[Low expression of activin A in mouse and human embryonic teratocarcinoma cells].

PubMed

Gordeeva, O F

2014-01-01

TGFP3 family factors play an important role in regulating the balance of self-renewal and differentiation of mouse and human pluripotent stem and embryonic teratocarcinoma cells. The expression patterns of TGFbeta family signaling ligands and functional roles of these signaling pathways differ significantly in mouse and human embryonic stem cells, but the activity and functional role of these factors in mouse and human embryonic teratocarcinoma cells were not sufficiently investigated. Comparative quantitative real-time PCR analysis of the expression of TGF@[beta] family factors in mouse embryonic stem, embryonic germ, and embryonic teratocarcinoma cells showed that embryonic teratocarcinoma cells express lower ActivinA than pluripotent stem cells but similar levels of factors Nodal, Lefty 1, TGFbeta1, BMP4, and GDF3. In human nullipotent embryonic teratocarcinoma PA-1 cells, most factors of the TGFbeta family (ACTIVINA, NODAL, LEFTY 1, BMP4, and GDF3) are expressed at lower levels than in human embryonic stem cells: Thus, in mouse and human nullipotent teratocarcinoma cells, theexpression of ActivinA is significantly reduced com- pared ivith embryonic stem cells. Presumably, these differences may be associated with changes in the functional activity of the respective signaling pathways and deregulation of proliferative and antiproliferative mechanisms in embryonic teratocarcinoma cells.

Expression of insulin-like growth factor-1 and proliferating cell nuclear antigen in human pulp cells of teeth with complete and incomplete root development.

PubMed

Caviedes-Bucheli, J; Canales-Sánchez, P; Castrillón-Sarria, N; Jovel-Garcia, J; Alvarez-Vásquez, J; Rivero, C; Azuero-Holguín, M M; Diaz, E; Munoz, H R

2009-08-01

To quantify the expression of insulin-like growth factor-1 (IGF-1) and proliferating cell nuclear antigen (PCNA) in human pulp cells of teeth with complete or incomplete root development, to support the specific role of IGF-1 in cell proliferation during tooth development and pulp reparative processes. Twenty six pulp samples were obtained from freshly extracted human third molars, equally divided in two groups according to root development stage (complete or incomplete root development). All samples were processed and immunostained to determine the expression of IGF-1 and PCNA in pulp cells. Sections were observed with a light microscope at 80x and morphometric analyses were performed to calculate the area of PCNA and IGF-1 immunostaining using digital image software. Mann-Whitney's test was used to determine statistically significant differences between groups (P < 0.05) for each peptide and the co-expression of both. Expression of IGF-1 and PCNA was observed in all human pulp samples with a statistically significant higher expression in cells of pulps having complete root development (P = 0.0009). Insulin-like growth factor-1 and PCNA are expressed in human pulp cells, with a significant greater expression in pulp cells of teeth having complete root development.

The Evolution of Human Cells in Terms of Protein Innovation

PubMed Central

Sardar, Adam J.; Oates, Matt E.; Fang, Hai; Forrest, Alistair R.R.; Kawaji, Hideya; Gough, Julian; Rackham, Owen J.L.

2014-01-01

Humans are composed of hundreds of cell types. As the genomic DNA of each somatic cell is identical, cell type is determined by what is expressed and when. Until recently, little has been reported about the determinants of human cell identity, particularly from the joint perspective of gene evolution and expression. Here, we chart the evolutionary past of all documented human cell types via the collective histories of proteins, the principal product of gene expression. FANTOM5 data provide cell-type–specific digital expression of human protein-coding genes and the SUPERFAMILY resource is used to provide protein domain annotation. The evolutionary epoch in which each protein was created is inferred by comparison with domain annotation of all other completely sequenced genomes. Studying the distribution across epochs of genes expressed in each cell type reveals insights into human cellular evolution in terms of protein innovation. For each cell type, its history of protein innovation is charted based on the genes it expresses. Combining the histories of all cell types enables us to create a timeline of cell evolution. This timeline identifies the possibility that our common ancestor Coelomata (cavity-forming animals) provided the innovation required for the innate immune system, whereas cells which now form the brain of human have followed a trajectory of continually accumulating novel proteins since Opisthokonta (boundary of animals and fungi). We conclude that exaptation of existing domain architectures into new contexts is the dominant source of cell-type–specific domain architectures. PMID:24692656

ZIP8 expression in human proximal tubule cells, human urothelial cells transformed by Cd+2 and As+3 and in specimens of normal human urothelium and urothelial cancer

PubMed Central

2012-01-01

Background ZIP8 functions endogenously as a Zn+2/HCO3- symporter that can also bring cadmium (Cd+2) into the cell. It has also been proposed that ZIP8 participates in Cd-induced testicular necrosis and renal disease. In this study real-time PCR, western analysis, immunostaining and fluorescent localization were used to define the expression of ZIP8 in human kidney, cultured human proximal tubule (HPT) cells, normal and malignant human urothelium and Cd+2 and arsenite (As+3) transformed urothelial cells. Results It was shown that in the renal system both the non-glycosylated and glycosylated form of ZIP8 was expressed in the proximal tubule cells with localization of ZIP8 to the cytoplasm and cell membrane; findings in line with previous studies on ZIP8. The studies in the bladder were the first to show that ZIP8 was expressed in normal urothelium and that ZIP8 could be localized to the paranuclear region. Studies in the UROtsa cell line confirmed a paranuclear localization of ZIP8, however addition of growth medium to the cells increased the expression of the protein in the UROtsa cells. In archival human samples of the normal urothelium, the expression of ZIP8 was variable in intensity whereas in urothelial cancers ZIP8 was expressed in 13 of 14 samples, with one high grade invasive urothelial cancer showing no expression. The expression of ZIP8 was similar in the Cd+2 and As+3 transformed UROtsa cell lines and their tumor transplants. Conclusion This is the first study which shows that ZIP8 is expressed in the normal urothelium and in bladder cancer. In addition the normal UROtsa cell line and its transformed counterparts show similar expression of ZIP8 compared to the normal urothelium and the urothelial cancers suggesting that the UROtsa cell line could serve as a model system to study the expression of ZIP8 in bladder disease. PMID:22550998

Differentiation and Transplantation of Human Embryonic Stem Cell-Derived Hepatocytes

PubMed Central

Basma, Hesham; Soto-Gutiérrez, Alejandro; Yannam, Govardhana Rao; Liu, Liping; Ito, Ryotaro; Yamamoto, Toshiyuki; Ellis, Ewa; Carson, Steven D.; Sato, Shintaro; Chen, Yong; Muirhead, David; Navarro-Álvarez, Nalu; Wong, Ron; Roy-Chowdhury, Jayanta; Platt, Jeffrey L.; Mercer, David F.; Miller, John D.; Strom, Stephen C.; Kobayashi, Noaya; Fox, Ira J.

2009-01-01

Background & Aims The ability to obtain unlimited numbers of human hepatocytes would improve development of cell-based therapies for liver diseases, facilitate the study of liver biology and improve the early stages of drug discovery. Embryonic stem cells are pluripotent, can potentially differentiate into any cell type and could therefore be developed as a source of human hepatocytes. Methods To generate human hepatocytes, human embryonic stem cells were differentiated by sequential culture in fibroblast growth factor 2 and human Activin-A, hepatocyte growth factor, and dexamethasone. Functional hepatocytes were isolated by sorting for surface asialoglycoprotein receptor expression. Characterization was performed by real-time PCR, imunohistochemistry, immunoblot, functional assays and transplantation. Results Embryonic stem cell-derived hepatocytes expressed liver-specific genes but not genes representing other lineages, secreted functional human liver-specific proteins similar to those of primary human hepatocytes and demonstrated human hepatocyte cytochrome P450 metabolic activity. Serum from rodents given injections of embryonic stem cell-derived hepatocytes contained significant amounts of human albumin and alpha-1-antitrypsin. Colonies of cytokeratin-18 and human albumin-expressing cells were present in the livers of recipient animals. Conclusion Human embryonic stem cells can be differentiated into cells with many characteristics of primary human hepatocytes. Hepatocyte-like cells can be enriched and recovered based on asialoglycoprotein receptor expression and could potentially be used in drug discovery research and developed as therapeutics. PMID:19026649

Expression and function of Allergin-1 on human primary mast cells.

PubMed

Nagai, Kei; Tahara-Hanaoka, Satoko; Morishima, Yuko; Tokunaga, Takahiro; Imoto, Yoshimasa; Noguchi, Emiko; Kanemaru, Kazumasa; Imai, Masamichi; Shibayama, Shiro; Hizawa, Nobuyuki; Fujieda, Shigeharu; Yamagata, Kunihiro; Shibuya, Akira

2013-01-01

Mast cells (MC) play an important role in allergic and non-allergic immune responses. Activation of human MC is modulated by several cell surface inhibitory receptors, including recently identified Allergin-1 expressed on both human and mouse MC. Although Allergin-1 suppresses IgE-mediated, mast cell-dependent anaphylaxis in mice, the expression profile and function of Allergin-1 on human primary MC remains undetermined. Here, we established a seven-color flow cytometry method for assessing expression and function of a very small number of human primary MC. We show that Allergin-1S1, a splicing isoform of Allergin-1, is predominantly expressed on human primary MC in both bronchoalveolar lavage (BAL) fluid and nasal scratching specimens. Moreover, Allergin-1S1 inhibits IgE-mediated activation from human primary MC in BAL fluid. These results indicate that Allergin-1 on human primary MC exhibits similar characteristics as mouse Allergin-1 in the expression profile and function.

Porcine Knock-in Fibroblasts Expressing hDAF on α-1,3-Galactosyltransferase (GGTA1) Gene Locus.

PubMed

Kim, Ji Woo; Kim, Hye-Min; Lee, Sang Mi; Kang, Man-Jong

2012-10-01

The Galactose-α1,3-galactose (α1,3Gal) epitope is responsible for hyperacute rejection in pig-to-human xenotransplantation. Human decay-accelerating factor (hDAF) is a cell surface regulatory protein that serves as a complement inhibitor to protect self cells from complement attack. The generation of α1,3-galactosyltransferase (GGTA1) knock-out pigs expressing DAF is a necessary step for their use as organ donors for humans. In this study, we established GGTA1 knock-out cell lines expressing DAF from pig ear fibroblasts for somatic cell nuclear transfer. hDAF expression was detected in hDAF knock-in heterozygous cells, but not in normal pig cells. Expression of the GGTA1 gene was lower in the knock-in heterozygous cell line compared to the normal pig cell. Knock-in heterozygous cells afforded more effective protection against cytotoxicity with human serum than with GGTA1 knock-out heterozygous and control cells. These cell lines may be used in the production of GGTA1 knock-out and DAF expression pigs for xenotransplantation.

Spatiotemporal analysis of putative notochordal cell markers reveals CD24 and keratins 8, 18, and 19 as notochord‐specific markers during early human intervertebral disc development

PubMed Central

Rodrigues‐Pinto, Ricardo; Berry, Andrew; Piper‐Hanley, Karen; Hanley, Neil; Richardson, Stephen M.

2016-01-01

ABSTRACT In humans, the nucleus pulposus (NP) is composed of large vacuolated notochordal cells in the fetus but, soon after birth, becomes populated by smaller, chondrocyte‐like cells. Although animal studies indicate that notochord‐derived cells persist in the adult NP, the ontogeny of the adult human NP cell population is still unclear. As such, identification of unique notochordal markers is required. This study was conducted to determine the spatiotemporal expression of putative human notochordal markers to aid in the elucidation of the ontogeny of adult human NP cells. Human embryos and fetuses (3.5–18 weeks post‐conception (WPC)) were microdissected to isolate the spine anlagens (notochord and somites/sclerotome). Morphology of the developing IVD was assessed using hematoxylin and eosin. Expression of keratin (KRT) 8, KRT18, KRT19, CD24, GAL3, CD55, BASP1, CTGF, T, CD90, Tie2, and E‐cadherin was assessed using immunohistochemistry. KRT8, KRT18, KRT19 were uniquely expressed by notochordal cells at all spine levels at all stages studied; CD24 was expressed at all stages except 3.5 WPC. While GAL3, CD55, BASP1, CTGF, and T were expressed by notochordal cells at specific stages, they were also co‐expressed by sclerotomal cells. CD90, Tie2, and E‐cadherin expression was not detectable in developing human spine cells at any stage. This study has identified, for the first time, the consistent expression of KRT8, KRT18, KRT19, and CD24 as human notochord‐specific markers during early IVD development. Thus, we propose that these markers can be used to help ascertain the ontogeny of adult human NP cells. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. J Orthop Res 34:1327–1340, 2016. PMID:26910849

Spatiotemporal analysis of putative notochordal cell markers reveals CD24 and keratins 8, 18, and 19 as notochord-specific markers during early human intervertebral disc development.

PubMed

Rodrigues-Pinto, Ricardo; Berry, Andrew; Piper-Hanley, Karen; Hanley, Neil; Richardson, Stephen M; Hoyland, Judith A

2016-08-01

In humans, the nucleus pulposus (NP) is composed of large vacuolated notochordal cells in the fetus but, soon after birth, becomes populated by smaller, chondrocyte-like cells. Although animal studies indicate that notochord-derived cells persist in the adult NP, the ontogeny of the adult human NP cell population is still unclear. As such, identification of unique notochordal markers is required. This study was conducted to determine the spatiotemporal expression of putative human notochordal markers to aid in the elucidation of the ontogeny of adult human NP cells. Human embryos and fetuses (3.5-18 weeks post-conception (WPC)) were microdissected to isolate the spine anlagens (notochord and somites/sclerotome). Morphology of the developing IVD was assessed using hematoxylin and eosin. Expression of keratin (KRT) 8, KRT18, KRT19, CD24, GAL3, CD55, BASP1, CTGF, T, CD90, Tie2, and E-cadherin was assessed using immunohistochemistry. KRT8, KRT18, KRT19 were uniquely expressed by notochordal cells at all spine levels at all stages studied; CD24 was expressed at all stages except 3.5 WPC. While GAL3, CD55, BASP1, CTGF, and T were expressed by notochordal cells at specific stages, they were also co-expressed by sclerotomal cells. CD90, Tie2, and E-cadherin expression was not detectable in developing human spine cells at any stage. This study has identified, for the first time, the consistent expression of KRT8, KRT18, KRT19, and CD24 as human notochord-specific markers during early IVD development. Thus, we propose that these markers can be used to help ascertain the ontogeny of adult human NP cells. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. J Orthop Res 34:1327-1340, 2016. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc.

Noninvasive method for assessing the human circadian clock using hair follicle cells

PubMed Central

Akashi, Makoto; Soma, Haruhiko; Yamamoto, Takuro; Tsugitomi, Asuka; Yamashita, Shiko; Yamamoto, Takuya; Nishida, Eisuke; Yasuda, Akio; Liao, James K.; Node, Koichi

2010-01-01

A thorough understanding of the circadian clock requires qualitative evaluation of circadian clock gene expression. Thus far, no simple and effective method for detecting human clock gene expression has become available. This limitation has greatly hampered our understanding of human circadian rhythm. Here we report a convenient, reliable, and less invasive method for detecting human clock gene expression using biopsy samples of hair follicle cells from the head or chin. We show that the circadian phase of clock gene expression in hair follicle cells accurately reflects that of individual behavioral rhythms, demonstrating that this strategy is appropriate for evaluating the human peripheral circadian clock. Furthermore, using this method, we indicate that rotating shift workers suffer from a serious time lag between circadian gene expression rhythms and lifestyle. Qualitative evaluation of clock gene expression in hair follicle cells, therefore, may be an effective approach for studying the human circadian clock in the clinical setting. PMID:20798039

Adult human pancreas-derived cells expressing stage-specific embryonic antigen 4 differentiate into Sox9-expressing and Ngn3-expressing pancreatic ducts in vivo.

PubMed

Lee, Song; Lee, Chan Mi; Kim, Song Cheol

2016-11-11

Tissue-specific stem/progenitor cells are found in various adult tissues and may have the capacity for lineage-specific differentiation, facilitating applications in autologous transplantation. Stage-specific embryonic antigen 4 (SSEA-4), an early embryonic glycolipid antigen, is expressed in cells derived from adult human pancreas exocrine tissue. Here, we examined the characteristics and lineage-specific differentiation capacity of SSEA-4 + cells. Human adult partial pancreas tissues were obtained from different donors and cultured in vitro. SSEA-4 + and CA19-9 + cells were isolated from adult human pancreas exocrine cells using magnetic-activated cell sorting, and gene expression was validated by quantitative polymerase chain reaction. To confirm in-vivo differentiation, SSEA-4 + and CA19-9 + cells were transplanted into the dorsal subcutaneous region of mice. Finally, morphological features of differentiated areas were confirmed by immunostaining and morphometric analysis. SSEA-4-expressing cells were detected in isolated pancreas exocrine cells from adult humans. These SSEA-4 + cells exhibited coexpression of CA19-9, a marker of pancreatic duct cells, but not amylase expression, as shown by immunostaining and flow cytometry. SSEA-4 + cells exhibited higher relative expression of Oct4, Nanog, Klf4, Sox2, and c-Myc mRNAs than CA19-9 + cells. Pancreatic intralobular ducts (PIDs) were generated from SSEA-4 + or CA19-9 + cells in vivo at 5 weeks after transplantation. However, newly formed PIDs from CA19-9 + cells were less abundant and showed an incomplete PID morphology. In contrast, newly formed PIDs from SSEA-4 + cells were abundant in the transplanted area and showed a crowded morphology, typical of PIDs. Sox9 and Ngn3, key transcription factors associated with pancreatic development and regeneration, were expressed in PIDs from SSEA-4 + cells. SSEA-4-expressing cells in the adult human pancreas may have the potential for regeneration of the pancreas and may be used as a source of stem/progenitor cells for pancreatic cell lineage-specific differentiation.

Expression pattern of circadian genes and steroidogenesis-related genes after testosterone stimulation in the human ovary.

PubMed

Chen, Minghui; Xu, Yanwen; Miao, Benyu; Zhao, Hui; Luo, Lu; Shi, Huijuan; Zhou, Canquan

2016-09-10

Previous studies have shown that circadian genes might be involved in the development of polycystic ovarian syndrome (PCOS). Hyperandrogenism is a hallmark feature of PCOS. However, the effect of hyperandrogenism on circadian gene expression in human granulosa cells is unknown, and the general expression pattern of circadian genes in the human ovary is unclear. Expression of the circadian proteins CLOCK and PER2 in human ovaries was observed by immunohistochemistry. The mRNA expression patterns of the circadian genes CLOCK, PER2, and BMAL1, and the steroidogenesis-related genes STAR, CYP11A1, HSD3B2, and CYP19A1 in cultured human luteinized granulosa cells were analyzed over the course of 48 h after testosterone treatment by quantitative polymerase chain reaction. Immunostaining of CLOCK and PER2 protein was detected in the granulosa cells of dominant antral follicles but was absent in the primordial, primary, or preantral follicles of human ovaries. After testosterone stimulation, expression of PER2 showed an oscillating pattern, with two peaks occurring at the 24th and 44th hours; expression of CLOCK increased significantly to the peak at the 24th hour, whereas expression of BMAL1 did not change significantly over time in human luteinized granulosa cells. Among the four steroidogenesis-related genes evaluated, only STAR displayed an oscillating expression pattern with two peaks occurring at the 24th and 40th hours after testosterone stimulation. Circadian genes are expressed in the dominant antral follicles of the human ovary. Oscillating expression of the circadian gene PER2 can be induced by testosterone in human granulosa cells in vitro. Expression of STAR also displayed an oscillating pattern after testosterone stimulation. Our results indicate a potential relationship between the circadian clock and steroidogenesis in the human ovary, and demonstrate the effect of testosterone on circadian gene expression in granulosa cells.

TGF-β1 downregulates StAR expression and decreases progesterone production through Smad3 and ERK1/2 signaling pathways in human granulosa cells.

PubMed

Fang, Lanlan; Chang, Hsun-Ming; Cheng, Jung-Chien; Leung, Peter C K; Sun, Ying-Pu

2014-11-01

Regulation of progesterone production in granulosa cells is important for normal reproductive functions. Steroidogenic acute regulatory protein (StAR) is recognized as the key regulatory protein involved in the rate-limiting step of steroidogenesis. TGF-β1 protein is detected in human follicular fluid, and TGF-β1 and its receptors are expressed in human granulosa cells. However, the functional role of TGF-β1 in the regulation of StAR expression and progesterone production in human granulosa cells remains unknown. Our objective was to investigate the effects of TGF-β1 on StAR expression and progesterone production in human granulosa cells. SVOG cells are human granulosa cells that were obtained from women undergoing in vitro fertilization and immortalized with SV40 large T antigen. SVOG cells were used to investigate the effects of TGF-β1 on StAR expression and progesterone production at an academic research center. Levels of mRNA and protein were examined by RT-qPCR and western blotting, respectively. The accumulation levels of progesterone were measured by enzyme-linked immunosorbent assay (ELISA). TGF-β1 treatment downregulated StAR expression and decreased progesterone production. The suppressive effects of TGF-β1 on StAR expression and progesterone production were abolished by the inhibition of TGF-β type I receptor. In addition, treatment with TGF-β1 activated the Smad2/3 and ERK1/2 signaling pathways. The inhibition of the Smad3 and ERK1/2 signaling pathways attenuated the TGF-β1-induced downregulation of StAR expression and progesterone production. TGF-β1 downregulated StAR expression and decreased progesterone production by activating the Smad3 and ERK1/2 signaling pathways in human granulosa cells.

