stained preparations of xanthelasma specimens demonstrated significantly more intense chronic lymphocytic infiltrate when compared with the control blepharoplasty tissues (p < 0.001). Immunohistochemical studies revealed more intense CD3+ T cell and CD163+ histiocytic infiltrate (11% vs. 5%; p = 0.02 and 28% vs. 5%; p = 0.003, respectively) and increased expression of cyclooxygenase-1 (44% vs. 20% expressing cells; p < 0.001 and 21% vs. 9% strongly expressing cells; p = 0.008) and inducible nitric oxide synthase (43% vs. 26% expressing cells; p = 0.03 and 42% vs. 25% strongly expressing cells; p = 0.02) in xanthelasma specimens compared with control tissues. The inflammatory milieu in xanthelasma appears to be analogous to descriptions of the early stages of cardiac atherosclerotic plaque formation. These findings may contribute to the understanding of xanthelasma pathogenesis and to the development of potential targeted therapies.

Production and Breeding of Transgenic Cloned Pigs Expressing Human CD73.

PubMed

Lee, Seung-Chan; Lee, Haesun; Oh, Keon Bong; Hwang, In-Sul; Yang, Hyeon; Park, Mi-Ryung; Ock, Sun-A; Woo, Jae-Seok; Im, Gi-Sun; Hwang, Seongsoo

2017-06-01

One of the reasons to causing blood coagulation in the tissue of xenografted organs was known to incompatibility of the blood coagulation and anti-coagulation regulatory system between TG pigs and primates. Thus, overexpression of human CD73 (hCD73) in the pig endothelial cells is considered as a method to reduce coagulopathy after pig-to-non-human-primate xenotransplantation. This study was performed to produce and breed transgenic pigs expressing hCD73 for the studies immune rejection responses and could provide a successful application of xenotransplantation. The transgenic cells were constructed an hCD73 expression vector under control porcine Icam2 promoter (pIcam2-hCD73) and established donor cell lines expressing hCD73. The numbers of transferred reconstructed embryos were 127 ± 18.9. The pregnancy and delivery rate of surrogates were 8/18 (44%) and 3/18 (16%). The total number of delivered cloned pigs were 10 (2 alive, 7 mummy, and 1 died after birth). Among them, three live hCD73-pigs were successfully delivered by Caesarean section, but one was dead after birth. The two hCD73 TG cloned pigs had normal reproductive ability. They mated with wild type (WT) MGH (Massachusetts General Hospital) female sows and produced totally 16 piglets. Among them, 5 piglets were identified as hCD73 TG pigs. In conclusion, we successfully generated the hCD73 transgenic cloned pigs and produced their litters by natural mating. It can be possible to use a mate for the production of multiple transgenic pigs such as α-1,3-galactosyltransferase knock-out /hCD46 for xenotransplantation.

Somatic isoform of angiotensin I-converting enzyme in the pathology of testicular germ cell tumors.

PubMed

Franke, F E; Pauls, K; Kerkman, L; Steger, K; Klonisch, T; Metzger, R; Alhenc-Gelas, F; Burkhardt, E; Bergmann, M; Danilov, S M

2000-12-01

Retained fetal expression of angiotensin I-converting enzyme (ACE, CD143) has recently been shown in intratubular germ cell neoplasms (IGCN) and invasive germ cell tumors (GCT), suggesting the somatic isoform (sACE) as a characteristic component of neoplastic germ cells. We analyzed the distribution of sACE in 159 testicular GCT, including 87 IGCN. sACE protein was determined by immunohistochemistry (MAb CG2) on routinely formalin-fixed and paraffin-embedded tissue sections, supplemented by mRNA expression analysis using in situ hybridization. These data were compared with those obtained by germ cell/placental alkaline phosphatases (PIAP; MAbs PL8-F6 and 8A9) employing an uniform score system for the evaluation of immunoreactivity (IRS; possible values from 0 to 12). Expression of sACE and PIAP was found in all 87 analyzed IGCN (IRS > 4, median IRS of 12). Heterogeneous staining patterns were not related to the type of adjacent GCT but correlated with low expression in adjacent seminomas (P =.032 for sACE; P =.005 for PIAP). Both sACE and PIAP often showed a decreased and more heterogeneous but still moderate expression in 91 classic seminomas (median IRS of 8) and were completely absent in tumor cells of spermatocytic seminomas. Despite all similarities, we found sACE and PIAP differently regulated during GCT progression. This was documented by a well-preserved expression of either sACE or PIAP or both in all classic seminomas, low PIAP immunoreactivity in metastasis of seminomas, and completely diverging expression patterns in nonseminomatous GCT. Our findings underline the close molecular relationship between IGCN and seminoma, and suggest sACE as an appropriate marker for seminomatous differentiated tumors. HUM PATHOL 31:1466-1476. Copyright 2000 by W.B. Saunders Company

Activated Leukocyte Cell Adhesion Molecule Expression and Shedding in Thyroid Tumors

PubMed Central

Miccichè, Francesca; Da Riva, Luca; Fabbi, Marina; Pilotti, Silvana; Mondellini, Piera; Ferrini, Silvano; Canevari, Silvana; Pierotti, Marco A.; Bongarzone, Italia

2011-01-01

Activated leukocyte cell adhesion molecule (ALCAM, CD166) is expressed in various tissues, cancers, and cancer-initiating cells. Alterations in expression of ALCAM have been reported in several human tumors, and cell adhesion functions have been proposed to explain its association with cancer. Here we documented high levels of ALCAM expression in human thyroid tumors and cell lines. Through proteomic characterization of ALCAM expression in the human papillary thyroid carcinoma cell line TPC-1, we identified the presence of a full-length membrane-associated isoform in cell lysate and of soluble ALCAM isoforms in conditioned medium. This finding is consistent with proteolytically shed ALCAM ectodomains. Nonspecific agents, such as phorbol myristate acetate (PMA) or ionomycin, provoked increased ectodomain shedding. Epidermal growth factor receptor stimulation also enhanced ALCAM secretion through an ADAM17/TACE-dependent pathway. ADAM17/TACE was expressed in the TPC-1 cell line, and ADAM17/TACE silencing by specific small interfering RNAs reduced ALCAM shedding. In addition, the CGS27023A inhibitor of ADAM17/TACE function reduced ALCAM release in a dose-dependent manner and inhibited cell migration in a wound-healing assay. We also provide evidence for the existence of novel O-glycosylated forms and of a novel 60-kDa soluble form of ALCAM, which is particularly abundant following cell stimulation by PMA. ALCAM expression in papillary and medullary thyroid cancer specimens and in the surrounding non-tumoral component was studied by western blot and immunohistochemistry, with results demonstrating that tumor cells overexpress ALCAM. These findings strongly suggest the possibility that ALCAM may have an important role in thyroid tumor biology. PMID:21364949

Acute UV irradiation increases heparan sulfate proteoglycan levels in human skin.

PubMed

Jung, Ji-Yong; Oh, Jang-Hee; Kim, Yeon Kyung; Shin, Mi Hee; Lee, Dayae; Chung, Jin Ho

2012-03-01

Glycosaminoglycans are important structural components in the skin and exist as various proteoglycan forms, except hyaluronic acid. Heparan sulfate (HS), one of the glycosaminoglycans, is composed of repeated disaccharide units, which are glucuronic acids linked to an N-acetyl-glucosamine or its sulfated forms. To investigate acute ultraviolet (UV)-induced changes of HS and HS proteoglycans (HSPGs), changes in levels of HS and several HSPGs in male human buttock skin were examined by immunohistochemistry and real-time quantitative polymerase chain reaction (qPCR) after 2 minimal erythema doses (MED) of UV irradiation (each n = 4-7). HS staining revealed that 2 MED of UV irradiation increased its expression, and staining for perlecan, syndecan-1, syndecan-4, CD44v3, and CD44 showed that UV irradiation increased their protein levels. However, analysis by real-time qPCR showed that UV irradiation did not change mRNA levels of CD44 and agrin, and decreased perlecan and syndecan-4 mRNA levels, while increased syndecan-1 mRNA level. As HS-synthesizing or -degrading enzymes, exostosin-1 and heparanase mRNA levels were increased, but exostosin-2 was decreased by UV irradiation. UV-induced matrix metalloproteinase-1 expression was confirmed for proper experimental conditions. Acute UV irradiation increases HS and HSPG levels in human skin, but their increase may not be mediated through their transcriptional regulation.

Hierarchy of stroma-derived factors in supporting growth of stroma-dependent hemopoietic cells: membrane-bound SCF is sufficient to confer stroma competence to epithelial cells.