[Effect of human oviductal embryotrophic factors on gene expression of mouse preimplantation embryos].

PubMed

Yao, Yuan-Qing; Lee, Kai-Fai; Xu, Jia-Seng; Ho, Pak-Chung; Yeung, Shu-Biu

2007-09-01

To investigate the effect of embryotrophic factors (ETF) from human oviductal cells on gene expression of mouse early developmental embryos and discuss the role of fallopian tube in early development of embryos. ETF was isolated from conditioned medium of human oviductal cell line by sequential liquid chromatographic systems. Mouse embryos were treated by ETF in vitro. Using differential display RT-PCR, the gene expression of embryos treated by ETF was compared with embryos without ETF treatment. The differentially expressed genes were separated, re-amplified, cloned and sequenced. Gene expression profiles of embryos with ETF treatment was different from embryos without this treatment. Eight differentially expressed genes were cloned and sequenced. These genes functioned in RNA degradation, synthesis, splicing, protein trafficking, cellular differentiation and embryo development. Embryotrophic factors from human oviductal cells affect gene expression of early developmental embryos. The human oviductal cells play wide roles in early developmental stages of embryos.

Comparison of the Gene Expression Profiles of Human Hematopoietic Stem Cells between Humans and a Humanized Xenograft Model.

PubMed

Matsuzawa, Hideyuki; Matsushita, Hiromichi; Yahata, Takashi; Tanaka, Masayuki; Ando, Kiyoshi

2017-04-20

The aim of this study is to evaluate the feasibility of NOD/Shi-scid-IL2Rγ null (NOG) mice transplanted with human CD34 + /CD38 - /Lin -/low hematopoietic cells from cord blood (CB) as an experimental model of the gene expression in human hematopoiesis. We compared the gene expressions of human CD34 + /CD38 - /Lin -/low cells from human bone marrow (BM) and in xenograft models. The microarray data revealed that 25 KEGG pathways were extracted from the comparison of human CD34 + /CD38 - /Lin -/low HSCs between CB and BM, and that 17 of them--which were mostly related to cellular survival, RNA metabolism and lymphoid development--were shared with the xenograft model. When the probes that were commonly altered in CD34 + /CD38 - /Lin -/low cells from both human and xenograft BM were analyzed, most of them, including the genes related hypoxia, hematopoietic differentiation, epigenetic modification, translation initiation, and RNA degradation, were downregulated. These alterations of gene expression suggest a reduced differentiation capacity and likely include key alterations of gene expression for settlement of CB CD34 + /CD38 - /Lin -/low cells in BM. Our findings demonstrate that the xenograft model of human CB CD34 + /CD38 - /Lin -/low cells using NOG mice was useful, at least in part, for the evaluation of the gene expression profile of human hematopoietic stem cells.

Widespread Non-Hematopoietic Tissue Distribution by Transplanted Human Progenitor Cells with High Aldehyde Dehydrogenase Activity

PubMed Central

Hess, David A.; Craft, Timothy P.; Wirthlin, Louisa; Hohm, Sarah; Zhou, Ping; Eades, William C.; Creer, Michael H.; Sands, Mark S.; Nolta, Jan A.

2011-01-01

Transplanted adult progenitor cells distribute to peripheral organs and can promote endogenous cellular repair in damaged tissues. However, development of cell-based regenerative therapies has been hindered by the lack of pre-clinical models to efficiently assess multiple organ distribution and difficulty defining human cells with regenerative function. After transplantation into beta-glucuronidase (GUSB)-deficient NOD/SCID/MPSVII mice, we characterized the distribution of lineage depleted human umbilical cord blood-derived cells purified by selection using high aldehyde dehydrogenase activity (ALDH) with CD133 co-expression. ALDHhi or ALDHhiCD133+ cells produced robust hematopoietic reconstitution, and variable levels of tissue distribution in multiple organs. GUSB+ donor cells that co-expressed human (HLA-A,B,C) and hematopoietic (CD45+) cell surface markers were the primary cell phenotype found adjacent to the vascular beds of several tissues, including islet and ductal regions of mouse pancreata. In contrast, variable phenotypes were detected in the chimeric liver, with HLA+/CD45+ cells demonstrating robust GUSB expression adjacent to blood vessels, and CD45−/HLA− cells with diluted GUSB expression predominant in the liver parenchyma. However, true non-hematopoietic human (HLA+/CD45−) cells were rarely detected in other peripheral tissues, suggesting that these GUSB+/HLA−/CD45− cells in the liver were a result of downregulated human surface marker expression in vivo, not widespread seeding of non-hematopoietic cells. However, relying solely on continued expression of cell surface markers, as employed in traditional xenotransplantation models, may underestimate true tissue distribution. ALDH-expressing progenitor cells demonstrated widespread and tissue-specific distribution of variable cellular phenotypes, indicating that these adult progenitor cells should be explored in transplantation models of tissue damage. PMID:18055447

SLAM family member 8 is involved in oncogenic KIT-mediated signaling in human mastocytosis.

PubMed

Sugimoto, Akihiko; Kataoka, Tatsuki R; Ueshima, Chiyuki; Takei, Yusuke; Kitamura, Kyohei; Hirata, Masahiro; Nomura, Takashi; Haga, Hironori

2018-03-02

The signaling lymphocytic activation molecule family member 8 (SLAMF8)/CD353 is a member of the CD2 family of proteins. Its ligand has not been identified. SLAMF8 is expressed by macrophages and suppresses cellular functions. No study has yet explored SLAMF8 expression or function in human mastocytosis, which features oncogenic KIT-mediated proliferation of human mast cells. SLAMF8 protein was expressed in human mastocytosis cells, immunohistochemically. SLAMF8 expression was also evident in the human mast cell lines, HMC1.2 (expressing oncogenic KIT) and LAD2 (expressing wild-type KIT) cells. SLAMF8-knockdown significantly reduced the KIT-mediated growth of HMC1.2 cells but not that of LAD2 cells. SLAMF8-knockdown HMC1.2 cells exhibited significant attenuation of SHP-2 activation and oncogenic KIT-mediated RAS-RAF-ERK signaling. An interaction between SLAMF8 and SHP-2 was confirmed in HMC1.2 cells and all pathological mastocytosis specimens examined (19 of 19 cases, 100%). Thus, SLAMF8 is involved in oncogenic KIT-mediated RAS-RAF-ERK signaling and the subsequent growth of human neoplastic mast cells mediated by SHP-2. SLAMF8 is a possible therapeutic target in human mastocytosis patients. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Increased ICAM-1 Expression in Transformed Human Oral Epithelial Cells: Molecular Mechanism and Functional Role in Peripheral Blood Mononuclear Cell Adhesion and Lymphokine-Activated-Killer Cell Cytotoxicity

PubMed Central

Huang, George T.-J.; Zhang, Xinli; Park, No-Hee

2012-01-01

The intercellular adhesion molecule-1 (ICAM-1, CD54) serves as a counter-receptor for the β2-integrins, LFA-1 and Mac-1, which are expressed on leukocytes. Although expression of ICAM-1 on tumor cells has a role in tumor progression and development, information on ICAM-1 expression and its role in oral cancer has not been established. Normal human oral keratinocytes (NHOK), human papilloma virus (HPV)-immortalized human oral keratinocyte lines (HOK-16B, HOK-18A, and HOK-18C), and six human oral neoplastic cell lines (HOK-16B-BaP-T1, SCC-4, SCC-9, HEp-2, Tu-177 and 1483) were used to study ICAM-1 expression and its functional role in vitro. Our results demonstrated that NHOK express negligible levels of ICAM-1, whereas immortalized human oral keratinocytes and cancer cells express significantly higher levels of ICAM-1, except for HOK-16B-BaP-T1 and HEp-2. Altered mRNA half-lives did not fully account for the increased accumulation of ICAM-1 mRNA. Adhesion of peripheral blood mononuclear cells (PBMC) to epithelial cells correlated with cell surface ICAM-1 expression levels. This adhesion was inhibited by antibodies specific for either ICAM-1 or LFA-1/Mac-1, suggesting a role for these molecules in adhesion. In contrast, lymphokine-activated-killer (LAK) cell cytotoxic killing of epithelial cells did not correlate with ICAM-1 levels or with adhesion. Nonetheless, within each cell line, blocking of ICAM-1 or LFA-1/Mac-1 reduced LAK cells killing, suggesting that ICAM-1 is involved in mediating this killing. PMID:10938387

Gene therapy for human ovarian cancer cells using efficient expression of Fas gene combined with γδT cells.

PubMed

Lin, Jiajing; Zeng, Dingyuan; He, Hongying; Tan, Guangping; Lan, Ying; Jiang, Fuyan; Sheng, Shuting

2017-10-01

Low tissue specificity and efficiency of exogenous gene expression are the two major obstacles in tumor‑targeted gene therapy. The Fas cell surface death receptor (Fas)/Fas ligand pathway is one of the primary pathways responsible for the regulation of cell apoptosis. The aim of the present study was to explore whether the regulation of tumor specific promoters and a two‑step transcriptional amplification system (TSTA) assured efficient, targeted expression of their downstream Fas gene in human ovarian cancer cells, and to assess the killing effect of γδT cells on these cells with high Fas expression. Three shuttle plasmids containing different control elements of the human telomerase reverse transcriptase (hTERT) promoter and/or TSTA were constructed and packaged into adenovirus 5 (Ad5) vectors for the expression of exogenous Fas gene. The human ovarian cancer cell line SKOV3 and a control human embryonic lung fibroblast cell line were transfected with Ad5‑hTERT‑Fas or Ad5‑hTERT‑TSTA‑Fas. Fas mRNA and protein expression were examined by reverse transcription‑quantitative polymerase chain reaction and western blot analysis. γδT lymphocytes were isolated, cultured and mixed at different ratios with SKOV3 cells with Fas expression in order to assess the killing effect of γδT cells. hTERT promoter induced the specific expression of FAS gene in SKOV3 cells, and the TSTA strategy increased FAS expression by 14.2‑fold. The killing effect of γδT cells increased with the expression level of Fas and the effector‑target cell ratio. The killing rate for SKOV3 cells with high FAS expression was 72.5% at an effector‑target cell ratio of 40:1. The regulators of hTERT promoter and TSTA assure the efficient and targeted expression of their downstream Fas gene in SKOV3 cells. The killing effect of γδT cells for ovarian cancer cells with relatively high Fas expression was improved.

Expression cloning of human B cell immunoglobulins.

PubMed

Wardemann, Hedda; Kofer, Juliane

2013-01-01

The majority of lymphomas originate from B cells at the germinal center stage or beyond. Preferential selection of B cell clones by a limited set of antigens has been suggested to drive lymphoma development. However, little is known about the specificity of the antibodies expressed by lymphoma cells, and the role of antibody-specificity in lymphomagenesis remains elusive. Here, we describe a strategy to characterize the antibody reactivity of human B cells. The approach allows the unbiased characterization of the human antibody repertoire on a single cell level through the generation of recombinant monoclonal antibodies from single primary human B cells of defined origin. This protocol offers a detailed description of the method starting from the flow cytometric isolation of single human B cells, to the RT-PCR-based amplification of the expressed Igh, Igκ, and Igλ chain genes, and Ig gene expression vector cloning for the in vitro production of monoclonal antibodies. The strategy may be used to obtain information on the clonal evolution of B cell lymphomas by single cell Ig gene sequencing and on the antibody reactivity of human lymphoma B cells.

Physiological role of urothelial cancer-associated one long noncoding RNA in human skeletogenic cell differentiation.

PubMed

Ishikawa, Takanori; Nishida, Takashi; Ono, Mitsuaki; Takarada, Takeshi; Nguyen, Ha Thi; Kurihara, Shinnosuke; Furumatsu, Takayuki; Murase, Yurika; Takigawa, Masaharu; Oohashi, Toshitaka; Kamioka, Hiroshi; Kubota, Satoshi

2018-06-01

A vast number of long-noncoding RNAs (lncRNA) are found expressed in human cells, which RNAs have been developed along with human evolution. However, the physiological functions of these lncRNAs remain mostly unknown. In the present study, we for the first time uncovered the fact that one of such lncRNAs plays a significant role in the differentiation of chondrocytes and, possibly, of osteoblasts differentiated from mesenchymal stem cells, which cells eventually construct the human skeleton. The urothelial cancer-associated 1 (UCA1) lncRNA is known to be associated with several human malignancies. Firstly, we confirmed that UCA1 was expressed in normal human chondrocytes, as well as in a human chondrocytic cell line; whereas it was not detected in human bone marrow mesenchymal stem cells (hBMSCs). Of note, although UCA1 expression was undetectable in hBMSCs, it was markedly induced along with the differentiation toward chondrocytes, suggesting its critical role in chondrogenesis. Consistent with this finding, silencing of the UCA1 gene significantly repressed the expression of chondrogenic genes in human chondrocytic cells. UCA1 gene silencing and hyper-expression also had a significant impact on the osteoblastic phenotype in a human cell line. Finally, forced expression of UCA1 in a murine chondrocyte precursor, which did not possess a UCA1 gene, overdrove its differentiation into chondrocytes. These results indicate a physiological and important role of this lncRNA in the skeletal development of humans, who require more sustained endochondral ossification and osteogenesis than do smaller vertebrates. © 2017 Wiley Periodicals, Inc.

SPARC Gene Expression is Repressed in Human Urothelial Cells (UROtsa) Exposed to or Malignantly Transformed by Cadmium or Arsenite

PubMed Central

Larson, Jennifer; Yasmin, Tahmina; Sens, Donald A.; Zhou, Xu Dong; Sens, Mary Ann; Garrett, Scott H.; Dunlevy, Jane R.; Cao, Ling; Somji, Seema

2010-01-01

SPARC belongs to a class of extracellular matrix-associated proteins that have counteradhesive properties. The ability of SPARC to modulate cell-cell and cell-matrix interactions provides a strong rationale for studies designed to determine its expression in cancer. The objective of this study was to determine if SPARC expression was altered in cadmium (Cd+2) and arsenite (As+3) induced bladder cancer and if these alterations were present in archival specimens of human bladder cancer. The expression of SPARC was determined in human parental UROtsa cells, their Cd+2 and As+3 transformed counterparts and derived tumors, and in archival specimens of human bladder cancer using a combination of real time reverse transcriptase polymerase chain reaction, western blotting, immunofluoresence localization and immunohistochemical staining. It was demonstrated that SPARC expression was down-regulated in Cd+2 and As+3 transformed UROtsa cells. In addition, the malignant epithelial component of tumors derived from these cell lines were also down-regulated for SPARC expression, but the stromal cells recruited to these tumors was highly reactive for SPARC. This finding was shown to translate to specimens of human bladder cancer where tumor cells were SPARC negative, but stromal cells were positive. Acute exposure of UROtsa cells to both cadmium and arsenite reduced the expression of SPARC through a mechanism that did not involve changes in DNA methylation or histone acetylation. These studies suggest that environmental exposure to As+3 or Cd+2 can alter cell-cell and cell-matrix interactions in normal urothelial cells through a reduction in the expression of SPARC. The SPARC associated loss of cell-cell and cell-matrix contacts may participate in the multi-step process of bladder carcinogenesis. PMID:20837119

Expression of Apg-1, a member of the Hsp110 family, in the human testis and sperm.

PubMed

Nonoguchi, K; Tokuchi, H; Okuno, H; Watanabe, H; Egawa, H; Saito, K; Ogawa, O; Fujita, J

2001-06-01

Apg-1 encodes a heat shock protein belonging to the Hsp110 family and is inducible by a 32 degrees C to 39 degrees C heat shock in somatic cells. In mouse testicular germ cells Apg-1 mRNA is constitutively expressed depending on the developmental stage. As human Apg-1 has recently been identified, the expression of Apg-1 in the human testis and sperm was investigated. Expression and heat-inducibility of Apg-1 in the human testicular germ cell tumor cell line, NEC8, was analyzed. Using an antimouse Apg-1 antibody, expression of Apg-1 in the human testis and sperm was examined by western blotting after confirmation of the specificity of the antibody. The cells expressing Apg-1 in the testis were further determined by immunohistochemistry. Slight induction of Apg-1 mRNA was detected in NEC8 cells after 32 degrees C to 39 degrees C temperature shift. In the human testis, the antibody specifically recognized Apg-1, which was absent in the testis without germ cells (Sertoli-cell-only syndrome) or arrested at spermatogonia. Spermatocytes and spermatids, but not testicular somatic cells, were positively stained with the anti-Apg-1 antibody. By western blot analysis, Apg-1 was detected in the preparation enriched for sperm from normal volunteers and infertile patients, but not from azoospermia patients. Apg-1 is developmentally expressed in human testicular germ cells and sperm, suggesting its role in spermatogenesis and fertilization. Identification of substrates for Apg-1 chaperone activity will help elucidate its function.

Gab3 is required for human colorectal cancer cell proliferation

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

Xiang, Shihao; Wang, Na; Hui, Pingping

Here, we focused on the potential function of Gab3, an uncommon Gab family protein, in human colorectal cancer (CRC) cells. We found that Gab3 was only expressed in human colon cancer tissues as well as in established (HCT-116 and HT-29 lines) and primary human CRC cells. It was however absent in normal human colon cancer tissues and in FHC colon epithelial cells. Knockdown of Gab3 by targeted-shRNAs inhibited proliferation of the CRC cells. Reversely, exogenous over-expression of Gab3 promoted CRC cell proliferation. At the signaling level, Gab3 co-precipitated with p85 and SHP2 in CRC cells, which was required for subsequentmore » Akt and Erk activation. Gab3 shRNA knockdown inhibited Akt and Erk activation, yet Gab3 over-expression augmented it. In vivo, HCT-116 xenograft tumor growth in severe combined immune deficient (SCID) mice was suppressed following expressing Gab3 shRNAs. Meanwhile, Akt and Erk activation in Gab3 shRNA-expressing tumors was also largely inhibited. Together, our results suggest that Gab3 expression in CRC cells is important for Akt-Erk activation and cell proliferation. - Highlights: • Gab3 is only expressed in colorectal cancer (CRC) cells, but not in colon epithelial cells. • Gab3 shRNA knockdown inhibits CRC cell proliferation. • Exogenous over-expression of Gab3 promotes HCT-116 cell proliferation. • Gab3 co-precipitates with p85 and SHP2 to mediate Akt and Erk activation in CRC cells. • HCT-116 tumor growth in SCID mice is suppressed with expression of Gab3 shRNAs.« less

Reprogramming of the MHC-I and its regulation by NFκB in human-induced pluripotent stem cells.

PubMed

Pick, Marjorie; Ronen, Daniel; Yanuka, Ofra; Benvenisty, Nissim

2012-12-01

The immunogenicity of human pluripotent stem cells plays a major role in their potential use in the clinic. We show that, during their reprogramming, human-induced pluripotent stem (iPS) cells downregulate expression of human leukocyte antigen (HLA)-A/B/C and β2 microglobulin (β2M), the two components of major histocompatibility complex-I (MHC-I). MHC-I expression in iPS cells can be restored by differentiation or treatment with interferon-gamma (IFNγ). To analyze the molecular mechanisms that regulate the expression of the MHC-I molecules in human iPS cells, we searched for correlation between the expression of HLA-A/B/C and β2M and the expression of transcription factors that bind to the promoter of these genes. Our results show a significant positive correlation between MHC-I expression and expression of the nuclear factors, nuclear factor kappa B 1 (NFκB1) and RelA, at the levels of RNA, protein and was confirmed by chromatin binding. Concordantly, we detected robust levels of NFκB1 and RelA proteins in the nucleus of somatic cells but not in the iPS cell derived from them. Overexpression of NFκB1 and RelA in undifferentiated pluripotent stem cells led to induction in expression of MHC-I, whereas silencing NFκB1 and RelA by small hairpin RNA decreased the expression of β2M after IFNγ treatment. Our data point to the critical role of NFκB proteins in regulating the MHC-I expression in human pluripotent stem cells. Copyright © 2012 AlphaMed Press.

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

Differentiation of Human Dental Stem Cells Reveal a Role for microRNA-218

PubMed Central

Gay, Isabel; Cavender, Adriana; Peto, David; Sun, Zhao; Speer, Aline; Cao, Huojun; Amendt, Brad A.