PubMed

Friel, Jutta; Itoh, Katsuhiko; Bergholz, Ulla; Jücker, Manfred; Stocking, Carol; Harrison, Paul; Ostertag, Wolfram

2002-03-01

Hemopoiesis takes place in a microenvironment where hemopoietic cells are closely associated with stroma by various interactions. Stroma coregulates the proliferation and differentiation of hemopoietic cells. Stroma-hemopoietic-cell contact can be supported by locally produced membrane associated growth factors. The stroma derived growth factor, stem cell factor (SCF) is important in hemopoiesis. We examined the different biological interactions of membrane bound and soluble SCF with human hemopoietic cells expressing the SCF receptor, c-kit. To analyze the function of the SCF isoforms in inducing the proliferation of hemopoietic TF1 or Cord blood (CB) CD34+ cells we used stroma cell lines that differ in their presentation of no SCF, membrane SCF, or soluble SCF. We established a new coculture system using an epithelial cell line that excludes potential interfering effects with other known stroma encoded hemopoietic growth factors. We show that soluble SCF, in absence of membrane-bound SCF, inhibits long term clonal growth of primary or established CD34+ hemopoietic cells, whereas membrane-inserted SCF "dominantly" induces long term proliferation of these cells. We demonstrate a hierarchy of these SCF isoforms in the interaction of stroma with hemopoietic TF1 cells. Membrane-bound SCF is "dominant" over soluble SCF, whereas soluble SCF acts epistatically in interacting with hemopoietic cells compared with other stroma derived factors present in SCF deficient stroma. A hierarchy of stroma cell lines can be arranged according to their presentation of membrane SCF or soluble SCF. In our model system, membrane-bound SCF expression is sufficient to confer stroma properties to an epithelial cell line but soluble SCF does not.

CD44 fucosylation on mesenchymal stem cell enhances homing and macrophage polarization in ischemic kidney injury.

PubMed

Chou, Kang-Ju; Lee, Po-Tsang; Chen, Chien-Liang; Hsu, Chih-Yang; Huang, Wei-Chieh; Huang, Chien-Wei; Fang, Hua-Chang

2017-01-01

The lack of homing ability possibly reduces the healing potential of bone-marrow-derived mesenchymal stem cells (MSCs). Therefore, transforming native CD44 on MSCs into a hematopoietic cell E-/L-selectin ligand (HCELL) that possesses potent E-selectin affinity might enhance the homing and regenerative abilities of MSCs. Through fucosyltransferase VI (FTVI) transfection, MSCs were fucosylated on N-glycans of CD44 to become HCELL positive, thus interacting with E-selectin on injured endothelial cells. HCELL expression facilitated MSC homing in kidneys within 24h after injury and reduced lung stasis. An in vitro adhesion assay revealed that transfection enhanced the association between MSCs and hypoxic endothelial cells. In mice treated with HCELL-positive MSCs, the injured kidneys exhibited clusters of homing MSCs, whereas MSCs were rarely observed in mouse kidneys treated with HCELL-negative MSCs. Most MSCs were initially localized at the renal capsule, and some MSCs later migrated inward between tubules. Most homing MSCs were in close contact with inflammatory cells without tubular transdifferentiation. Furthermore, HCELL-positive MSCs substantially alleviated renal injury, partly by enhancing the polarization of infiltrating macrophages. In conclusion, engineering the glycan of CD44 on MSCs through FTVI transfection might enhance renotropism and the regenerating ability of MSCs in ischemic kidney injury. Copyright © 2016 Elsevier Inc. All rights reserved.

Hyaluronan injection in murine osteoarthritis prevents TGFbeta 1-induced synovial neovascularization and fibrosis and maintains articular cartilage integrity by a CD44-dependent mechanism

PubMed Central

2012-01-01

Introduction The mechanism by which intra-articular injection of hyaluronan (HA) ameliorates joint pathology is unknown. Animal studies have shown that HA can reduce synovial activation, periarticular fibrosis and cartilage erosion; however, its specific effects on the different cell types involved remain unclear. We have used the TTR (TGFbeta1 injection and Treadmill Running) model of murine osteoarthritis (OA), which exhibits many OA-like changes, including synovial activation, to examine in vivo tissue-specific effects of intra-articular HA. Methods The kinetics of clearance of fluorotagged HA from joints was examined with whole-body imaging. Naïve and treated knee joints were examined macroscopically for cartilage erosion, meniscal damage and fibrosis. Quantitative histopathology was done with Safranin O for cartilage and with Hematoxylin & Eosin for synovium. Gene expression in joint tissues for Acan, Col1a1, Col2a1, Col3a1, Col5a1, Col10a1, Adamts5 and Mmp13 was done by quantitative PCR. The abundance and distribution of aggrecan, collagen types I, II, III, V and X, ADAMTS5 and MMP13 were examined by immunohistochemistry. Results Injected HA showed a half-life of less than 2 h in the murine knee joint. At the tissue level, HA protected against neovascularization and fibrosis of the meniscus/synovium and maintained articular cartilage integrity in wild-type but not in Cd44 knockout mice. HA injection enhanced the expression of chondrogenic genes and proteins and blocked that of fibrogenic/degradative genes and proteins in cartilage/subchondral bone, whereas it blocked activation of both groups in meniscus/synovium. In all locations it reduced the expression/protein for Mmp13 and blocked Adamts5 expression but not its protein abundance in the synovial lining. Conclusions The injection of HA, 24 h after TGFbeta1 injection, inhibited the cascade of OA-like joint changes seen after treadmill use in the TTR model of OA. In terms of mechanism, tissue protection by HA injection was abrogated by Cd44 ablation, suggesting that interaction of the injected HA with CD44 is central to its protective effects on joint tissue remodeling and degeneration in OA progression. PMID:22721434

Expression of metallothionein protein in the lungs of Wistar rats and C57 and DBA mice exposed to cadmium oxide fumes.

PubMed

McKenna, I M; Gordon, T; Chen, L C; Anver, M R; Waalkes, M P

1998-12-01

Chronic exposure to inhaled cadmium (Cd) has been shown to induce lung tumors in rats (Wistar strain) but not in mice (NMRI strain). The protein metallothionein (MT) plays an important role in Cd detoxification, and it has been suggested that differential inducibility of pulmonary MT may lead to interspecies susceptibility differences to inhaled Cd. Interstrain differences in the pulmonary response of the MT gene to Cd stimuli have not been examined in rats or mice. We compared pulmonary MT expression in Wistar Furth (WF) rats with that in DBA and C57 mice, following a single 3-h exposure to CdO fumes containing 1 mg Cd/m3. Induction of the MT gene was assessed by the levels of MT-I and MT-II transcripts, MT-protein content, and number of MT-labeled alveolar and bronchiolar epithelial cells immediately after Cd exposure and 1, 3, and 5 days later. Control animals were exposed to air/argon furnace gases. We observed differential intra- and interspecies inducibility of the MT gene in the lung following Cd inhalation. DBA mice exhibited greater levels of MT-mRNA, mainly for the MT-I isoform, MT-protein content, and number of MT positive cells relative to C57 mice. WF rats showed lower transcription and translation responses of the MT gene upon Cd stimuli than C57 mice. The present results, in concert with our previous findings of higher lung cell proliferation in Cd-exposed C57 relative to DBA mice, predict greater susceptibility of C57 to the carcinogenic effects of inhaled Cd. Furthermore, the low transcriptional and translation responses of the MT gene to Cd stimuli in WF rats might explain the higher susceptibility of this rat strain to develop malignant lung tumors after chronic exposure to Cd via inhalation. Parallel to our findings in mice, differences in the responsiveness of lung MT gene may exist across rat strains. Thus intraspecies genetic variability in pulmonary MT may influence the susceptibility of rats or mice to lung carcinogenesis induced by inhalation of Cd compounds. Copyright 1998 Academic Press.

Differential gene expression of CYP3A isoforms in equine liver and intestines.

PubMed

Tydén, E; Löfgren, M; Pegolo, S; Capolongo, F; Tjälve, H; Larsson, P

2012-12-01

Recently, seven CYP3A isoforms - CYP3A89, CYP3A93, CYP3A94, CYP3A95, CYP3A96, CYP3A97 and CYP129 - have been isolated from the horse genome. In this study, we have examined the hepatic and intestinal gene expression of these CYP3A isoforms using TaqMan probes. We have also studied the enzyme activity using luciferin-isopropyl acetal (LIPA) as a substrate. The results show a differential gene expression of the CYP3A isoforms in the liver and intestines in horses. In the liver, CYP3A89, CYP3A94, CYP3A96 and CYP3A97 were highly expressed, while in the intestine there were only two dominating isoforms, CYP3A93 and CYP3A96. The isoform CYP3A129 was not detected in the liver or the intestine, although this gene consists of a complete set of exons and should therefore code for a functional protein. It is possible that this gene is expressed in tissues other than the liver and intestines. In the intestine, both CYP3A96 and CYP3A93 showed the highest gene expression in the duodenum and the proximal parts of the jejunum. This correlated with a high protein expression in these tissues. Studies of the enzyme activity showed the same K(m) for the LIPA substrate in the liver and the intestine, while the maximum velocity (V(max)) in the liver was higher than in the intestine. Our finding of a differential gene expression of the CYP3A isoforms in the liver and the intestines contributes to a better understanding of drug metabolism in horses. © 2012 Blackwell Publishing Ltd.