2013-01-01

Background Regeneration of the lost periodontium is the ultimate goal of periodontal therapy. Advances in tissue engineering have demonstrated the multilineage potential and plasticity of adult stem cells located in the periodontal apparatus. However, it remains unclear how epigenetic mechanisms controlling signals determine tissue specification and cell lineage decisions. To date, no data is available on micro-RNAs (miRNAs) activity behind human-derived dental stem cells. Methods In this study, we isolated periodontal ligament stem cells (PDLSCs), dental pulp stem cells (DPSCs), and gingival stem cells (GSCs) from extracted third molars; human bone marrow stem cells (BMSCs) were used as a positive control. The expression of OCT4A and NANOG was confirmed in these undifferentiated cells. All cells were cultured under osteogenic inductive conditions and RUNX2 expression was analyzed as a marker of mineralized tissue differentiation. A miRNA expression profile was obtained at baseline and after osteogenic induction in all cell types. Results RUNX2 expression demonstrated the successful osteogenic induction of all cell types, which was confirmed by alizarin red stain. The analysis of 765 miRNAs demonstrated a shift in miRNA expression occurred in all four stem cell types, including a decrease in hsa-mir-218 across all differentiated cell populations. Hsa-mir-218 targets RUNX2 and decreases RUNX2 expression in undifferentiated human dental stem cells (DSCs). DSC mineralized tissue type differentiation is associated with a decrease in hsa-mir-218 expression. Conclusions These data reveal a miRNA regulated pathway for the differentiation of human DSCs and a select network of human microRNAs that control DSC osteogenic differentiation. PMID:23662917

Development and validation of primary human myometrial cell culture models to study pregnancy and labour.

PubMed

Mosher, Andrea A; Rainey, Kelly J; Bolstad, Seunghwa S; Lye, Stephen J; Mitchell, Bryan F; Olson, David M; Wood, Stephen L; Slater, Donna M

2013-01-01

The development of the in vitro cell culture model has greatly facilitated the ability to study gene expression and regulation within human tissues. Within the human uterus, the upper (fundal) segment and the lower segment may provide distinct functions throughout pregnancy and during labour. We have established primary cultured human myometrial cells, isolated from both upper and lower segment regions of the pregnant human uterus, and validated them for the purpose of studying human pregnancy and labour. The specific objectives of this study were to monitor the viability and characterize the expression profile using selected cellular, contractile and pregnancy associated markers in the primary cultured human myometrial cells. Labour has been described as an inflammatory process; therefore, the ability of these cells to respond to an inflammatory stimulus was also investigated. Myometrial cells isolated from paired upper segment (US) and lower segment (LS) biopsies, obtained from women undergoing Caesarean section deliveries at term prior to the onset of labour, were used to identify expression of; α smooth muscle actin, calponin, caldesmon, connexin 43, cyclo-oxygenase-2 (COX-2), oxytocin receptor, tropomyosin and vimentin, by RT-PCR and/or immunocytochemistry. Interleukin (IL)-1β was used to treat cells, subsequently expression of COX-2 mRNA and release of interleukin-8 (CXCL8), were measured. ANOVA followed by Bonferroni's multiple comparisons test was performed. We demonstrate that US and LS human myometrial cells stably express all markers examined to at least passage ten (p10). Connexin 43, COX-2 and vimentin mRNA expression were significantly higher in LS cells compared to US cells. Both cell populations respond to IL-1β, demonstrated by a robust release of CXCL8 and increased expression of COX-2 mRNA from passage one (p1) through to p10. Isolated primary myometrial cells maintain expression of smooth muscle and pregnancy-associated markers and retain their ability to respond to an inflammatory stimulus. These distinct myometrial cell models will provide a useful tool to investigate mechanisms underlying the process of human labour and the concept of functional regionalization of the pregnant uterus.

Tetraspanin CD9 modulates human lymphoma cellular proliferation via histone deacetylase activity

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

Herr, Michael J.; Department of Medicine, The University of Tennessee Health Science Center, Memphis, TN 38163; Department of Molecular Sciences, The University of Tennessee Health Science Center, Memphis, TN 38163

2014-05-16

Highlights: • CD9 is differentially expressed in human Burkitt’s lymphoma cells. • We found that CD9 expression promotes these cells proliferation. • CD9 expression also increases HDAC activity. • HDAC inhibition decreased both cell proliferation and importantly CD9 expression. • CD9 may dictate HDAC efficacy and play a role in HDAC regulation. - Abstract: Non-Hodgkin Lymphoma (NHL) is a type of hematological malignancy that affects two percent of the overall population in the United States. Tetraspanin CD9 is a cell surface protein that has been thoroughly demonstrated to be a molecular facilitator of cellular phenotype. CD9 expression varies in twomore » human lymphoma cell lines, Raji and BJAB. In this report, we investigated the functional relationship between CD9 and cell proliferation regulated by histone deacetylase (HDAC) activity in these two cell lines. Introduction of CD9 expression in Raji cells resulted in significantly increased cell proliferation and HDAC activity compared to Mock transfected Raji cells. The increase in CD9–Raji cell proliferation was significantly inhibited by HDAC inhibitor (HDACi) treatment. Pretreatment of BJAB cells with HDAC inhibitors resulted in a significant decrease in endogenous CD9 mRNA and cell surface expression. BJAB cells also displayed decreased cell proliferation after HDACi treatment. These results suggest a significant relationship between CD9 expression and cell proliferation in human lymphoma cells that may be modulated by HDAC activity.« less

High expression of B7-H6 in human glioma tissues promotes tumor progression.

PubMed

Jiang, Tianwei; Wu, Wei; Zhang, Huasheng; Zhang, Xiangsheng; Zhang, Dingding; Wang, Qiang; Huang, Lei; Wang, Ye; Hang, Chunhua

2017-06-06

B7-H6, a new member of B7-family ligand, also known as NCR3LG1, plays an important role in NK cells mediated immune responses. Many studies have shown that it is highly expressed in various human cancers, and its expression levels are significantly associated with cancer patients' clinicopathological parameters and postoperative prognoses. But, still the exact role of B7-H6 expression in human glioma remains elusive. In the present study, we have characterized the B7-H6 expression in the human glioma tissues as well as glioma cell lines, U87 and U251. We observed that B7-H6 was highly expressed in the human glioma tissues, and its expression was significantly associated with cancer progression. By using the RNA interference technology, we successfully ablated B7-H6 expression in human glioma cell lines to further study its contribution towards various biological features of this malignancy. Our study identified that the B7-H6 knockdown in U87 and U251 glioma cells significantly suppressed cell proliferation, migration, invasion, and enhanced apoptosis along with induction of cell cycle arrest. It thus suggested that B7-H6 play an important role in the regulation of the biological behavior of these glioma cells. However, the detailed mechanism of B7-H6 mediated regulation of glioma cancer cell transformation and its prognostic value merits further investigation.

Bombesin-like peptide receptors in human bronchial epithelial cells.

PubMed

Kane, M A; Toi-Scott, M; Johnson, G L; Kelley, K K; Boose, D; Escobedo-Morse, A

1996-01-01

Northern blot and RNAse protection assays previously failed to detect bombesin-like peptide (BLP) receptors in normal human lung tissue, but by RT/PCR cultured human bronchial epithelial (HBE) cells expressed all three BLP receptor subtypes, predominantly neuromedin B (NMB) receptor. By RT/PCR, we found expression of all three BLP receptor subtypes by human lung tissue and confirmed NMB receptor expression in six out of six HBE samples. However, transformed HBE BEAS B2B cells expressed only gastrin-releasing peptide (GRP) receptors; saturable, high-affinity (Kd = 3.5 nM) specific [125I]GRP binding confirmed functional GRP receptor, with M(r) = 75 kDa and immunologic cross-reactivity with GRP receptor from human small-cell lung carcinoma (SCLC) NCI-H345 cells. Altered regulation of BLP receptors may accompany transformation of normal lung cells to cancer.

The influence of rAAV2-mediated SOX2 delivery into neonatal and adult human RPE cells; a comparative study.

PubMed

Ezati, Razie; Etemadzadeh, Azadeh; Soheili, Zahra-Soheila; Samiei, Shahram; Ranaei Pirmardan, Ehsan; Davari, Malihe; Najafabadi, Hoda Shams

2018-02-01

Cell replacement is a promising therapy for degenerative diseases like age-related macular degeneration (AMD). Since the human retina lacks regeneration capacity, much attention has been directed toward persuading for cells that can differentiate into retinal neurons. In this report, we have investigated reprogramming of the human RPE cells and concerned the effect of donor age on the cellular fate as a critical determinant in reprogramming competence. We evaluated the effect of SOX2 over-expression in human neonatal and adult RPE cells in cultures. The coding region of human SOX2 gene was cloned into adeno-associated virus (AAV2) and primary culture of human neonatal/adult RPE cells were infected by recombinant virus. De-differentiation of RPE to neural/retinal progenitor cells was investigated by quantitative real-time PCR and ICC for neural/retinal progenitor cells' markers. Gene expression analysis showed 80-fold and 12-fold over-expression for SOX2 gene in infected neonatal and adult hRPE cells, respectively. The fold of increase for Nestin in neonatal and adult hRPE cells was 3.8-fold and 2.5-fold, respectively. PAX6 expression was increased threefold and 2.5-fold in neonatal/adult treated cultures. Howbeit, we could not detect rhodopsin, and CHX10 expression in neonatal hRPE cultures and expression of rhodopsin in adult hRPE cells. Results showed SOX2 induced human neonatal/adult RPE cells to de-differentiate toward retinal progenitor cells. However, the increased number of PAX6, CHX10, Thy1, and rhodopsin positive cells in adult hRPE treated cultures clearly indicated the considerable generation of neuro-retinal terminally differentiated cells. © 2017 Wiley Periodicals, Inc.

The T-Cell Oncogene Tal2 Is a Target of PU.1 and Upregulated during Osteoclastogenesis

PubMed Central

Courtial, Nadine; Mücke, Christian; Herkt, Stefanie; Kolodziej, Stephan; Hussong, Helge; Lausen, Jörn

2013-01-01

Transcription factors play a crucial role in regulating differentiation processes during human life and are important in disease. The basic helix-loop-helix transcription factors Tal1 and Lyl1 play a major role in the regulation of gene expression in the hematopoietic system and are involved in human leukemia. Tal2, which belongs to the same family of transcription factors as Tal1 and Lyl1, is also involved in human leukaemia. However, little is known regarding the expression and regulation of Tal2 in hematopoietic cells. Here we show that Tal2 is expressed in hematopoietic cells of the myeloid lineage. Interestingly, we found that usage of the Tal2 promoter is different in human and mouse cells. Two promoters, hP1 and hP2 drive Tal2 expression in human erythroleukemia K562 cells, however in mouse RAW cells only the mP1 promoter is used. Furthermore, we found that Tal2 expression is upregulated during oesteoclastogenesis. We show that Tal2 is a direct target gene of the myeloid transcription factor PU.1, which is a key transcription factor for osteoclast gene expression. Strikingly, PU.1 binding to the P1 promoter is conserved between mouse and human, but PU.1 binding to P2 was only detected in human K562 cells. Additionally, we provide evidence that Tal2 influences the expression of the osteoclastic differentiation gene TRACP. These findings provide novel insight into the expression control of Tal2 in hematopoietic cells and reveal a function of Tal2 as a regulator of gene expression during osteoclast differentiation. PMID:24086757

Sulforaphane inhibits hypoxia-induced HIF-1α and VEGF expression and migration of human colon cancer cells.

PubMed

Kim, Dong Hwan; Sung, Bokyung; Kang, Yong Jung; Hwang, Seong Yeon; Kim, Min Jeong; Yoon, Jeong-Hyun; Im, Eunok; Kim, Nam Deuk

2015-12-01

The effects of sulforaphane (a natural product commonly found in broccoli) was investigated on hypoxia inducible factor-1α (HIF-1α) expression in HCT116 human colon cancer cells and AGS human gastric cancer cells. We found that hypoxia-induced HIF-1α protein expression in HCT116 and AGS cells, while treatment with sulforaphane markedly and concentration-dependently inhibited HIF-1α expression in both cell lines. Treatment with sulforaphane inhibited hypoxia-induced vascular endothelial growth factor (VEGF) expression in HCT116 cells. Treatment with sulforaphane modulated the effect of hypoxia on HIF-1α stability. However, degradation of HIF-1α by sulforaphane was not mediated through the 26S proteasome pathway. We also found that the inhibition of HIF-1α by sulforaphane was not mediated through AKT and extracellular signal-regulated kinase phosphorylation under hypoxic conditions. Finally, hypoxia-induced HCT116 cell migration was inhibited by sulforaphane. These data suggest that sulforaphane may inhibit human colon cancer progression and cancer cell angiogenesis by inhibiting HIF-1α and VEGF expression. Taken together, these results indicate that sulforaphane is a new and potent chemopreventive drug candidate for treating patients with human colon cancer.

A fully human chimeric antigen receptor with potent activity against cancer cells but reduced risk for off-tumor toxicity

PubMed Central

Song, De-Gang; Ye, Qunrui; Poussin, Mathilde; Liu, Lin; Figini, Mariangela; Powell, Daniel J.

2015-01-01

Chimeric antigen receptors (CARs) can redirect T cells against antigen-expressing tumors in an HLA-independent manner. To date, various CARs have been constructed using mouse single chain antibody variable fragments (scFvs) of high affinity that are immunogenic in humans and have the potential to mediate “on-target” toxicity. Here, we developed and evaluated a fully human CAR comprised of the human C4 folate receptor-alpha (αFR)-specific scFv coupled to intracellular T cell signaling domains. Human T cells transduced to express the C4 CAR specifically secreted proinflammatory cytokine and exerted cytolytic functions when cultured with αFR-expressing tumors in vitro. Adoptive transfer of C4 CAR T cells mediated the regression of large, established human ovarian cancer in a xenogeneic mouse model. Relative to a murine MOv19 scFv-based αFR CAR, C4 CAR T cells mediated comparable cytotoxic tumor activity in vitro and in vivo but had lower affinity for αFR protein and exhibited reduced recognition of normal cells expressing low levels of αFR. Thus, T cells expressing a fully human CAR of intermediate affinity can efficiently kill antigen-expressing tumors in vitro and in vivo and may overcome issues of transgene immunogenicity and “on-target off-tumor” toxicity that plague trials utilizing CARs containing mouse-derived, high affinity scFvs. PMID:26101914

Glucagon Like Peptide-1 Receptor Expression in the Human Thyroid Gland

PubMed Central

Gier, Belinda; Butler, Peter C.; Lai, Chi K.; Kirakossian, David; DeNicola, Matthew M.

2012-01-01

Background: Glucagon like peptide-1 (GLP-1) mimetic therapy induces medullary thyroid neoplasia in rodents. We sought to establish whether C cells in human medullary thyroid carcinoma, C cell hyperplasia, and normal human thyroid express the GLP-1 receptor. Methods: Thyroid tissue samples with medullary thyroid carcinoma (n = 12), C cell hyperplasia (n = 9), papillary thyroid carcinoma (n = 17), and normal human thyroid (n = 15) were evaluated by immunofluorescence for expression of calcitonin and GLP-1 receptors. Results: Coincident immunoreactivity for calcitonin and GLP-1 receptor was consistently observed in both medullary thyroid carcinoma and C cell hyperplasia. GLP-1 receptor immunoreactivity was also detected in 18% of papillary thyroid carcinoma (three of 17 cases). Within normal human thyroid tissue, GLP-1 receptor immunoreactivity was found in five of 15 of the examined cases in about 35% of the total C cells assessed. Conclusions: In humans, neoplastic and hyperplastic lesions of thyroid C cells express the GLP-1 receptor. GLP-1 receptor expression is detected in 18% papillary thyroid carcinomas and in C cells in 33% of control thyroid lobes. The consequence of long-term pharmacologically increased GLP-1 signaling on these GLP-1 receptor-expressing cells in the thyroid gland in humans remains unknown, but appropriately powered prospective studies to exclude an increase in medullary or papillary carcinomas of the thyroid are warranted. PMID:22031513

Species-specific vesicular monoamine transporter 2 (VMAT2) expression in mammalian pancreatic beta cells: implications for optimising radioligand-based human beta cell mass (BCM) imaging in animal models

PubMed Central

Hartwig, N. R.; Kalmbach, N.; Klietz, M.; Anlauf, M.; Eiden, L. E.; Weihe, E.

2014-01-01

Aims/hypothesis Imaging of beta cell mass (BCM) is a major challenge in diabetes research. The vesicular monoamine transporter 2 (VMAT2) is abundantly expressed in human beta cells. Radiolabelled analogues of tetrabenazine (TBZ; a low-molecular-weight, cell-permeant VMAT2-selective ligand) have been employed for pancreatic islet imaging in humans. Since reports on TBZ-based VMAT2 imaging in rodent pancreas have been fraught with confusion, we compared VMAT2 gene expression patterns in the mouse, rat, pig and human pancreas, to identify appropriate animal models with which to further validate and optimise TBZ imaging in humans. Methods We used a panel of highly sensitive VMAT2 antibodies developed against equivalently antigenic regions of the transporter from each species in combination with immunostaining for insulin and species-specific in situ hybridisation probes. Individual pancreatic islets were obtained by laser-capture microdissection and subjected to analysis of mRNA expression of VMAT2. Results The VMAT2 protein was not expressed in beta cells in the adult pancreas of common mouse or rat laboratory strains, in contrast to its expression in beta cells (but not other pancreatic endocrine cell types) in the pancreas of pigs and humans. VMAT2- and tyrosine hydroxylase co-positive (catecholaminergic) innervation was less abundant in humans than in rodents. VMAT2-positive mast cells were identified in the pancreas of all species. Conclusions/interpretation Primates and pigs are suitable models for TBZ imaging of beta cells. Rodents, because of a complete lack of VMAT2 expression in the endocrine pancreas, are a ‘null’ model for assessing interference with BCM measurements by VMAT2-positive mast cells and sympathetic innervation in the pancreas. PMID:23404442

Expression of the oncoprotein gankyrin and phosphorylated retinoblastoma protein in human testis and testicular germ cell tumor.

PubMed

Ando, Satoshi; Matsuoka, Taeko; Kawai, Koji; Sugita, Shintaro; Joraku, Akira; Kojima, Takahiro; Suetomi, Takahiro; Miyazaki, Jun; Fujita, Jun; Nishiyama, Hiroyuki

2014-10-01

The oncoprotein, gankyrin, is known to facilitate cell proliferation through phosphorylation and degradation of retinoblastoma protein. In the present study, we evaluated the expression of gankyrin and phosphorylated retinoblastoma protein in human testis and testicular germ cell tumors. The effects of suppression of gankyrin by locked nucleic acid on phosphorylation status of retinoblastoma and cell proliferation were analyzed using western blot analysis and testicular tumor cell line NEC8. The expressions of gankyrin, retinoblastoma and retinoblastoma protein were analyzed in 93 testicular germ cell tumor samples and five normal human testis by immunohistochemistry. The retinoblastoma protein expression was determined using an antibody to retinoblastoma protein, Ser795. Gankyrin was expressed in NEC8 cells as well as a normal human testis and testicular tumors. Suppression of gankyrin by locked nucleic acid led to suppression of retinoblastoma protein and cell proliferation in NEC8 cells. Immunohistochemistry of normal testis showed that gankyrin is expressed dominantly in spermatocytes. In testicular germ cell tumors, high expressions of gankyrin and phosphorylated-retinoblastoma protein were observed in seminoma and embryonal carcinoma, whereas the expressions of both proteins were weak in histological subtypes of non-seminoma. Growing teratoma and testicular malignant transformation tissues expressed phosphorylated-retinoblastoma protein strongly, but gankyrin faintly. Gankyrin is dominantly expressed in normal spermatocytes and seminoma/embryonal carcinoma, and its expression correlates well with retinoblastoma protein expression except in the growing teratoma and testicular malignant transformation cases. These data provide new insights into the molecular mechanisms of normal spermatogenesis and pathogenesis of testicular germ cell tumors. © 2014 The Japanese Urological Association.

Comparative analysis of TCDD-induced AhR-mediated gene expression in human, mouse and rat primary B cells

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

Kovalova, Natalia, E-mail: kovalova@msu.edu

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental pollutant that activates the aryl hydrocarbon receptor (AhR) resulting in altered gene expression. In vivo, in vitro, and ex vivo studies have demonstrated that B cells are directly impaired by TCDD, and are a sensitive target as evidenced by suppression of antibody responses. The window of sensitivity to TCDD-induced suppression of IgM secretion among mouse, rat and human B cells is similar. Specifically, TCDD must be present within the initial 12 h post B cell stimulation, indicating that TCDD disrupts early signaling network(s) necessary for B lymphocyte activation and differentiation. Therefore, we hypothesized thatmore » TCDD treatment across three different species (mouse, rat and human) triggers a conserved, B cell-specific mechanism that is involved in TCDD-induced immunosuppression. RNA sequencing (RNA-Seq) was used to identify B cell-specific orthologous genes that are differentially expressed in response to TCDD in primary mouse, rat and human B cells. Time course studies identified TCDD-elicited differential expression of 515 human, 2371 mouse and 712 rat orthologous genes over the 24-h period. 28 orthologs were differentially expressed in response to TCDD in all three species. Overrepresented pathways enriched in all three species included cytokine-cytokine receptor interaction, ECM-receptor interaction, focal adhesion, regulation of actin cytoskeleton and pathways in cancer. Differentially expressed genes functionally associated with cell-cell signaling in humans, immune response in mice, and oxidation reduction in rats. Overall, these results suggest that despite the conservation of the AhR and its signaling mechanism, TCDD elicits species-specific gene expression changes. - Highlights: • Kovalova TAAP Highlights Nov. 2016 • RNA-Seq identified TCDD-induced gene expression in PWM-activated primary B cells. • TCDD elicited differential expression of 515 human, 2371 mouse and 712 rat orthologs. • 28 orthologs were differentially expressed in response to TCDD in all three species. • TCDD elicits mostly species-specific gene expression changes in activated B cells.« less

Analysis of gene expression profile induced by EMP-1 in esophageal cancer cells using cDNA Microarray

PubMed Central

Wang, Hai-Tao; Kong, Jian-Ping; Ding, Fang; Wang, Xiu-Qin; Wang, Ming-Rong; Liu, Lian-Xin; Wu, Min; Liu, Zhi-Hua

2003-01-01

AIM: To obtain human esophageal cancer cell EC9706 stably expressed epithelial membrane protein-1 (EMP-1) with integrated eukaryotic plasmid harboring the open reading frame (ORF) of human EMP-1, and then to study the mechanism by which EMP-1 exerts its diverse cellular action on cell proliferation and altered gene profile by exploring the effect of EMP-1. METHODS: The authors first constructed pcDNA3.1/myc-his expression vector harboring the ORF of EMP-1 and then transfected it into human esophageal carcinoma cell line EC9706. The positive clones were analyzed by Western blot and RT-PCR. Moreover, the cell growth curve was observed and the cell cycle was checked by FACS technique. Using cDNA microarray technology, the authors compared the gene expression pattern in positive clones with control. To confirm the gene expression profile, semi-quantitative RT-PCR was carried out for 4 of the randomly picked differentially expressed genes. For those differentially expressed genes, classification was performed according to their function and cellular component. RESULTS: Human EMP-1 gene can be stably expressed in EC9706 cell line transfected with human EMP-1. The authors found the cell growth decreased, among which S phase was arrested and G1 phase was prolonged in the transfected positive clones. By cDNA microarray analysis, 35 genes showed an over 2.0 fold change in expression level after transfection, with 28 genes being consistently up-regulated and 7 genes being down-regulated. Among the classified genes, almost half of the induced genes (13 out of 28 genes) were related to cell signaling, cell communication and particularly to adhesion. CONCLUSION: Overexpression of human EMP-1 gene can inhibit the proliferation of EC9706 cell with S phase arrested and G1 phase prolonged. The cDNA microarray analysis suggested that EMP-1 may be one of regulators involved in cell signaling, cell communication and adhesion regulators. PMID:12632483

Analysis of gene expression profile induced by EMP-1 in esophageal cancer cells using cDNA Microarray.

PubMed

Wang, Hai-Tao; Kong, Jian-Ping; Ding, Fang; Wang, Xiu-Qin; Wang, Ming-Rong; Liu, Lian-Xin; Wu, Min; Liu, Zhi-Hua

2003-03-01

To obtain human esophageal cancer cell EC9706 stably expressed epithelial membrane protein-1 (EMP-1) with integrated eukaryotic plasmid harboring the open reading frame (ORF) of human EMP-1, and then to study the mechanism by which EMP-1 exerts its diverse cellular action on cell proliferation and altered gene profile by exploring the effect of EMP-1. The authors first constructed pcDNA3.1/myc-his expression vector harboring the ORF of EMP-1 and then transfected it into human esophageal carcinoma cell line EC9706. The positive clones were analyzed by Western blot and RT-PCR. Moreover, the cell growth curve was observed and the cell cycle was checked by FACS technique. Using cDNA microarray technology, the authors compared the gene expression pattern in positive clones with control. To confirm the gene expression profile, semi-quantitative RT-PCR was carried out for 4 of the randomly picked differentially expressed genes. For those differentially expressed genes, classification was performed according to their function and cellular component. Human EMP-1 gene can be stably expressed in EC9706 cell line transfected with human EMP-1. The authors found the cell growth decreased, among which S phase was arrested and G1 phase was prolonged in the transfected positive clones. By cDNA microarray analysis, 35 genes showed an over 2.0 fold change in expression level after transfection, with 28 genes being consistently up-regulated and 7 genes being down-regulated. Among the classified genes, almost half of the induced genes (13 out of 28 genes) were related to cell signaling, cell communication and particularly to adhesion. Overexpression of human EMP-1 gene can inhibit the proliferation of EC9706 cell with S phase arrested and G1 phase prolonged. The cDNA microarray analysis suggested that EMP-1 may be one of regulators involved in cell signaling, cell communication and adhesion regulators.

Mesenchymal stem cells derived from human exocrine pancreas express transcription factors implicated in beta-cell development.

PubMed

Baertschiger, Reto M; Bosco, Domenico; Morel, Philippe; Serre-Beinier, Veronique; Berney, Thierry; Buhler, Leo H; Gonelle-Gispert, Carmen

2008-07-01

Transplantation of in vitro generated islets or insulin-producing cells represents an attractive option to overcome organ shortage. The aim of this study was to isolate, expand, and characterize cells from human exocrine pancreas and analyze their potential to differentiate into beta cells. Fibroblast-like cells growing out of human exocrine tissue were characterized by flow cytometry and by their capacity to differentiate into mesenchymal cell lineages. During cell expansion and after differentiation toward beta cells, expression of transcription factors of endocrine pancreatic progenitors was analyzed by reverse transcription polymerase chain reaction. Cells emerged from 14/18 human pancreatic exocrine fractions and were expanded up to 40 population doublings. These cells displayed surface antigens similar to mesenchymal stem cells from bone marrow. A culture of these cells in adipogenic and chondrogenic differentiation media allowed differentiation into adipocyte- and chondrocyte-like cells. During expansion, cells expressed transcription factors implicated in islet development such as Isl1, Nkx2.2, Nkx6.1, nestin, Ngn3, Pdx1, and NeuroD. Activin A and hepatocyte growth factor induced an expression of insulin, glucagon, and glucokinase. Proliferating cells with characteristics of mesenchymal stem cells and endocrine progenitors were isolated from exocrine tissue. Under specific conditions, these cells expressed little insulin. Human pancreatic exocrine tissue might thus be a source of endocrine cell progenitors.

The gene expression profile of non-cultured, highly purified human adipose tissue pericytes: Transcriptomic evidence that pericytes are stem cells in human adipose tissue

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

Silva Meirelles, Lindolfo da, E-mail: lindolfomeirelles@gmail.com; Laboratory for Stem Cells and Tissue Engineering, PPGBioSaúde, Lutheran University of Brazil, Av. Farroupilha 8001, 92425-900 Canoas, RS; Deus Wagatsuma, Virgínia Mara de

Pericytes (PCs) are a subset of perivascular cells that can give rise to mesenchymal stromal cells (MSCs) when culture-expanded, and are postulated to give rise to MSC-like cells during tissue repair in vivo. PCs have been suggested to behave as stem cells (SCs) in situ in animal models, although evidence for this role in humans is lacking. Here, we analyzed the transcriptomes of highly purified, non-cultured adipose tissue (AT)-derived PCs (ATPCs) to detect gene expression changes that occur as they acquire MSC characteristics in vitro, and evaluated the hypothesis that human ATPCs exhibit a gene expression profile compatible with anmore » AT SC phenotype. The results showed ATPCs are non-proliferative and express genes characteristic not only of PCs, but also of AT stem/progenitor cells. Additional analyses defined a gene expression signature for ATPCs, and revealed putative novel ATPC markers. Almost all AT stem/progenitor cell genes differentially expressed by ATPCs were not expressed by ATMSCs or culture-expanded ATPCs. Genes expressed by ATMSCs but not by ATPCs were also identified. These findings strengthen the hypothesis that PCs are SCs in vascularized tissues, highlight gene expression changes they undergo as they assume an MSC phenotype, and provide new insights into PC biology. - Highlights: • Non-cultured adipose tissue-derived human pericytes (ncATPCs) exhibit a distinctive gene expression signature. • ncATPCs express key adipose tissue stem cell genes previously described in vivo in mice. • ncATPCs express message for anti-proliferative and antiangiogenic molecules. • Most ncATPC-specific transcripts are absent in culture-expanded pericytes or ATMSCs • Gene expression changes ncATPCs undergo as they acquire a cultured ATMSC phenotype are pointed out.« less

Possible Involvement of Human Mast Cells in the Establishment of Pregnancy via Killer Cell Ig-Like Receptor 2DL4.

PubMed

Ueshima, Chiyuki; Kataoka, Tatsuki R; Hirata, Masahiro; Sugimoto, Akihiko; Iemura, Yoshiki; Minamiguchi, Sachiko; Nomura, Takashi; Haga, Hironori

2018-06-01

The involvement of mast cells in the establishment of pregnancy is unclear. Herein, we found that human mast cells are present in the decidual tissues of parous women and expressed a human-specific protein killer cell Ig-like receptor (KIR) 2DL4, a receptor for human leukocyte antigen G expressed on human trophoblasts. In contrast, decreased numbers of decidual mast cells and reduced KIR2DL4 expression were observed in these cells of infertile women who had undergone long-term corticosteroid treatment. Co-culture of the human mast cell line, LAD2, and human trophoblast cell line, HTR-8/SVneo, accelerated the migration and tube formation of HTR-8/SVneo cells in a KIR2DL4-dependent manner. These observations suggest the possible involvement of human mast cells in the establishment of pregnancy via KIR2DL4 and that long-term corticosteroid treatment may cause infertility by influencing the phenotypes of decidual mast cells. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Osthole inhibits proliferation and induces apoptosis in human osteosarcoma cells.

PubMed

Ding, Yong; Lu, Xiongwei; Hu, Xiaopeng; Ma, Jie; Ding, Huan

2014-02-01

The purpose of this study was to investigate the effect of osthole on osteosarcoma cell proliferation and apoptosis. Cell counting Kit-8 assay was performed to establish the effects of osthole on osteosarcoma MG-63 cell proliferation. Annexin V-FITC/PI was performed to analyze the apoptotic rate of the cells. The inhibitory effects of osthole on the expression of BCL-2, BAX, and caspase-3 were detected by Western blotting. Osthole inhibited the growth of human osteosarcoma MG-63 cells by inhibiting cell proliferation and induced cell apoptosis. Western blotting demonstrated that osthole downregulated the expressions of BCL-2 and caspase-3 and upregulated the expression of BAX in human osteosarcoma cells. Osthole can inhibit osteosarcoma cell proliferation and induced apoptosis effectively in a dose-dependent manner through downregulating the expression of BCL-2 and caspase-3 proteins levels and upregulating the expression of BAX proteins levels.

Growth factors FGF8 and FGF2 and their receptor FGFR1, transcriptional factors Msx-1 and MSX-2, and apoptotic factors p19 and RIP5 participate in the early human limb development.

PubMed

Becic, Tina; Kero, Darko; Vukojevic, Katarina; Mardesic, Snjezana; Saraga-Babic, Mirna

2018-04-01

The expression pattern of fibroblast growth factors FGF8 and FGF2 and their receptor FGFR1, transcription factors MSX-1 and MSX-2, as well as cell proliferation (Ki-67) and cell death associated caspase-3, p19 and RIP5 factors were analyzed in histological sections of eight 4th-9th-weeks developing human limbs by immunohistochemistry and semi-thin sectioning. Increasing expression of all analyzed factors (except FGF8) characterized both the multilayered human apical ectodermal ridge (AER), sub-ridge mesenchyme (progress zone) and chondrocytes in developing human limbs. While cytoplasmic co-expression of MSX-1 and MSX-2 was observed in both limb epithelium and mesenchyme, p19 displayed strong cytoplasmic expression in non-proliferating cells. Nuclear expression of Ki-67 proliferating cells, and partly of MSX-1 and MSX-2 was detected in the whole limb primordium. Strong expression of factors p19 and RIP5, both in the AER and mesenchyme of human developing limbs indicates their possible involvement in control of cell senescence and cell death. In contrast to animal studies, expression of FGFR1 in the surface ectoderm and p19 in the whole limb primordium might reflect interspecies differences in limb morphology. Expression of FGF2 and downstream RIP5 gene, and transcription factors Msx-1 and MSX-2 did not show human-specific changes in expression pattern. Based on their spatio-temporal expression during human limb development, our study indicates role of FGFs and Msx genes in stimulation of cell proliferation, limb outgrowth, digit elongation and separation, and additionally MSX-2 in control of vasculogenesis. The cascade of orchestrated gene expressions, including the analyzed developmental factors, jointly contribute to the complex human limb development. Copyright © 2018 Elsevier GmbH. All rights reserved.

GRP-induced up-regulation of Hsp72 promotes CD16+/94+ natural killer cell binding to colon cancer cells causing tumor cell cytolysis.

PubMed

Taglia, Lauren; Matusiak, Damien; Benya, Richard V

2008-01-01

Gastrin-releasing peptide (GRP) and its receptor (GRPR) are not normally expressed by epithelial cells lining the adult human colon. However post malignant transformation both GRP and its receptor are aberrantly expressed in the colon where we have previously shown they act to retard metastasis by enhancing tumor cell attachment to the extracellular matrix. In the present study, we show that GRP signaling via its cognate receptor when both are aberrantly expressed in human colon cancer cells causes heat shock protein 72 (Hsp72) to be expressed. We show that GRP/GRPR induces expression of Hsp72 by signaling via focal adhesion kinase. When expressed, Hsp72 promotes the binding of CD16+ and CD94+ natural killer cells, resulting in tumor cell cytolysis. These findings demonstrate the presence of a novel mechanism whereby aberrantly expressed GRP/GRPR in human colorectal cancer attenuates tumor progression and may promote a favorable outcome.

Altering calcium influx for selective destruction of breast tumor.

PubMed

Yu, Han-Gang; McLaughlin, Sarah; Newman, Mackenzie; Brundage, Kathleen; Ammer, Amanda; Martin, Karen; Coad, James

2017-03-04

Human triple-negative breast cancer has limited therapeutic choices. Breast tumor cells have depolarized plasma membrane potential. Using this unique electrical property, we aim to develop an effective selective killing of triple-negative breast cancer. We used an engineered L-type voltage-gated calcium channel (Cec), activated by membrane depolarization without inactivation, to induce excessive calcium influx in breast tumor cells. Patch clamp and flow cytometry were used in testing the killing selectivity and efficiency of human breast tumor cells in vitro. Bioluminescence and ultrasound imaging were used in studies of human triple-negative breast cancer cell MDA-MB-231 xenograft in mice. Histological staining, immunoblotting and immunohistochemistry were used to investigate mechanism that mediates Cec-induced cell death. Activating Cec channels expressed in human breast cancer MCF7 cells produced enormous calcium influx at depolarized membrane. Activating the wild-type Cav1.2 channels expressed in MCF7 cells also produced a large calcium influx at depolarized membrane, but this calcium influx was diminished at the sustained membrane depolarization due to channel inactivation. MCF7 cells expressing Cec died when the membrane potential was held at -10 mV for 1 hr, while non-Cec-expressing MCF7 cells were alive. MCF7 cell death was 8-fold higher in Cec-expressing cells than in non-Cec-expressing cells. Direct injection of lentivirus containing Cec into MDA-MB-231 xenograft in mice inhibited tumor growth. Activated caspase-3 protein was detected only in MDA-MB-231 cells expressing Cec, along with a significantly increased expression of activated caspase-3 in xenograft tumor treated with Cec. We demonstrated a novel strategy to induce constant calcium influx that selectively kills human triple-negative breast tumor cells.

Expression pattern of RAGE and IGF-1 in the human fetal ovary and ovarian serous carcinoma.

PubMed

Poljicanin, Ana; Filipovic, Natalija; Vukusic Pusic, Tanja; Soljic, Violeta; Caric, Ana; Saraga-Babic, Mirna; Vukojevic, Katarina

2015-01-01

The expression pattern of RAGE and IGF-1 proteins in different ovarian cell lineages was histologically analyzed in six fetal, nine adult human ovaries, and nine serous ovarian carcinomas (OSC) using immunohistochemical methods. Mild expression of IGF-1 in ovarian surface epithelium (Ose) and oocytes in the 15-week human ovaries increased to moderate or strong in the stromal cells, oocytes and follicular cells in week 22. Occasional mild RAGE expression was observed in Ose during week 15, while strong expression characterized primordial follicles in week 22. In the reproductive human ovary, IGF-1 was mildly to moderately expressed in all ovarian cell lineages except in theca cells of the tertiary follicle where IGF-1 was negative. RAGE was strongly positive in the granulosa cells and some theca cells of the tertiary follicle, while negative to mildly positive in all cells of the secondary follicle. In the postmenopausal human ovary IGF-1 and RAGE were mildly expressed in Ose and stroma. In OSC, cells were strongly positive to IGF-1 and RAGE, except for some negative stromal cells. Different levels of IGF-1 and RAGE co-expression characterized fetal ovarian cells during development. In reproductive ovaries, IGF-1 and RAGE were co-localized in the granulosa and theca interna cells of tertiary follicles, while in postmenopausal ovaries and OSC, IGF-1 and RAGE were co-localized in Ose and OSC cells respectively. Our results indicate that intracellular levels of IGF-1 and RAGE protein might regulate the final destiny of the ovarian cell populations prior and during folliculogenesis, possibly controlling the metastatic potential of OSC as well. Copyright © 2015. Published by Elsevier GmbH.

Radiosensitization by inhibiting STAT1 in renal cell carcinoma.

PubMed

Hui, Zhouguang; Tretiakova, Maria; Zhang, Zhongfa; Li, Yan; Wang, Xiaozhen; Zhu, Julie Xiaohong; Gao, Yuanhong; Mai, Weiyuan; Furge, Kyle; Qian, Chao-Nan; Amato, Robert; Butler, E Brian; Teh, Bin Tean; Teh, Bin S

2009-01-01

Renal cell carcinoma (RCC) has been historically regarded as a radioresistant malignancy, but the molecular mechanism underlying its radioresistance is not understood. This study investigated the role of signal transducer and activator of transcription 1 (STAT1), a transcription factor downstream of the interferon-signaling pathway, in radioresistant RCC. The expressions of STAT1 and STAT3 in 164 human clear cell RCC samples, 47 papillary RCC samples, and 15 normal kidney tissue samples were examined by microarray expression profiling and immunohistochemistry. Western blotting was performed to evaluate the total and phosphorylated STAT1 expression in CRL-1932 (786-O) (human clear cell RCC), SKRC-39 (human papillary RCC), CCL-116 (human fibroblast), and CRL-1441 (G-401) (human Wilms tumor). STAT1 was reduced or inhibited by fludarabine and siRNA, respectively, and the effects on radiation-induced cell death were investigated using clonogenic assays. STAT1 expression, but not STAT3 expression, was significantly greater in human RCC samples (p = 1.5 x 10(-8) for clear cell; and p = 3.6 x 10(-4) for papillary). Similarly, the expression of STAT1 was relatively greater in the two RCC cell lines. STAT1 expression was reduced by both fludarabine and siRNA, significantly increasing the radiosensitivity in both RCC cell lines. This is the first study reporting the overexpression of STAT1 in human clear cell and papillary RCC tissues. Radiosensitization in RCC cell lines was observed by a reduction or inhibition of STAT1 signaling, using fludarabine or siRNA. Our data suggest that STAT1 may play a key role in RCC radioresistance and manipulation of this pathway may enhance the efficacy of radiotherapy.

[Expression of calponin and P63 in human submandibular glands].

PubMed

Lu, Yu-he; Gao, Yan

2007-02-01

To observe the expression of new myoepithelial cell markers calponin and P63 in human submandibular glands. Calponin and P63 antigen in routinely processed human submandibular gland tissues were immunohistochemically demonstrated by monoclonal antibodies to calponin and P63. Calponin expressed around all acinus and intercalated ducts as linear or punctuate pattern. Positive staining was also noted in peripheral area of some thin striated ducts that connect to intercalated ducts. Subulate or trigonal calponin expression was sometimes seen between the duct dells of striated ducts. P63 expressed mainly in the nucleus of the basal cells of excretory duct. Calponin is an ideal gland. P63 labels mainly the basal cells of excretory duct. marker for myoepithelial cells of human submandibular

Effect of dexamethasone on expression of glucocorticoid receptor in human monocyte cell line THP-1.

PubMed

Li, Bo; Bai, Xiangjun; Wanh, Haiping

2006-01-01

The effect of dexamethasone with different concentrations and different stimulating periods on the expression of glucocorticoid receptors (GRalpha, GRbeta) protein was investigated in human monocyte cell line THP-1. The cultured human monocyte line THP-1 cells were stimulated by dexamethasone with different concentrations and different periods. The expression of GRalpha and GRbeta protein was detected by Western blotting. The results showed that the expression of GRalpha and GRbeta was detected in the THP-1 cells. The quantity of GRalpha expression was reduced by dexamethasone under the same concentration with the prolongation of the stimulating periods. The quantity of GRbeta expression was increased by dexamethasone treatment in a time- and dose-dependent manner. It was concluded that dexamethasone stimulation time-dependently reduced the GRalpha expression in THP-1 cells. Dexamethasone stimulation time- and dose-dependently increased the GRbeta expression in THP-1 cells. The expression of GRalpha and GRbeta was regulated by glucocorticoid.

Human T lymphocytes express N-methyl-D-aspartate receptors functionally active in controlling T cell activation

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

Miglio, Gianluca; Varsaldi, Federica; Lombardi, Grazia

2005-12-30

The aim of this study was to investigate the expression and the functional role of N-methyl-D-aspartate (NMDA) receptors in human T cells. RT-PCR analysis showed that human resting peripheral blood lymphocytes (PBL) and Jurkat T cells express genes encoding for both NR1 and NR2B subunits: phytohemagglutinin (PHA)-activated PBL also expresses both these genes and the NR2A and NR2D genes. Cytofluorimetric analysis showed that NR1 expression increases as a consequence of PHA (10 {mu}g/ml) treatment. D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5), and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine [(+)-MK 801], competitive and non-competitive NMDA receptor antagonists, respectively, inhibited PHA-induced T cell proliferation, whereas they did not affect IL-2 (10more » U/ml)-induced proliferation of PHA blasts. These effects were due to the prevention of T cell activation (inhibition of cell aggregate formation and CD25 expression), but not to cell cycle arrest or death. These results demonstrate that human T lymphocytes express NMDA receptors, which are functionally active in controlling cell activation.« less

Alternative splicing of the tyrosinase gene transcript in normal human melanocytes and lymphocytes.

PubMed

Fryer, J P; Oetting, W S; Brott, M J; King, R A

2001-11-01

We have identified and isolated ectopically expressed tyrosinase transcripts in normal human melanocytes and lymphocytes and in a human melanoma (MNT-1) cell line to establish a baseline for the expression pattern of this gene in normal tissue. Tyrosinase mRNA from human lymphoblastoid cell lines was reverse transcribed and amplified using specific "nested" primers. This amplification yielded eight identifiable transcripts; five that resulted from alternative splicing patterns arising from the utilization of normal and alternative splice sequences. Identical splicing patterns were found in transcripts from human primary melanocytes in culture and a melanoma cell line, indicating that lymphoblastoid cell lines provide an accurate reflection of transcript processing in melanocytes. Similar splicing patterns have also been found with murine melanocyte tyrosinase transcripts. Our results demonstrate that alternative splicing of human tyrosinase gene transcript produces a number of predictable and identifiable transcripts, and that human lymphoblastoid cell lines provide a source of ectopically expressed transcripts that can be used to study the biology of tyrosinase gene expression in humans.

Aquaporin 3 expression in human fetal membranes and its up-regulation by cyclic adenosine monophosphate in amnion epithelial cell culture.

PubMed

Wang, Shengbiao; Amidi, Fataneh; Beall, Marie; Gui, Lizhen; Ross, Michael G

2006-04-01

The cell membrane water channel protein aquaporins (AQPs) may be important in regulating the intramembranous (IM) pathway of amniotic fluid (AF) resorption. The objective of the present study was to determine whether aquaporin 3 (AQP3) is expressed in human fetal membranes and to further determine if AQP3 expression in primary human amnion cell culture is regulated by second-messenger cyclic adenosine monophosphate (cAMP). AQP3 expression in human fetal membranes of normal term pregnancy was studied by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC). To determine the effect of cAMP on AQP3 expression, primary human amnion cell cultures were treated in either heat-inactivated medium alone (control), or heat-inactivated medium containing: (1) SP-cAMP, a membrane-permeable and phosphodiesterase resistant cAMP agonist, or (2) forskolin, an adenylate cyclase stimulator. Total RNA was isolated and multiplex real-time RT-PCR employed for relative quantitation of AQP3 expression. We detected AQP3 expression in placenta, chorion, and amnion using RT-PCR. Using IHC, we identified AQP3 protein expression in placenta syncytiotrophoblasts and cytotrophoblasts, chorion cytotrophoblasts, and amnion epithelia. In primary amnion epithelial cell culture, AQP3 mRNA significantly increased at 2 hours following forskolin or SP-cAMP, remained elevated at 10 hours following forskolin, and returned to baseline levels by 20 hours following treatment. This study provides evidence of AQP3 expression in human fetal membranes and demonstrates that AQP3 expression in primary human amnion cell culture is up-regulated by second-messenger cAMP. As AQP3 is permeable to water, urea, and glycerol, modulation of its expression in fetal membranes may contribute to AF homeostasis.

Epidermal growth factor increases LRF/Pokemon expression in human prostate cancer cells.

PubMed

Aggarwal, Himanshu; Aggarwal, Anshu; Agrawal, Devendra K

2011-10-01

Leukemia/lymphoma related factor/POK erythroid myeloid ontogenic factor (LRF/Pokemon) is a member of the POK family of proteins that promotes oncogenesis in several forms of cancer. Recently, we found higher LRF expression in human breast and prostate carcinomas compared to the corresponding normal tissues. The aim of this study was to examine the regulation of LRF expression in human prostate cells. Epidermal growth factor (EGF) and its receptors mediate several tumorigenic cascades that regulate cell differentiation, proliferation, migration and survival of prostate cancer cells. There was significantly higher level of LRF expression in the nucleus of LNCaP and PC-3 cells than RWPE-1 cells. A significant increase in LRF expression was observed with increasing doses of EGF in more aggressive and androgen-sensitive prostate cancer cells suggesting that EGF signaling pathway is critical in upregulating the expression of LRF/Pokemon to promote oncogenesis. Copyright © 2011 Elsevier Inc. All rights reserved.

Epidermal Growth Factor Increases LRF/Pokemon Expression in Human Prostate Cancer Cells

PubMed Central

Aggarwal, Himanshu; Aggarwal, Anshu; Agrawal, Devendra K.

2011-01-01

Leukemia/lymphoma related factor/POK erythroid myeloid ontogenic factor (LRF/Pokemon) is a member of the POK family of proteins that promotes oncogenesis in several forms of cancer. Recently, we found higher LRF expression in human breast and prostate carcinomas compared to the corresponding normal tissues. The aim of this study was to examine the regulation of LRF expression in human prostate cells. Epidermal growth factor (EGF) and its receptors mediate several tumorigenic cascades that regulate cell differentiation, proliferation, migration and survival of prostate cancer cells. There was significantly higher level of LRF expression in the nucleus of LNCaP and PC-3 cells than RWPE-1 cells. A significant increase in LRF expression was observed with increasing doses of EGF in more aggressive and androgen-sensitive prostate cancer cells suggesting that EGF signaling pathway is critical in upregulating the expression of LRF/Pokemon to promote oncogenesis. PMID:21640721

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.

The Human Membrane Cofactor CD46 Is a Receptor for Species B Adenovirus Serotype 3

PubMed Central

Sirena, Dominique; Lilienfeld, Benjamin; Eisenhut, Markus; Kälin, Stefan; Boucke, Karin; Beerli, Roger R.; Vogt, Lorenz; Ruedl, Christiane; Bachmann, Martin F.; Greber, Urs F.; Hemmi, Silvio

2004-01-01

Many human adenovirus (Ad) serotypes use the coxsackie B virus-Ad receptor (CAR). Recently, CD46 was suggested to be a receptor of species B Ad serotype 11 (Ad11), Ad14, Ad16, Ad21, Ad35, and Ad50. Using Sindbis virus-mediated cDNA library expression, we identify here the membrane cofactor protein CD46 as a surface receptor of species B Ad3. All four major CD46 transcripts and one minor CD46 transcript expressed in nucleated human cells were isolated. Rodent BHK cells stably expressing the BC1 form of CD46 bound radiolabeled Ad3 with a dissociation constant of 0.3 nM, identical to that of CD46-positive HeLa cells expressing twice as many Ad3 binding sites. Pull-down experiments with recombinant Ad3 fibers and a soluble form of the CD46 extracellular domain linked to the Fc portion of human immunoglobulin G (CD46ex-Fc) indicated direct interactions of the Ad3 fiber knob with CD46ex-Fc but not CARex-Fc (Fc-linked extracellular domain of CAR). Ad3 colocalized with cell surface CD46 in both rodent and human cells at the light and electron microscopy levels. Anti-CD46 antibodies and CD46ex-Fc inhibited Ad3 binding to CD46-expressing BHK cells more than 10-fold and to human cells 2-fold. In CD46-expressing BHK cells, wild-type Ad3 and a chimeric Ad consisting of the Ad5 capsid and the Ad3 fiber elicited dose-dependent cytopathic effects and transgene expression, albeit less efficiently than in human cells. Together, our results show that all of the major splice forms of CD46 are predominant and functional binding sites of Ad3 on CD46-expressing rodent and human cells but may not be the sole receptor of species B Ads on human cells. These results have implications for understanding viral pathogenesis and therapeutic gene delivery. PMID:15078926

The human membrane cofactor CD46 is a receptor for species B adenovirus serotype 3.

PubMed

Sirena, Dominique; Lilienfeld, Benjamin; Eisenhut, Markus; Kälin, Stefan; Boucke, Karin; Beerli, Roger R; Vogt, Lorenz; Ruedl, Christiane; Bachmann, Martin F; Greber, Urs F; Hemmi, Silvio

2004-05-01

Many human adenovirus (Ad) serotypes use the coxsackie B virus-Ad receptor (CAR). Recently, CD46 was suggested to be a receptor of species B Ad serotype 11 (Ad11), Ad14, Ad16, Ad21, Ad35, and Ad50. Using Sindbis virus-mediated cDNA library expression, we identify here the membrane cofactor protein CD46 as a surface receptor of species B Ad3. All four major CD46 transcripts and one minor CD46 transcript expressed in nucleated human cells were isolated. Rodent BHK cells stably expressing the BC1 form of CD46 bound radiolabeled Ad3 with a dissociation constant of 0.3 nM, identical to that of CD46-positive HeLa cells expressing twice as many Ad3 binding sites. Pull-down experiments with recombinant Ad3 fibers and a soluble form of the CD46 extracellular domain linked to the Fc portion of human immunoglobulin G (CD46ex-Fc) indicated direct interactions of the Ad3 fiber knob with CD46ex-Fc but not CARex-Fc (Fc-linked extracellular domain of CAR). Ad3 colocalized with cell surface CD46 in both rodent and human cells at the light and electron microscopy levels. Anti-CD46 antibodies and CD46ex-Fc inhibited Ad3 binding to CD46-expressing BHK cells more than 10-fold and to human cells 2-fold. In CD46-expressing BHK cells, wild-type Ad3 and a chimeric Ad consisting of the Ad5 capsid and the Ad3 fiber elicited dose-dependent cytopathic effects and transgene expression, albeit less efficiently than in human cells. Together, our results show that all of the major splice forms of CD46 are predominant and functional binding sites of Ad3 on CD46-expressing rodent and human cells but may not be the sole receptor of species B Ads on human cells. These results have implications for understanding viral pathogenesis and therapeutic gene delivery.

GATA3 Inhibits Lysyl Oxidase Mediated Metastases of Human Basal Triple-Negative Breast Cancer Cells

PubMed Central

Chu, Isabel M.; Michalowski, Aleksandra M.; Hoenerhoff, Mark; Szauter, Kornelia M.; Luger, Dror; Sato, Misako; Flanders, Kathy; Oshima, Akira; Csiszar, Katalin; Green, Jeffrey E.

2011-01-01

Discovery of mechanisms that impede the aggressive and metastatic phenotype of human basal triple-negative type breast cancers (BTNBC) could provide novel targets for therapy for this form of breast cancer that has a relatively poor prognosis. Previous studies have demonstrated that the expression of GATA3, the master transcriptional regulator of mammary luminal differentiation, can reduce the tumorigenicity and metastatic propensity of the human BTNBC MDA-MB-231 cell line (MB231), although the mechanism for reduced metastases was not elucidated. We demonstrate through gene expression profiling that GATA3 expression in 231 cells resulted in the dramatic reduction in the expression of Lysyl oxidase (LOX), a metastasis-promoting matrix remodeling protein, in part, through methylation of the LOX promoter. Suppression of LOX expression by GATA3 was further confirmed in the BTNBC Hs578T cell line. Conversely, reduction of GATA3 expression by siRNA in luminal BT474 cells increased LOX expression. Reconstitution of LOX expression in 231-GATA3 cells restored metastatic propensity. A strong inverse association between high LOX and low GATA3 expression was confirmed in a panel of 51 human breast cancer cell lines. Similarly, human breast cancer microarray data demonstrated that high LOX/low GATA3 expression is associated with the BTNBC subtype of breast cancer and poor patient prognosis. Expression of GATA3 reprograms BTNBC to a less aggressive phenotype and inhibits a major mechanism of metastasis through inhibition of LOX. Induction of GATA3 in BTNBC cells or novel approaches that inhibit LOX expression or activity could be important strategies for treating BTNBC. PMID:21892208

Cell Type-Specific Chromatin Signatures Underline Regulatory DNA Elements in Human Induced Pluripotent Stem Cells and Somatic Cells.

PubMed

Zhao, Ming-Tao; Shao, Ning-Yi; Hu, Shijun; Ma, Ning; Srinivasan, Rajini; Jahanbani, Fereshteh; Lee, Jaecheol; Zhang, Sophia L; Snyder, Michael P; Wu, Joseph C

2017-11-10

Regulatory DNA elements in the human genome play important roles in determining the transcriptional abundance and spatiotemporal gene expression during embryonic heart development and somatic cell reprogramming. It is not well known how chromatin marks in regulatory DNA elements are modulated to establish cell type-specific gene expression in the human heart. We aimed to decipher the cell type-specific epigenetic signatures in regulatory DNA elements and how they modulate heart-specific gene expression. We profiled genome-wide transcriptional activity and a variety of epigenetic marks in the regulatory DNA elements using massive RNA-seq (n=12) and ChIP-seq (chromatin immunoprecipitation combined with high-throughput sequencing; n=84) in human endothelial cells (CD31 + CD144 + ), cardiac progenitor cells (Sca-1 + ), fibroblasts (DDR2 + ), and their respective induced pluripotent stem cells. We uncovered 2 classes of regulatory DNA elements: class I was identified with ubiquitous enhancer (H3K4me1) and promoter (H3K4me3) marks in all cell types, whereas class II was enriched with H3K4me1 and H3K4me3 in a cell type-specific manner. Both class I and class II regulatory elements exhibited stimulatory roles in nearby gene expression in a given cell type. However, class I promoters displayed more dominant regulatory effects on transcriptional abundance regardless of distal enhancers. Transcription factor network analysis indicated that human induced pluripotent stem cells and somatic cells from the heart selected their preferential regulatory elements to maintain cell type-specific gene expression. In addition, we validated the function of these enhancer elements in transgenic mouse embryos and human cells and identified a few enhancers that could possibly regulate the cardiac-specific gene expression. Given that a large number of genetic variants associated with human diseases are located in regulatory DNA elements, our study provides valuable resources for deciphering the epigenetic modulation of regulatory DNA elements that fine-tune spatiotemporal gene expression in human cardiac development and diseases. © 2017 American Heart Association, Inc.

Expression of Voltage-Gated Sodium Channel Nav1.8 in Human Prostate Cancer is Associated with High Histological Grade

PubMed Central

Suy, Simeng; Hansen, Todd P.; Auto, Heather D.; Kallakury, Bhaskar V.S.; Dailey, Vernon; Danner, Malika; MacArthur, Linda; Zhang, Ying; Miessau, Matthew J.; Collins, Sean P.; Brown, Milton L.

2013-01-01

Voltage-gated sodium (Nav) channels are required for impulse conductance in excitable tissues. Navs have been linked to human cancers, including prostate. The expression and distribution of Nav isoforms (Nav1.1-Nav1.9) in human prostate cancer are not well established. Here, we evaluated the expression of these isoforms and investigated the expression of Nav1.8 in human prostate cancer tissues. Nav1.8 was highly expressed in all examined cells. Expression of Nav1.1, Nav1.2, and Nav1.9 were high in DU-145, PC-3 and PC-3M cells compared to LNCaP (hormone-dependent), C4-2, C4-2B, and CWR22Rv-1 cells. Nav1.5 and Nav1.6 were expressed in all cells examined. Nav1.7 expression was absent in PC-3M and CWR22Rv-1, but expressed in the other cells examined. Immunohistochemistry revealed intensive Nav1.8 staining correlated with more advanced pathologic stage of disease. Increased intensity of nuclear Nav1.8 correlated with increased Gleason grade. Our results revealed that Nav1.8 is universally expressed in human prostate cancer cells. Nav1.8 expression statistically correlated with pathologic stage (P=0.04) and Gleason score (P=0.01) of human prostate tissue specimens. The aberrant nuclear localization of Nav1.8 with advanced prostate cancer tissues warrant further investigation into use of Nav1.8 as a potential biomarker to differentiate between early and advanced disease. PMID:24163825

Tissue-specific expression of human CD4 in transgenic mice.

PubMed Central

Gillespie, F P; Doros, L; Vitale, J; Blackwell, C; Gosselin, J; Snyder, B W; Wadsworth, S C

1993-01-01

The gene for the human CD4 glycoprotein, which serves as the receptor for human immunodeficiency virus type 1, along with approximately 23 kb of sequence upstream of the translational start site, was cloned. The ability of 5' flanking sequences to direct tissue-specific expression was tested in cell culture and in transgenic mice. A 5' flanking region of 6 kb was able to direct transcription of the CD4 gene in NIH 3T3 cells but did not result in detectable expression in the murine T-cell line EL4 or in four lines of transgenic mice. A larger 5' flanking region of approximately 23 kb directed high-level CD4 transcription in the murine T-cell line EL4 and in three independent lines of transgenic mice. Human CD4 expression in all tissues analyzed was tightly correlated with murine CD4 expression; the highest levels of human CD4 RNA expression were found in the thymus and spleen, with relatively low levels detected in other tissues. Expression of human CD4 protein in peripheral blood mononuclear cells was examined by flow cytometry in these transgenic animals and found to be restricted to the murine CD4+ subset of lymphocytes. Human CD4 protein, detected with an anti-human CD4 monoclonal antibody, was present on the surface of 45 to 50% of the peripheral blood mononuclear cells from all transgenic lines. Images PMID:8474453

[Cloning of human CD45 gene and its expression in Hela cells].

PubMed

Li, Jie; Xu, Tianyu; Wu, Lulin; Zhang, Liyun; Lu, Xiao; Zuo, Daming; Chen, Zhengliang

2015-11-01

To clone human CD45 gene PTPRC and establish Hela cells overexpressing recombinant human CD45 protein. The intact cDNA encoding human CD45 amplified using RT-PCR from the total RNA extracted from peripheral blood mononuclear cells (PBMCs) of a healthy donor was cloned into pMD-18T vector. The CD45 cDNA fragment amplified from the pMD-18T-CD45 by PCR was inserted to the coding region of the PcDNA3.1-3xflag vector, and the resultant recombinant expression vector PcDNA3.1-3xflag-CD45 was transfected into Hela cells. The expression of CD45 in Hela cells was detected by flow cytometry and Western blotting, and the phosphastase activity of CD45 was quantified using an alkaline phosphatase assay kit. The cDNA fragment of about 3 900 bp was amplified from human PBMCs and cloned into pMD-18T vector. The recombinant expression vector PcDNA3.1-3xflag-CD45 was constructed, whose restriction maps and sequence were consistent with those expected. The expression of CD45 in transfected Hela cells was detected by flow cytometry and Western blotting, and the expressed recombinant CD45 protein in Hela cells showed a phosphastase activity. The cDNA of human CD45 was successfully cloned and effectively expressed in Hela cells, which provides a basis for further exploration of the functions of CD45.

The role of CD133 in normal human prostate stem cells and malignant cancer-initiating cells.

PubMed

Vander Griend, Donald J; Karthaus, Wouter L; Dalrymple, Susan; Meeker, Alan; DeMarzo, Angelo M; Isaacs, John T

2008-12-01

Resolving the specific cell of origin for prostate cancer is critical to define rational targets for therapeutic intervention and requires the isolation and characterization of both normal human prostate stem cells and prostate cancer-initiating cells (CIC). Single epithelial cells from fresh normal human prostate tissue and prostate epithelial cell (PrEC) cultures derived from them were evaluated for the presence of subpopulations expressing stem cell markers and exhibiting stem-like growth characteristics. When epithelial cell suspensions containing cells expressing the stem cell marker CD133+ are inoculated in vivo, regeneration of stratified human prostate glands requires inductive prostate stromal cells. PrEC cultures contain a small subpopulation of CD133+ cells, and fluorescence-activated cell sorting-purified CD133+ PrECs self-renew and regenerate cell populations expressing markers of transit-amplifying cells (DeltaNp63), intermediate cells (prostate stem cell antigen), and neuroendocrine cells (CD56). Using a series of CD133 monoclonal antibodies, attachment and growth of CD133+ PrECs requires surface expression of full-length glycosylated CD133 protein. Within a series of androgen receptor-positive (AR+) human prostate cancer cell lines, CD133+ cells are present at a low frequency, self-renew, express AR, generate phenotypically heterogeneous progeny negative for CD133, and possess an unlimited proliferative capacity, consistent with CD133+ cells being CICs. Unlike normal adult prostate stem cells, prostate CICs are AR+ and do not require functional CD133. This suggests that (a) AR-expressing prostate CICs are derived from a malignantly transformed intermediate cell that acquires "stem-like activity" and not from a malignantly transformed normal stem cell and (b) AR signaling pathways are a therapeutic target for prostate CICs.

Overexpression of stromal cell-derived factor 1 and its receptor CXCR4 induces autocrine/paracrine cell proliferation in human pituitary adenomas.

PubMed

Barbieri, Federica; Bajetto, Adriana; Stumm, Ralf; Pattarozzi, Alessandra; Porcile, Carola; Zona, Gianluigi; Dorcaratto, Alessandra; Ravetti, Jean-Louis; Minuto, Francesco; Spaziante, Renato; Schettini, Gennaro; Ferone, Diego; Florio, Tullio

2008-08-15

Hypothalamic or locally produced growth factors and cytokines control pituitary development, functioning, and cell division. We evaluated the expression of the chemokine stromal cell-derived factor 1 (SDF1) and its receptor CXCR4 in human pituitary adenomas and normal pituitary tissues and their role in cell proliferation. The expression of SDF1 and CXCR4 in 65 human pituitary adenomas and 4 human normal pituitaries was determined by reverse transcription-PCR, immunohistochemistry, and confocal immunofluorescence. The proliferative effect of SDF1 was evaluated in eight fibroblast-free human pituitary adenoma cell cultures. CXCR4 mRNA was expressed in 92% of growth hormone (GH)-secreting pituitary adenomas (GHoma) and 81% of nonfunctioning pituitary adenomas (NFPA), whereas SDF1 was identified in 63% and 78% of GHomas and NFPAs, respectively. Immunostaining for CXCR4 and SDF1 showed a strong homogenous labeling in all tumoral cells in both GHomas and NFPAs. In normal tissues, CXCR4 and SDF1 were expressed only in a subset of anterior pituitary cells, with a lower expression of SDF1 compared with its cognate receptor. CXCR4 and SDF1 were not confined to a specific cell population in the anterior pituitary but colocalized with discrete subpopulations of GH-, prolactin-, and adrenocorticorticotropic hormone-secreting cells. Conversely, most of the SDF1-containing cells expressed CXCR4. In six of eight pituitary adenoma primary cultures, SDF1 induced a statistically significant increase in DNA synthesis that was prevented by the treatment with the CXCR4 antagonist AMD3100 or somatostatin. CXCR4 and SDF1 are overexpressed in human pituitary adenomas and CXCR4 activation may contribute to pituitary cell proliferation and, possibly, to adenoma development in humans.

Histone deacetylase inhibitors up-regulate LL-37 expression independent of toll-like receptor mediated signalling in airway epithelial cells.

PubMed

Liu, Quan; Liu, Juan; Roschmann, Kristina Irene Lisolette; van Egmond, Danielle; Golebski, Korneliusz; Fokkens, Wytske Johanna; Wang, Dehui; van Drunen, Cornelis Maria

2013-04-11

HDAC inhibitors have been proposed as anticancer agents. However, their roles in innate genes expression remain not well known. Cathelicidin LL-37 is one of the few human bactericidal peptides, but the regulation of histone acetylation on LL-37 expression in airway epithelium remains largely unknown. Therefore, we investigated the effects of two non-selective HDACi, trichostatin A (TSA) and sodium butyrate (SB), on the expression of the cathelicidin LL-37 in human airway epithelial cells. LL37 in human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells(PNEC) in response to HDAC inhibitors with or without poly (I:C) stimulation was assessed using real-time PCR and western blot. In parallel, IL-6 expression was evaluated by ELISA. Our results showed that HDAC inhibitors up-regulated LL-37 gene expression independent of poly (I:C) stimulation in PNEC as well as in NCI-H292 cells. HDAC inhibitors increased LL37 protein expression in NCI-H292 cells but not in PNEC. In addition, HDAC inhibitors significantly inhibited poly (I:C)-induced IL-6 production in both of the epithelial cells. In conclusion, HDAC inhibitors directly up-regulated LL-37 gene expression in human airway epithelial cells.

Histone deacetylase inhibitors up-regulate LL-37 expression independent of toll-like receptor mediated signalling in airway epithelial cells

PubMed Central

2013-01-01

HDAC inhibitors have been proposed as anticancer agents. However, their roles in innate genes expression remain not well known. Cathelicidin LL-37 is one of the few human bactericidal peptides, but the regulation of histone acetylation on LL-37 expression in airway epithelium remains largely unknown. Therefore, we investigated the effects of two non-selective HDACi, trichostatin A (TSA) and sodium butyrate (SB), on the expression of the cathelicidin LL-37 in human airway epithelial cells. LL37 in human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells(PNEC) in response to HDAC inhibitors with or without poly (I:C) stimulation was assessed using real-time PCR and western blot. In parallel, IL-6 expression was evaluated by ELISA. Our results showed that HDAC inhibitors up-regulated LL-37 gene expression independent of poly (I:C) stimulation in PNEC as well as in NCI-H292 cells. HDAC inhibitors increased LL37 protein expression in NCI-H292 cells but not in PNEC. In addition, HDAC inhibitors significantly inhibited poly (I:C)-induced IL-6 production in both of the epithelial cells. In conclusion, HDAC inhibitors directly up-regulated LL-37 gene expression in human airway epithelial cells. PMID:23577829

BetaIg-h3 is involved in the HAb18G/CD147-mediated metastasis process in human hepatoma cells.

PubMed

Tang, Juan; Zhou, Hong-wei; Jiang, Jian-li; Yang, Xiang-min; Li, Yu; Zhang, Hong-xin; Chen, Zhi-nan; Guo, Wei-ping

2007-03-01

HAb18G/CD147, a new hepatoma-associated antigen cloned and screened from human hepatocellular carcinoma cDNA library, is closely correlated with metastasis process in human hepatoma cells. In the present study we aimed to identify the pivotal molecules of the HAb18G/CD147 signal transduction pathway. The investigation showed that betaig-h3, a secretory extracellular matrix (ECM) protein, was upregulated in HAb18G/CD147-expressing human hepatoma T7721 cells and was downregulated by depressing HAb18G/CD147 expression. The expression of betaig-h3, upregulated in human hepatoma cells, was positively relative to the expression of HAb18G/CD147 in different human hepatoma cell lines. By overexpressing betaig-h3 in human SMMC-7721 hepatoma cells, we discovered that betaig-h3 promoted cell adhesion, invasion, and matrix metalloproteinase (MMP) secretion potential. HAb18G/CD147-induced invasion and metastasis potential of human hepatoma cells can be attenuated by antibodies specific for betaig-h3, and no significant differences on inhibitory effects were observed among T7721 cells incubated with antibodies for betaig-h3 or HAb18G/CD147 or both types together. Taken together, our study suggests that betaig-h3, regulated by the expression of HAb18G/CD147, is involved in the HAb18G/CD147 signal transduction pathway and mediates the HAb18G/CD147-induced invasion and metastasis process of human hepatoma cells.

Regulation of human genome expression and RNA splicing by human papillomavirus 16 E2 protein.

PubMed

Gauson, Elaine J; Windle, Brad; Donaldson, Mary M; Caffarel, Maria M; Dornan, Edward S; Coleman, Nicholas; Herzyk, Pawel; Henderson, Scott C; Wang, Xu; Morgan, Iain M

2014-11-01

Human papillomavirus 16 (HPV16) is causative in human cancer. The E2 protein regulates transcription from and replication of the viral genome; the role of E2 in regulating the host genome has been less well studied. We have expressed HPV16 E2 (E2) stably in U2OS cells; these cells tolerate E2 expression well and gene expression analysis identified 74 genes showing differential expression specific to E2. Analysis of published gene expression data sets during cervical cancer progression identified 20 of the genes as being altered in a similar direction as the E2 specific genes. In addition, E2 altered the splicing of many genes implicated in cancer and cell motility. The E2 expressing cells showed no alteration in cell growth but were altered in cell motility, consistent with the E2 induced altered splicing predicted to affect this cellular function. The results present a model system for investigating E2 regulation of the host genome. Copyright © 2014 Elsevier Inc. All rights reserved.

Human Naive T Cells Express Functional CXCL8 and Promote Tumorigenesis.

PubMed

Crespo, Joel; Wu, Ke; Li, Wei; Kryczek, Ilona; Maj, Tomasz; Vatan, Linda; Wei, Shuang; Opipari, Anthony W; Zou, Weiping

2018-05-25

Naive T cells are thought to be functionally quiescent. In this study, we studied and compared the phenotype, cytokine profile, and potential function of human naive CD4 + T cells in umbilical cord and peripheral blood. We found that naive CD4 + T cells, but not memory T cells, expressed high levels of chemokine CXCL8. CXCL8 + naive T cells were preferentially enriched CD31 + T cells and did not express T cell activation markers or typical Th effector cytokines, including IFN-γ, IL-4, IL-17, and IL-22. In addition, upon activation, naive T cells retained high levels of CXCL8 expression. Furthermore, we showed that naive T cell-derived CXCL8 mediated neutrophil migration in the in vitro migration assay, supported tumor sphere formation, and promoted tumor growth in an in vivo human xenograft model. Thus, human naive T cells are phenotypically and functionally heterogeneous and can carry out active functions in immune responses. Copyright © 2018 by The American Association of Immunologists, Inc.

Human scavenger receptor class B type I is expressed with cell-specific fashion in both initial and terminal site of reverse cholesterol transport.

PubMed

Nakagawa-Toyama, Yumiko; Hirano, Ken-ichi; Tsujii, Ken-ichi; Nishida, Makoto; Miyagawa, Jun-ichiro; Sakai, Naohiko; Yamashita, Shizuya

2005-11-01

The reverse cholesterol transport (RCT) is one of the major protective systems against atherosclerosis, in which high-density lipoprotein (HDL) removes cholesterol from lipid-laden cells and delivers it to the liver. Scavenger receptor class B type I (SR-BI) is a HDL receptor in the liver and adrenal glands and is involved in the selective uptake of cholesteryl ester from HDL, which has been extensively, analyzed using rodent models. However, the expression and regulation of the human homologue of this receptor are not known yet. We previously reported that this receptor is expressed in in vitro differentiated macrophages and its expression is up-regulated by the addition of modified lipoproteins into the medium [Hirano K, Yamashita S, Nakagawa Y, et al. Expression of human scavenger receptor class B type I in cultured human monocyte-derived macrophages and atherosclerotic lesions. Circ Res 1999;85:108-16]. In order to further investigate the physiological significance of this receptor in humans, we have performed extensive immunohistochemical analyses with specimens of the liver and adrenal glands as well as arteries with different stages of atherosclerotic lesions. In human liver and adrenal glands, a positive SR-BI immunoreactivity was detected in both hepatic and adrenal parenchymal cells as well as Kupffer cells. These parenchymal cells had a strong signal on the cell surface, whereas Kupffer cells showed a heterogeneous and punctate pattern. In human aorta and coronary arteries, SR-BI was highly expressed in atherosclerotic plaques, but not in non-atherosclerotic lesions. Double immunostaining revealed that SR-BI was expressed in a subpopulation of macrophages, of which staining pattern was similar to that observed in Kupffer cells. These data clearly demonstrated that SR-BI was expressed with cell-specific fashions in both the initial and terminal step of RCT in humans. Thus, SR-BI might be physiologically relevant and have distinct tissue-specific functions.

A Human-Specific α7-Nicotinic Acetylcholine Receptor Gene in Human Leukocytes: Identification, Regulation and the Consequences of CHRFAM7A Expression

PubMed Central

Costantini, Todd W; Dang, Xitong; Yurchyshyna, Maryana V; Coimbra, Raul; Eliceiri, Brian P; Baird, Andrew

2015-01-01

The human genome contains a variant form of the α7-nicotinic acetylcholine receptor (α7nAChR) gene that is uniquely human. This CHRFAM7A gene arose during human speciation and recent data suggests that its expression alters ligand tropism of the normally homopentameric human α7-AChR ligand-gated cell surface ion channel that is found on the surface of many different cell types. To understand its possible significance in regulating inflammation in humans, we investigated its expression in normal human leukocytes and leukocyte cell lines, compared CHRFAM7A expression to that of the CHRNA7 gene, mapped its promoter and characterized the effects of stable CHRFAM7A overexpression. We report here that CHRFAM7A is highly expressed in human leukocytes but that the levels of both CHRFAM7A and CHRNA7 mRNAs were independent and varied widely. To this end, mapping of the CHRFAM7A promoter in its 5′-untranslated region (UTR) identified a unique 1-kb sequence that independently regulates CHRFAM7A gene expression. Because overexpression of CHRFAM7A in THP1 cells altered the cell phenotype and modified the expression of genes associated with focal adhesion (for example, FAK, P13K, Akt, rho, GEF, Elk1, CycD), leukocyte transepithelial migration (Nox, ITG, MMPs, PKC) and cancer (kit, kitL, ras, cFos cyclinD1, Frizzled and GPCR), we conclude that CHRFAM7A is biologically active. Most surprisingly however, stable CHRFAM7A overexpression in THP1 cells upregulated CHRNA7, which, in turn, led to increased binding of the specific α7nAChR ligand, bungarotoxin, on the THP1 cell surface. Taken together, these data confirm the close association between CHRFAM7A and CHRNA7 expression, establish a biological consequence to CHRFAM7A expression in human leukocytes and support the possibility that this human-specific gene might contribute to, and/or gauge, a human-specific response to inflammation. PMID:25860877

Elevated expression of CD147 in patients with endometriosis and its role in regulating apoptosis and migration of human endometrial cells.

PubMed

Jin, Aihong; Chen, Hao; Wang, Chaoqun; Tsang, Lai Ling; Jiang, Xiaohua; Cai, Zhiming; Chan, Hsiao Chang; Zhou, Xiaping

2014-06-01

To examine the expression of CD147 in 60 human endometriosis lesions and how CD147 regulates migration and apoptosis in human uterine epithelial (HESs) cells. Experimental clinical study and laboratory-based investigation. Hospital and academic research center. Sixty women with chocolate cysts and 16 control women without endometriosis. Human uterine epithelial cells were treated with anti-CD147 antibody. Real-time polymerase chain reaction for detecting CD147 expression in 60 human endometriosis lesions; migration assay and CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) assay for cell functional investigation; Western blot for detecting protein levels; gelatin zymography for evaluating the activity of matrix metalloproteinase-2 (MMP-2) in cultured cells. Expression of CD147 was significantly higher in ectopic endometrial tissues from patients with endometriosis than in normal endometrial tissues. Interference with CD147 function led to decreased migration and cell viability in HESs cells. Surprisingly, MMP-2 expression and activity were not changed after treating HESs cells with anti-CD147 antibody. Further examination revealed that immunodepletion of CD147 induced apoptosis in HESs cells, leading to the activation of caspase 3 and poly(ADP-ribose) polymerase. The results of the present study suggest that abnormally high expression of CD147 in ovarian endometriosis lesions with enhanced cell survival (reduced apoptosis) and migration, in an MMP-2-independent manner, may underlie the progression of endometriosis in humans. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Genetically engineered humanized anti-ganglioside GM2 antibody against multiple organ metastasis produced by GM2-expressing small-cell lung cancer cells.

PubMed

Yamada, Tadaaki; Bando, Hideaki; Takeuchi, Shinji; Kita, Kenji; Li, Qi; Wang, Wei; Akinaga, Shiro; Nishioka, Yasuhiko; Sone, Saburo; Yano, Seiji

2011-12-01

Small-cell lung cancer (SCLC) grows rapidly and metastasizes to multiple organs. We examined the antimetastatic effects of the humanized anti-ganglioside GM2 (GM2) antibodies, BIW-8962 and KM8927, compared with the chimeric antibody KM966, in a SCID mouse model of multiple organ metastases induced by GM2-expressing SCLC cells. BIW-8962 and KM8927 induced higher antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity than KM966 against the GM2-expressing SCLC cell line SBC-3 in vitro. These humanized antibodies inhibited the production of multiple organ metastases, increased the number of apoptotic cells, and prolonged the survival of the SCID mice. Histological analyses using clinical specimens showed that SCLC cells expressed GM2. These findings suggest that humanized anti-GM2 antibodies could be therapeutically useful for controlling multiple organ metastases of GM2-expressing SCLC. © 2011 Japanese Cancer Association.

Fractalkine (CX3CL1), a new factor protecting β-cells against TNFα.

PubMed

Rutti, Sabine; Arous, Caroline; Schvartz, Domitille; Timper, Katharina; Sanchez, Jean-Charles; Dermitzakis, Emmanouil; Donath, Marc Y; Halban, Philippe A; Bouzakri, Karim

2014-10-01

We have previously shown the existence of a muscle-pancreas intercommunication axis in which CX3CL1 (fractalkine), a CX3C chemokine produced by skeletal muscle cells, could be implicated. It has recently been shown that the fractalkine system modulates murine β-cell function. However, the impact of CX3CL1 on human islet cells especially regarding a protective role against cytokine-induced apoptosis remains to be investigated. Gene expression was determined using RNA sequencing in human islets, sorted β- and non-β-cells. Glucose-stimulated insulin secretion (GSIS) and glucagon secretion from human islets was measured following 24 h exposure to 1-50 ng/ml CX3CL1. GSIS and specific protein phosphorylation were measured in rat sorted β-cells exposed to CX3CL1 for 48 h alone or in the presence of TNFα (20 ng/ml). Rat and human β-cell apoptosis (TUNEL) and rat β-cell proliferation (BrdU incorporation) were assessed after 24 h treatment with increasing concentrations of CX3CL1. Both CX3CL1 and its receptor CX3CR1 are expressed in human islets. However, CX3CL1 is more expressed in non-β-cells than in β-cells while its receptor is more expressed in β-cells. CX3CL1 decreased human (but not rat) β-cell apoptosis. CX3CL1 inhibited human islet glucagon secretion stimulated by low glucose but did not impact human islet and rat sorted β-cell GSIS. However, CX3CL1 completely prevented the adverse effect of TNFα on GSIS and on molecular mechanisms involved in insulin granule trafficking by restoring the phosphorylation (Akt, AS160, paxillin) and expression (IRS2, ICAM-1, Sorcin, PCSK1) of key proteins involved in these processes. We demonstrate for the first time that human islets express and secrete CX3CL1 and CX3CL1 impacts them by decreasing glucagon secretion without affecting insulin secretion. Moreover, CX3CL1 decreases basal apoptosis of human β-cells. We further demonstrate that CX3CL1 protects β-cells from the adverse effects of TNFα on their function by restoring the expression and phosphorylation of key proteins of the insulin secretion pathway.

Gemcitabine-induced CXCL8 expression counteracts its actions by inducing tumor neovascularization

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

Song, Yao; Baba, Tomohisa; Li, Ying-Yi

Patients with pancreatic ductal adenocarcinoma (PDAC) are frequently complicated with metastatic disease or locally advanced tumors, and consequently need chemotherapy. Gemcitabine is commonly used for PDAC treatment, but with limited efficacy. The capacity of gemcitabine to generate reactive oxygen species (ROS) in human pancreatic cancer cells, prompted us to examine its effects on the expression of pro-inflammatory cytokines and chemokines. We observed that gemcitabine enhanced selectively the expression of CXCL8 in human pancreatic cancer cells through ROS generation and NF-κB activation. In vitro blocking of CXCL8 failed to modulate gemcitabine-mediated inhibition of cell proliferation in human pancreatic cancer cells. Gemcitabine alsomore » enhanced CXCL8 expression in pancreatic cancer cells in xenografted tumor tissues. Moreover, anti-CXCL8 antibody treatment in vivo attenuated tumor formation as well as intra-tumoral vascularity in nude mice, which were transplanted with Miapaca-2 cells and treated with gemcitabine. Thus, gemcitabine-induced CXCL8 may counteract the drug through inducing neovascularization. - Highlights: • Gemcitabine induced CXCL8 expression in human pancreatic cancer cells. • CXCL8 expression required ROS generation and NF-κB activation. • CXCL8 did not affect in vitro proliferation of human pancreatic cancer cells. • CXCL8 in vivo counteracted gemcitabine by inducing neovascularization.« less

CREB-1 and AP-1 transcription factors JunD and Fra-2 regulate bone sialoprotein gene expression in human breast cancer cells.

PubMed

Detry, C; Lamour, V; Castronovo, V; Bellahcène, A

2008-02-01

Bone sialoprotein (BSP) expression is detected in a variety of human osteotropic cancers. High expression of BSP in breast and prostate primary carcinomas is associated with progression and bone metastases development. In this study, we examined the transcriptional regulation of BSP gene expression in MDA-MB-231 and MCF-7 human breast cancer cells compared with Saos-2 human osteoblast-like cells. BSP human promoter deletion analyses delineated a -56/-84 region, which comprises a cAMP response element (CRE) that was sufficient for maximal promoter activity in breast cancer cell lines. We found that the basic fibroblast growth factor response element (FRE) also located in the proximal promoter was a crucial regulator of human BSP promoter activity in Saos-2 but not in breast cancer cells. Promoter activity experiments in combination with DNA mobility shift assays demonstrated that BSP promoter activity is under the control of the CRE element, through CREB-1, JunD and Fra-2 binding, in MDA-MB-231, MCF-7 and in Saos-2 cells. Forskolin, a protein kinase A pathway activator, failed to enhance BSP transcriptional activity suggesting that CRE site behaves as a constitutive rather than an inducible element in these cell lines. Over-expression of JunD and Fra-2 increased BSP promoter activity and upregulated endogenous BSP protein expression in MCF-7 and Saos-2 cells while siRNA-mediated inhibition of both factors expression significantly reduced BSP protein level in MDA-MB-231. Collectively, these data provide with new transcriptional mechanisms, implicating CREB and AP-1 factors, that control BSP gene expression in breast cancer cells.

The Toxoplasma gondii ME-49 strain upregulates levels of A20 that inhibit NF-κB activation and promotes apoptosis in human leukaemia T-cell lines.

PubMed

Chen, Qian; Pang, Min-Hui; Ye, Xiao-Hong; Yang, Guang; Lin, Chen

2018-05-18

Acute T-lymphocyte leukaemia is a form of haematological malignancy with abnormal activation of NF-κB pathway, which results in high expression of A20 and ABIN1, which constitute a negative feedback mechanism for the regulation of NF-κB activation. Clinical studies have found that acute T-lymphocyte leukaemia patients are susceptible to Toxoplasma gondii infection; however, the effect of T. gondii on the proliferation and apoptosis of human leukaemia T-cells remains unclear. Here, we used the T. gondii ME-49 strain to infect human leukaemia T-cell lines Jurkat and Molt-4, to explore the effect of T. gondii on proliferation and apoptosis, which is mediated by NF-κB in human leukaemia T-cells. The Tunel assay was used to detect cell apoptosis. Cell Counting Kit-8 was used to detect cell proliferation viability. The apoptosis level and the expression level of NF-κB related proteins in human leukaemia T-cells were detected by flow cytometry and Western blotting. Western blotting analyses revealed that the T. gondii ME-49 strain increased the expression of A20 and decreased both ABIN1 expression and NF-κB p65 phosphorylation. By constructing a lentiviral-mediated shRNA to knockdown the A20 gene in Jurkat T-cells and Molt-4 T-cells, the apoptosis levels of the two cell lines decreased after T. gondii ME-49 infection, and levels of NF-κB p65 phosphorylation and ABIN1 were higher than in the non-konckdown group. After knockingdown ABIN1 gene expression by constructing the lentiviral-mediated shRNA and transfecting the recombinant expression plasmid containing the ABIN1 gene into two cell lines, apoptosis levels and cleaved caspase-8 expression increased or decreased in response to T. gondii ME-49 infection, respectively. Our data suggest that ABIN1 protects human leukaemia T-cells by allowing them to resist the apoptosis induced by T. gondii ME-49 and that the T. gondii ME-49 strain induces the apoptosis of human leukaemia T-cells via A20-mediated downregulation of ABIN1 expression.

Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells.

PubMed

Wang, Rui; Kozhaya, Lina; Mercer, Frances; Khaitan, Alka; Fujii, Hodaka; Unutmaz, Derya

2009-08-11

The molecules that define human regulatory T cells (Tregs) phenotypically and functionally remain to be fully characterized. We recently showed that activated human Tregs express mRNA for a transmembrane protein called glycoprotein A repetitions predominant (GARP, or LRRC32). Here, using a GARP-specific mAb, we demonstrate that expression of GARP on activated Tregs correlates with their suppressive capacity. However, GARP was not induced on T cells activated in the presence of TGFbeta, which expressed high levels of FOXP3 and lacked suppressive function. Ectopic expression of FOXP3 in conventional T cells was also insufficient for induction of GARP expression in most donors. Functionally, silencing GARP in Tregs only moderately attenuated their suppressive activity. CD25+ T cells sorted for high GARP expression displayed more potent suppressive activity compared with CD25+GARP- cells. Remarkably, CD25+GARP- T cells expanded in culture contained 3-5 fold higher IL-17-secreting cells compared with either CD25+GARP+ or CD25-GARP- cells, suggesting that high GARP expression can potentially discriminate Tregs from those that have switched to Th17 lineage. We also determined whether GARP expression correlates with FOXP3-expressing T cells in human immunodeficiency virus (HIV) -infected subjects. A subset of HIV+ individuals with high percentages of FOXP3+ T cells did not show proportionate increase in GARP+ T cells. This finding suggests that higher FOXP3 levels observed in these HIV+ individuals is possibly due to immune activation rather than to an increase in Tregs. Our findings highlight the significance of GARP both in dissecting duality of Treg/Th17 cell differentiation and as a marker to identify bona fide Tregs during diseases with chronic immune activation.

Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells

PubMed Central

Wang, Rui; Kozhaya, Lina; Mercer, Frances; Khaitan, Alka; Fujii, Hodaka; Unutmaz, Derya

2009-01-01

The molecules that define human regulatory T cells (Tregs) phenotypically and functionally remain to be fully characterized. We recently showed that activated human Tregs express mRNA for a transmembrane protein called glycoprotein A repetitions predominant (GARP, or LRRC32). Here, using a GARP-specific mAb, we demonstrate that expression of GARP on activated Tregs correlates with their suppressive capacity. However, GARP was not induced on T cells activated in the presence of TGFβ, which expressed high levels of FOXP3 and lacked suppressive function. Ectopic expression of FOXP3 in conventional T cells was also insufficient for induction of GARP expression in most donors. Functionally, silencing GARP in Tregs only moderately attenuated their suppressive activity. CD25+ T cells sorted for high GARP expression displayed more potent suppressive activity compared with CD25+GARP− cells. Remarkably, CD25+GARP− T cells expanded in culture contained 3–5 fold higher IL-17-secreting cells compared with either CD25+GARP+ or CD25−GARP− cells, suggesting that high GARP expression can potentially discriminate Tregs from those that have switched to Th17 lineage. We also determined whether GARP expression correlates with FOXP3-expressing T cells in human immunodeficiency virus (HIV) −infected subjects. A subset of HIV+ individuals with high percentages of FOXP3+ T cells did not show proportionate increase in GARP+ T cells. This finding suggests that higher FOXP3 levels observed in these HIV+ individuals is possibly due to immune activation rather than to an increase in Tregs. Our findings highlight the significance of GARP both in dissecting duality of Treg/Th17 cell differentiation and as a marker to identify bona fide Tregs during diseases with chronic immune activation. PMID:19666573

IL13Rα2 expression identifies tissue-resident IL-22 producing PLZF+ innate T cells in the human liver.

PubMed

Paquin-Proulx, Dominic; Greenspun, Benjamin C; Pasquet, Lise; Strunz, Benedikt; Aleman, Soo; Falconer, Karolin; Terabe, Masaki; Berzofsky, Jay A; Sandberg, Johan K; Melum, Espen; Nixon, Douglas F; Björkström, Niklas K

2018-04-20

Innate lymphocytes are selectively enriched in the liver where they have important roles in liver immunology. Murine studies have shown that type I NKT cells can promote liver inflammation whereas type II NKT cells have an anti-inflammatory role. In humans, type II NKT cells were found to accumulate in the gut during inflammation and IL13Rα2 was proposed as a marker for these cells. In the human liver, less is known about type I and II NKT cells. Here, we studied the phenotype and function of human liver T cells expressing IL13Rα2. We found that IL13Rα2 was expressed by around 1% of liver resident memory T cells but not on circulating T cells. In support of their innate-like T cell character, the IL13Rα2 + T cells had higher expression of PLZF compared to IL13Rα2 - T cells and possessed the capacity to produce IL-22. However, only a minority of human liver sulfatide-reactive type II NKT cells expressed IL13Rα2. Collectively, these findings suggest that IL13Rα2 identifies tissue-resident intrahepatic T cells with innate characteristics and the capacity to produce IL-22. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Heat shock protein-27 protects human bronchial epithelial cells against oxidative stress–mediated apoptosis: possible implication in asthma

PubMed Central

Merendino, Anna M.; Paul, Catherine; Vignola, Antonio M.; Costa, Maria A.; Melis, Mario; Chiappara, Giuseppina; Izzo, V.; Bousquet, J.; Arrigo, André-Patrick

2002-01-01

Inflammation of the human bronchial epithelium, as observed in asthmatics, is characterized by the selective death of the columnar epithelial cells, which desquamate from the basal cells. Tissue repair initiates from basal cells that resist inflammation. Here, we have evaluated the extent of apoptosis as well as the Hsp27 level of expression in epithelial cells from bronchial biopsy samples taken from normal and asthmatic subjects. Hsp27 is a chaperone whose expression protects against oxidative stress. We report that in asthmatic subjects the basal epithelium cells express a high level of Hsp27 but no apoptotic morphology. In contrast, apoptotic columnar cells are devoid of Hsp27 expression. Moreover, we observed a decreased resistance to hydrogen peroxide–induced apoptosis in human bronchial epithelial 16–HBE cells when they were genetically modified to express reduced levels of Hsp27. PMID:12482203

Gamma-glutamylcyclotransferase promotes the growth of human glioma cells by activating Notch-Akt signaling

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

Shen, Shang-Hang; Yu, Ning; Liu, Xi-Yao

Glioma as an aggressive type tumor is rapidly growing and has become one of the leading cause of cancer-related death worldwide. γ-Glutamylcyclotransferase (GGCT) has been shown as a diagnostic marker in various cancers. To reveal whether there is a correlation between GGCT and human glioma, GGCT expression in human glioma tissues and cell lines was first determined. We found that GGCT expression was up-regulated in human glioma tissues and cell lines. Further, we demonstrate that GGCT knockdown inhibits glioma cell T98G and U251 proliferation and colony formation, whereas GGCT overexpression leads to oppose effects. GGCT overexpression promotes the expression ofmore » Notch receptors and activates Akt signaling in glioma cells, and Notch-Akt signaling is activated in glioma tissues with high expression of GGCT. Finally, we show that inhibition of Notch-Akt signaling with Notch inhibitor MK-0752 blocks the effects of GGCT on glioma proliferation and colony formation. In conclusion, GGCT plays a critical role in glioma cell proliferation and may be a potential cancer therapeutic target. - Highlights: • GGCT expression is up-regulated in human glioma tissues and cell lines. • GGCT promotes glioma cell growth and colony formation. • GGCT promotes the activation of Notch-Akt signaling in glioma cells and tissues. • Notch inhibition blocks the role of GGCT in human glioma cells.« less

Molecular cloning of a human Ca2+-dependent cell-cell adhesion molecule homologous to mouse placental cadherin: its low expression in human placental tissues

PubMed Central

1989-01-01

P-cadherin is a subclass of Ca2+-dependent cell-cell adhesion molecules present in mouse placenta, where its localization suggests a function of connecting the embryo to the uterus (Nose, A., and M. Takeichi. 1986. J. Cell Biol. 103:2649-2658). We recently identified a human cadherin detected by an mAb capable of disrupting cell-cell adhesion of A-431 cells, and found that it was closely related immunochemically to mouse P-cadherin. Curiously, this cadherin was undetectable in human placenta by immunohistochemical examination (Shimoyama, Y., S. Hirohashi, S. Hirano, M. Noguchi, Y. Shimosato, M. Takeichi, and O. Abe. 1989. Cancer Res. 49:2128-2133). We here report the cloning and sequencing of cDNA clone encoding the human homologue of mouse P- cadherin. The deduced amino acid sequence of the human P-cadherin consists of 829 amino acid and shows striking homology with mouse P- cadherin. On Northern blot analysis, human P-cadherin was scarcely expressed in human placenta in contrast to mouse P-cadherin, which was abundantly expressed in mouse placenta throughout pregnancy, and it was shown that E-cadherin, but not P-cadherin, was the major cadherin molecule in human placenta. Moreover, NIH3T3 cells transfected with human P-cadherin cDNA expressed the functional cadherin molecule, which was identical to the cadherin we had previously identified using the mAb, showing that this molecule really does mediate cell-cell adhesion and that the cadherin we detected immunochemically is undoubtedly human P-cadherin. The results obtained in this study support the idea that P- cadherin plays little role, if any, in Ca2+-dependent cell-cell binding in human placental tissue at least after several weeks of pregnancy. PMID:2793940

In vitro generation of type-II pneumocytes can be initiated in human CD34(+) stem cells.

PubMed

Srikanth, Lokanathan; Venkatesh, Katari; Sunitha, Manne Mudhu; Kumar, Pasupuleti Santhosh; Chandrasekhar, Chodimella; Vengamma, Bhuma; Sarma, Potukuchi Venkata Gurunadha Krishna

2016-02-01

Human CD34(+) stem cells differentiated into type-II pneumocytes in Dulbecco's modified Eagle medium (DMEM) having hydrocortisone, insulin, fibroblast growth factor (FGF), epidermal growth factor (EGF) and bovine serum albumin (BSA), expressing surfactant proteins-B (SP-B) and C (SP-C), alkaline phosphatase (ALP) and lysozyme. FACS-enumerated pure CD34(+) cells, isolated from human peripheral blood, were cultured in DMEM and showed positive reaction with anti-human CD34 monoclonal antibodies in immunocytochemistry. These cells were cultured in DMEM having hydrocortisone, insulin, FGF, EGF and BSA (HIFEB-D) medium having an air-liquid interface. They differentiated into type-II pneumocytes with expression of SP-B and SP-C genes and disappearance of CD34 expression as assessed using real-time PCR. In reverse transcription-PCR amplicons showed 208 and 907 bp confirming SP-B and SP-C expressions. These cells expressed ALP with an activity of 1.05 ± 0.09 mM ml(-1) min(-1) and lysozyme that killed E. coli. The successful differentiation of human CD34(+) stem cells into type-II pneumocytes, and transplantation of such cells obtained from the patient's stem cell could be the futuristic approach to regenerate diseased lung alveoli.

Muscarinic receptor agonists stimulate matrix metalloproteinase 1-dependent invasion of human colon cancer cells

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

Raufman, Jean-Pierre, E-mail: jraufman@medicine.umaryland.edu; Cheng, Kunrong; Saxena, Neeraj

2011-11-18

Highlights: Black-Right-Pointing-Pointer Muscarinic receptor agonists stimulated robust human colon cancer cell invasion. Black-Right-Pointing-Pointer Anti-matrix metalloproteinase1 antibody pre-treatment blocks cell invasion. Black-Right-Pointing-Pointer Bile acids stimulate MMP1 expression, cell migration and MMP1-dependent invasion. -- Abstract: Mammalian matrix metalloproteinases (MMPs) which degrade extracellular matrix facilitate colon cancer cell invasion into the bloodstream and extra-colonic tissues; in particular, MMP1 expression correlates strongly with advanced colon cancer stage, hematogenous metastasis and poor prognosis. Likewise, muscarinic receptor signaling plays an important role in colon cancer; muscarinic receptors are over-expressed in colon cancer compared to normal colon epithelial cells. Muscarinic receptor activation stimulates proliferation, migration and invasionmore » of human colon cancer cells. In mouse intestinal neoplasia models genetic ablation of muscarinic receptors attenuates carcinogenesis. In the present work, we sought to link these observations by showing that MMP1 expression and activation plays a mechanistic role in muscarinic receptor agonist-induced colon cancer cell invasion. We show that acetylcholine, which robustly increases MMP1 expression, stimulates invasion of HT29 and H508 human colon cancer cells into human umbilical vein endothelial cell monolayers - this was abolished by pre-incubation with atropine, a non-selective muscarinic receptor inhibitor, and by pre-incubation with anti-MMP1 neutralizing antibody. Similar results were obtained using a Matrigel chamber assay and deoxycholyltaurine (DCT), an amidated dihydroxy bile acid associated with colon neoplasia in animal models and humans, and previously shown to interact functionally with muscarinic receptors. DCT treatment of human colon cancer cells resulted in time-dependent, 10-fold increased MMP1 expression, and DCT-induced cell invasion was also blocked by pre-treatment with anti-MMP1 antibody. This study contributes to understanding mechanisms underlying muscarinic receptor agonist-induced promotion of colon cancer and, more importantly, indicates that blocking MMP1 expression and activation has therapeutic promise to stop or retard colon cancer invasion and dissemination.« less

Tryptophanyl-tRNA synthetase mediates high-affinity tryptophan uptake into human cells.

PubMed

Miyanokoshi, Miki; Yokosawa, Takumi; Wakasugi, Keisuke

2018-06-01

The tryptophan (Trp) transport system has a high affinity and selectivity toward Trp, and has been reported to exist in both human and mouse macrophages. Although this system is highly expressed in interferon-γ (IFN-γ)-treated cells and indoleamine 2,3-dioxygenase 1 (IDO1)-expressing cells, its identity remains incompletely understood. Tryptophanyl-tRNA synthetase (TrpRS) is also highly expressed in IFN-γ-treated cells and also has high affinity and selectivity for Trp. Here, we investigated the effects of human TrpRS expression on Trp uptake into IFN-γ-treated human THP-1 monocytes or HeLa cells. Inhibition of human TrpRS expression by TrpRS-specific siRNAs decreased and overexpression of TrpRS increased Trp uptake into the cells. Of note, the TrpRS-mediated uptake system had more than hundred-fold higher affinity for Trp than the known System L amino acid transporter, promoted uptake of low Trp concentrations, and had very high Trp selectivity. Moreover, site-directed mutagenesis experiments indicated that Trp- and ATP-binding sites, but not tRNA-binding sites, in TrpRS are essential for TrpRS-mediated Trp uptake into the human cells. We further demonstrate that the addition of purified TrpRS to cell culture medium increases Trp uptake into cells. Taken together, our results reveal that TrpRS plays an important role in high-affinity Trp uptake into human cells. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Human T-lymphotropic virus type I tax regulates the expression of the human lymphotoxin gene.

PubMed

Tschachler, E; Böhnlein, E; Felzmann, S; Reitz, M S

1993-01-01

Human T-lymphotropic virus type-I (HTLV-I)-infected T-cell lines constitutively produce high levels of lymphotoxin (LT). To analyze the mechanisms that lead to the expression of LT in HTLV-I-infected cell lines, we studied regulatory regions of the human LT promoter involved in the activation of the human LT gene. As determined by deletional analysis, sequences between +137 and -116 (relative to the transcription initiation site) are sufficient to direct expression of a reporter gene in the HTLV-I-infected cell line MT-2. Site-directed mutation of a of the single kappa B-like motif present in the LT promoter region (positions -99 to -89) completely abrogated LT promoter activity in MT-2 cells, suggesting that this site plays a critical role in the activation of the human LT gene. Transfection of LT constructs into HTLV-I-uninfected and -unstimulated Jurkat and U937 cell lines showed little to no activity of the LT promoter. Cotransfection of the same constructs with a tax expression plasmid into Jurkat cells led to detectable promoter activity, which could be significantly increased by stimulation of the cells with phorbol myristate acetate (PMA). Similarly, cotransfection of the LT promoter constructs and the tax expression plasmid into U937 cells led to significant promoter activity upon stimulation with PMA. These data suggest that HTLV-I tax is involved in the upregulation of LT gene expression in HTLV-I-infected cells.

Multipotent human stromal cells isolated from cord blood, term placenta and adult bone marrow show distinct differences in gene expression pattern

PubMed Central

Matigian, Nicholas; Brooke, Gary; Zaibak, Faten; Rossetti, Tony; Kollar, Katarina; Pelekanos, Rebecca; Heazlewood, Celena; Mackay-Sim, Alan; Wells, Christine A.; Atkinson, Kerry

2014-01-01

Multipotent mesenchymal stromal cells derived from human placenta (pMSCs), and unrestricted somatic stem cells (USSCs) derived from cord blood share many properties with human bone marrow-derived mesenchymal stromal cells (bmMSCs) and are currently in clinical trials for a wide range of clinical settings. Here we present gene expression profiles of human cord blood-derived unrestricted somatic stem cells (USSCs), human placental-derived mesenchymal stem cells (hpMSCs), and human bone marrow-derived mesenchymal stromal cells (bmMSCs), all derived from four different donors. The microarray data are available on the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-TABM-880. Additionally, the data has been integrated into a public portal, www.stemformatics.org. Our data provide a resource for understanding the differences in MSCs derived from different tissues. PMID:26484151

Phospholipase C and perfringolysin O from Clostridium perfringens upregulate endothelial cell-leukocyte adherence molecule 1 and intercellular leukocyte adherence molecule 1 expression and induce interleukin-8 synthesis in cultured human umbilical vein endothelial cells.

PubMed Central

Bryant, A E; Stevens, D L

1996-01-01

Clostridium perfringens phospholipase C (PLC) and perfringolysin O (PFO) differentially induced human umbilical vein endothelial cell expression and synthesis of endothelial cell-leukocyte adherence molecule-1 (ELAM-1), intracellular leukocyte adherence molecule-1 (ICAM-1), and interleukin-8 (IL-8). PLC strongly induced expression of ELAM-1, ICAM-1, and IL-8, while PFO stimulated early ICAM-1 expression but did not promote ELAM-1 expression or IL-8 synthesis. PLC caused human umbilical vein endothelial cells to assume a fibroblastoid morphology, whereas PFO, in high concentrations or after prolonged low-dose toxin exposure, caused cell death. The toxin-induced expression of proadhesive and activational proteins and direct cytopathic effects may contribute to the leukostasis, vascular compromise, and capillary leak characteristics of C. perfringens gas gangrene. PMID:8557365

Human serum activates CIDEB-mediated lipid droplet enlargement in hepatoma cells

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

Singaravelu, Ragunath; National Research Council of Canada, Ottawa, Ontario K1A 0R6; Lyn, Rodney K.

Highlights: •Human serum induced differentiation of hepatoma cells increases cellular lipid droplet (LD) size. •The observed increase in LD size correlates with increased PGC-1α and CIDEB expression. •Induction of CIDEB expression correlates with rescue of VLDL secretion and loss of ADRP. •siRNA knockdown of CIDEB impairs the human serum mediated increase in LD size. •This system represents a cost-efficient model to study CIDEB’s role in lipid biology. -- Abstract: Human hepatocytes constitutively express the lipid droplet (LD) associated protein cell death-inducing DFFA-like effector B (CIDEB). CIDEB mediates LD fusion, as well as very-low-density lipoprotein (VLDL) maturation. However, there are limitedmore » cell culture models readily available to study CIDEB’s role in these biological processes, as hepatoma cell lines express negligible levels of CIDEB. Recent work has highlighted the ability of human serum to differentiate hepatoma cells. Herein, we demonstrate that culturing Huh7.5 cells in media supplemented with human serum activates CIDEB expression. This activation occurs through the induced expression of PGC-1α, a positive transcriptional regulator of CIDEB. Coherent anti-Stokes Raman scattering (CARS) microscopy revealed a correlation between CIDEB levels and LD size in human serum treated Huh7.5 cells. Human serum treatment also resulted in a rapid decrease in the levels of adipose differentiation-related protein (ADRP). Furthermore, individual overexpression of CIDEB was sufficient to down-regulate ADRP protein levels. siRNA knockdown of CIDEB revealed that the human serum mediated increase in LD size was CIDEB-dependent. Overall, our work highlights CIDEB’s role in LD fusion, and presents a new model system to study the PGC-1α/CIDEB pathway’s role in LD dynamics and the VLDL pathway.« less

Monocrotophos Induces the Expression of Xenobiotic Metabolizing Cytochrome P450s (CYP2C8 and CYP3A4) and Neurotoxicity in Human Brain Cells.

PubMed

Tripathi, Vinay Kumar; Kumar, Vivek; Pandey, Ankita; Vatsa, Pankhi; Dhasmana, Anupam; Singh, Rajat Pratap; Appikonda, Sri Hari Chandan; Hwang, Inho; Lohani, Mohtashim

2017-07-01

Expression of various cytochrome P450s (CYPs) in mammalian brain cells is well documented. However, such studies are hampered in neural/glial cells of human origin due to nonavailability of human brain cells. To address this issue, we investigated the expression and inducibility of CYP2C8 and CYP3A4 and their responsiveness against cyclophosphamide (CPA) and organophosphorus pesticide monocrotophos (MCP), a known developmental neurotoxicant in human neural (SH-SY5Y) and glial (U373-MG) cell lines. CPA induced significant expression of CYP2C8 and CYP3A4 in both types of cells in a time-dependent manner. Neural cell line exhibited relatively higher constitutive and inducible expression of CYPs than the glial cell line. MCP exposure alone could not induce the significant expression of CYPs, whereas the cells preexposed to CPA showed a significant response to MCP. Similar to the case of CPA induced expressions, neural cells were found to be more vulnerable than glial cells. Our data indicate differential expressions of CYPs in cultured human neural and glial cell lines. The findings were synchronized with protein ligand docking studies, which showed a significant modulatory capacity of MCP by strong interaction with CYP regulators-CAR and PXR. Similarly, the known CYP inducer CPA has also shown significant high docking scores with the two studied CYP regulators. We also observed a significant induction in reactive oxygen species (ROS), lipid peroxides (LPO), micronucleus (MN), chromosomal aberration (CA), and reduction in reduced glutathione (GSH) and catalase following the exposure of MCP. Moreover, the expressions of apoptotic markers such as caspase-3, caspase-9, Bax, and p53 were significantly upregulated, whereas the levels of antiapoptotic marker, Bcl2, was downregulated after the exposure of MCP in both cell lines. These findings confirm the involvement of ROS-mediated oxidative stress, which subsequently triggers apoptosis pathways in both human neural (SH-SY5Y) and glial (U373-MG) cell lines following the exposure of MCP.

The human leukocyte antigen G promotes trophoblast fusion and β-hCG production through the Erk1/2 pathway in human choriocarcinoma cell lines

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

Wang, Ji-meng; State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101; Zhao, Hong-xi

2013-05-10

Highlights: •HLA-G expression promotes BeWo cells fusion and fusogenic gene expression. •HLA-G is capable of inducing β-hCG production in human choriocarcinoma cell lines. •Up-regulation of β-hCG production by HLA-G is mediated via the Erk1/2 pathway. -- Abstract: The human leukocyte antigen G (HLA-G) is expressed on the fetal–maternal interface and plays a role in protecting fetal-derived trophoblasts from the maternal immune response, allowing trophoblasts to invade the uterus. However, HLA-G also possesses immune suppressing-independent functions. We found that HLA-G expressing BeWo choriocarcinoma cells increased cell–cell fusion compared to control BeWo cells under forskolin treatment. Regardless of forskolin treatment, the expressionmore » of fusogenic gene mRNAs, including syncytin-1, the transcription factor glial cell missing 1 (Gcm1), and beta human chorionic gonadotropin (β-hCG) were elevated. HLA-G up-regulates β-hCG production in human choriocarcinoma cells because HLA-G knockdown in JEG-3 cells induces a dramatic decrease in β-hCG compared with control cells. The defect in β-hCG production in HLA-G knocked-down cells could not be completely overcome by stimulating hCG production through increasing intracellular cAMP levels. HLA-G expressing cells have increased phosphorylation levels for extracellular signal-regulated kinase1/2 (Erk1/2) in BeWo cells. The Erk1/2 pathway is inactivated after the inhibition of HLA-G expression in JEG-3 cells. Finally, Erk1/2 inhibition was able to suppress the increased hCG production induced by HLA-G expression. Together, these data suggest novel roles for HLA-G in regulating β-hCG production via the modulation of the Erk1/2 pathway and by inducing trophoblast cell fusion.« less

Methylation of DACT2 accelerates esophageal cancer development by activating Wnt signaling

PubMed Central

Zhang, Meiying; Linghu, Enqiang; Zhan, Qimin; He, Tao; Cao, Baoping; Brock, Malcolm V.; Herman, James G.; Xiang, Rong; Guo, Mingzhou

2016-01-01

Esophageal cancer is one of the most common malignancies worldwide. DACT2 is frequently methylated in human lung, hepatic, gastric and thyroid cancers. The methylation status and function of DACT2 remain to be elucidated in human esophageal cancer. Ten esophageal cancer cell lines, 42 cases of dysplasia and 126 cases of primary esophageal cancer samples were analyzed in this study. The expression of DACT2 was detected in YES2 cells, while reduced DACT2 expression levels were found in TE8 and KYSE70 cells, and complete loss of DACT2 expression was found in KYSE30, KYSE140, KYSE150, KYSE410, KYSE450, TE3 and TE7 cells. Loss of expression or reduced expression of DACT2 correlated with promoter region hypermethylation in esophageal cancer cells. Restoration of DACT2 expression was induced by 5-aza-2′-deoxycytidine. In human primary esophageal squamous carcinoma, 69% (87/126) of samples were methylated. Methylation of DACT2 was significantly associated with tumor stage and metastasis (P < 0.01, P < 0.05). DACT2 suppressed colony formation, cell migration and invasion in esophageal cancer cells, and it also suppressed esophageal cancer cell xenograft growth. DACT2 inhibited Wnt signaling in human esophageal cancer cells. In conclusion, DACT2 is frequently methylated in human esophageal cancer and its expression is regulated by promoter region methylation. DACT2 suppresses esophageal cancer growth by inhibiting Wnt signaling. PMID:26919254

Trans-species comparison of PPAR and RXR expression by rat and human urothelial tissues.

PubMed

Chopra, Bikramjit; Hinley, Jennifer; Oleksiewicz, Martin B; Southgate, Jennifer

2008-04-01

Because some investigational peroxisome proliferator-activated receptors (PPAR) agonists cause tumors in the lower urinary tract of rats, we compared normal human and rat urothelium in terms of PPAR and retinoid X receptor (RXR) expression and proliferation-associated phenotypes. In situ, few human but most rat urothelial cells were Ki67 positive, indicating fundamental differences in cell cycle control. Rat and human urothelia expressed all 3 PPAR and the RXRalpha and RXRbeta isoforms in a predominantly nuclear localization, indicating that they may be biologically active. However, immunolocalization differences were observed between species. First, whereas PPARalpha and PPARbeta/delta were expressed throughout the human bladder or ureteric urothelium, in the rat urothelium PPARalpha was primarily, and PPARbeta/delta exclusively, restricted to superficial cells. Second, RXRbeta was restricted to intermediate and superficial layers of the human urothelium but tended to be absent from the rat superficial cells. Third, PPARgamma expression was present throughout the urothelia of both species but was most intense in the superficial human urothelium. Species differences were also observed in the expression of PPAR and RXR isoforms between cultured rat and human urothelial cells and in the smooth muscle. Our findings highlight the unique coexpression of multiple PPAR and RXR isoforms by urothelium and suggest that species differences in PPAR function between rat and human urothelia may be explored in an in vitro setting.

Effects of exogenous human leptin on heat shock protein 70 expression in MCF-7 breast cancer cells and breast carcinoma of nude mice xenograft model.

PubMed

Xue, Rong-quan; Gu, Jun-chao; Yu, Wei; Wang, Yu; Zhang, Zhong-tao; Ma, Xue-mei

2012-02-01

It is important to identify the multiple sites of leptin activity in obese women with breast cancer. In this study, we examined the effect of exogenous human leptin on heat shock protein 70 (HSP70) expression in MCF-7 human breast cancer cells and in a breast carcinoma xenograft model of nude mice. We cultured MCF-7 human breast cancer cells and established nude mice bearing xenografts of these cells, and randomly divided them into experimental and control groups. The experimental group was treated with human leptin, while the control group was treated with the same volume of normal saline. A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay was developed to quantify the mRNA expression of HSP70 in the MCF-7 human breast cancer cells and in tumor tissues. Western blotting analysis was applied to quantify the protein expression of HSP70 in the MCF-7 cells. Immunohistochemical staining was done to assess the positive rate of HSP70 expression in the tumor tissues. Leptin activated HSP70 in a dose-dependent manner in vitro: leptin upregulated significantly the expression of HSP70 at mRNA and protein levels in MCF-7 human breast cancer cells (P < 0.001). There was no significant difference in expression of HSP70 mRNA in the implanted tumors between the leptin-treated group and the control group (P > 0.05). Immunohistochemical staining revealed no significant difference in tumor HSP70 expression between the leptin-treated group and the control group (P > 0.05). A nude mouse xenograft model can be safely and efficiently treated with human leptin by subcutaneous injections around the tumor. HSP70 may be target of leptin in breast cancer. Leptin can significantly upregulate the expression of HSP70 in a dose-dependent manner in vitro.

Molecular expression in transfected corneal endothelial cells

NASA Astrophysics Data System (ADS)

Wang, Fan; Miao, Zhuang; Lu, Chengwei; Hao, Jilong

2017-10-01

To investigate the capability of human corneal endothelial cells serving as immunological cells. Expression of HLA-DP, -DQ, -DR, CD40, CD80, and CD86 was determined by immunohistochemical methods. Meanwhile, purified peripheral blood mononuclear cells were cocultured with human corneal endothelial cells which were pre-treated with and without -IFN respectively, activation of lymphocytes was determined by FACS analysis. In coculture system, T lymphocyte was activated by corneal endothelial cells, HLA-DP, -DQ, -DR and CD40 expression were increased by - IFN induction. Costimulatory molecular CD80 was shown on the endothelial cells. Human corneal endothelial cells were assumed to be involved in the corneal transplantation rejection process as potential antigen presenting cells.

Towards immunotherapy with redirected T cells in a large animal model: Ex vivo activation, expansion, and genetic modification of canine T cells

PubMed Central

Mata, Melinda; Vera, Juan; Gerken, Claudia; Rooney, Cliona M.; Miller, Tasha; Pfent, Catherine; Wang, Lisa L.; Wilson-Robles, Heather M.; Gottschalk, Stephen

2014-01-01

Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) has shown promising anti-tumor activity in early phase clinical studies, especially for hematological malignancies. However, most preclinical models do not reliably mimic human disease. We reasoned that developing an adoptive T-cell therapy approach for spontaneous osteosarcoma (OS) occurring in dogs would more closely reproduce the condition in human cancer. To generate CAR-expressing canine T cells we developed expansion and transduction protocols that allow for the generation of sufficient numbers of CAR-expressing canine T cells for future clinical studies in dogs within 2 weeks of ex vivo culture. To evaluate the functionality of CAR-expressing canine T cells we targeted HER2-positive OS. We demonstrate that canine OS is positive for HER2, and that canine T cells expressing a HER2-specific CAR with human-derived transmembrane and CD28.ζ signaling domains recognize and kill HER2-positive canine OS cell lines in an antigen-dependent manner. To reduce the potential immunogenicity of the CAR we evaluated a CAR with canine-derived transmembrane and signaling domains, and found no functional difference between human and canine CARs. Hence, we have successfully developed a strategy to generate CAR-expressing canine T cells for future preclinical studies in dogs. Testing T-cell therapies in an immunocompetent, outbred animal model may improve our ability to predict their safety and efficacy prior to conducting studies in humans. PMID:25198528

Single-cell gene expression analysis reveals diversity among human spermatogonia.

PubMed

Neuhaus, N; Yoon, J; Terwort, N; Kliesch, S; Seggewiss, J; Huge, A; Voss, R; Schlatt, S; Grindberg, R V; Schöler, H R

2017-02-10

Is the molecular profile of human spermatogonia homogeneous or heterogeneous when analysed at the single-cell level? Heterogeneous expression profiles may be a key characteristic of human spermatogonia, supporting the existence of a heterogeneous stem cell population. Despite the fact that many studies have sought to identify specific markers for human spermatogonia, the molecular fingerprint of these cells remains hitherto unknown. Testicular tissues from patients with spermatogonial arrest (arrest, n = 1) and with qualitatively normal spermatogenesis (normal, n = 7) were selected from a pool of 179 consecutively obtained biopsies. Gene expression analyses of cell populations and single-cells (n = 105) were performed. Two OCT4-positive individual cells were selected for global transcriptional capture using shallow RNA-seq. Finally, expression of four candidate markers was assessed by immunohistochemistry. Histological analysis and blood hormone measurements for LH, FSH and testosterone were performed prior to testicular sample selection. Following enzymatic digestion of testicular tissues, differential plating and subsequent micromanipulation of individual cells was employed to enrich and isolate human spermatogonia, respectively. Endpoint analyses were qPCR analysis of cell populations and individual cells, shallow RNA-seq and immunohistochemical analyses. Unexpectedly, single-cell expression data from the arrest patient (20 cells) showed heterogeneous expression profiles. Also, from patients with normal spermatogenesis, heterogeneous expression patterns of undifferentiated (OCT4, UTF1 and MAGE A4) and differentiated marker genes (BOLL and PRM2) were obtained within each spermatogonia cluster (13 clusters with 85 cells). Shallow RNA-seq analysis of individual human spermatogonia was validated, and a spermatogonia-specific heterogeneous protein expression of selected candidate markers (DDX5, TSPY1, EEF1A1 and NGN3) was demonstrated. The heterogeneity of human spermatogonia at the RNA and protein levels is a snapshot. To further assess the functional meaning of this heterogeneity and the dynamics of stem cell populations, approaches need to be developed to facilitate the repeated analysis of individual cells. Our data suggest that heterogeneous expression profiles may be a key characteristic of human spermatogonia, supporting the model of a heterogeneous stem cell population. Future studies will assess the dynamics of spermatogonial populations in fertile and infertile patients. RNA-seq data is published in the GEO database: GSE91063. This work was supported by the Max Planck Society and the Deutsche Forschungsgemeinschaft DFG-Research Unit FOR 1041 Germ Cell Potential (grant numbers SCHO 340/7-1, SCHL394/11-2). The authors declare that there is no conflict of interest. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

KITENIN is associated with tumor progression in human gastric cancer.

PubMed

Ryu, Ho-Seong; Park, Young-Lan; Park, Su-Jin; Lee, Ji-Hee; Cho, Sung-Bum; Lee, Wan-Sik; Chung, Ik-Joo; Kim, Kyung-Keun; Lee, Kyung-Hwa; Kweon, Sun-Seog; Joo, Young-Eun

2010-09-01

KAI1 COOH-terminal interacting tetraspanin (KITENIN) promotes tumor cell migration, invasion and metastasis in colon, bladder, head and neck cancer. The aims of current study were to evaluate whether KITENIN affects tumor cell behavior in human gastric cancer cell line and to document the expression of KITENIN in a well-defined series of gastric tumors, including complete long-term follow-up, with special reference to patient prognosis. To evaluate the impact of KITENIN knockdown on behavior of a human gastric cancer cell line, AGS, migration, invasion and proliferation assays using small-interfering RNA were performed. The expression of activator protein-1 (AP-1) target genes and AP-1 transcriptional activity were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and luciferase reporter assay. The expression of KITENIN and AP-1 target genes by RT-PCR and Western blotting or immunohistochemistry was also investigated in human gastric cancer tissues. The knockdown of KITENIN suppressed tumor cell migration, invasion and proliferation in AGS cells. The mRNA expression of matrix metalloproteinase-1 (MMP-1), MMP-3, cyclooxygenase-2 (COX-2), and CD44 was reduced by knockdown of KITENIN in AGS. AP-1 transcriptional activity was significantly decreased by knockdown of KITENIN in AGS cells. KITENIN expression was significantly increased in human cancer tissues at RNA and protein levels. Expression of MMP-1, MMP-3, COX-2 and CD44 were significantly increased in human gastric cancer tissues. Immunostaining of KITENIN was predominantly identified in the cytoplasm of cancer cells. Expression of KITENIN was significantly associated with tumor size, Lauren classification, depth of invasion, lymph node metastasis, tumor stage and poor survival. These results indicate that KITENIN plays an important role in human gastric cancer progression by AP-1 activation.

Expression and function of activin receptors in human endometrial adenocarcinoma cells.

PubMed

Tanaka, Tetsuji; Toujima, Saori; Otani, Tsutomu; Minami, Sawako; Yamoto, Mareo; Umesaki, Naohiko

2003-09-01

Menstrual cycle-dependent expressions of activin A in normal human endometrial tissues have been reported. Expression of activin receptor mRNAs and increased activin A production were also observed in human endometrial adenocarcinoma tissues, suggesting that activin A might enhance cell proliferation and inhibit apoptotic signaling in endometrial cancer cells. In this study, we have examined the effects of activin A on cell proliferation, anticancer drug-induced apoptosis and Fas-mediated apoptosis in 3 differentiated human endometrial adenocarcinoma cell lines, namely HEC-1, HHUA and Ishikawa. Flow cytometric analyses revealed moderate expressions of all 4 types of activin receptor subunits on the cell surfaces of the 3 cell lines. The proliferations of the 3 endometrial cancer cells were completely unaffected by activin A, whereas it suppressed the cell proliferation of a human ovarian endometrioid adenocarcinoma cell line, OVK-18, in a dose-dependent manner. Moreover, activin A did not affect the apoptotic changes in the 3 endometrial adenocarcinoma cells treated with 4 different anticancer drugs, namely CDDP, paclitaxel, etoposide and SN38. The apoptotic changes in HHUA cells treated with anti-Fas IgM were also unaffected by activin A. These results indicate that the increased activin A production in human endometrial adenocarcinoma tissues in vivo may not stimulate carcinoma cell proliferation or inhibit apoptotic signaling in carcinoma cells. Insensitivity to the usual growth suppression signals induced by activin A might be one of the mechanisms of immortality of human endometrial adenocarcinoma cells.

MICA/B expression is inhibited by unfolded protein response and associated with poor prognosis in human hepatocellular carcinoma.

PubMed

Fang, Liang; Gong, Jiuyu; Wang, Ying; Liu, Rongrong; Li, Zengshan; Wang, Zhe; Zhang, Yun; Zhang, Chunmei; Song, Chaojun; Yang, Angang; Ting, Jenny P-Y; Jin, Boquan; Chen, Lihua

2014-09-18

MICA/B are major ligands for NK cell activating receptor NKG2D and previous studies showed that the serum level of soluble MICA (sMICA) is an independent prognostic factor for advanced human hepatocellular carcinoma. However, the correlation between cellular MICA/B expression pattern and human hepatocellular carcinoma progression has not been well explored. The unfolded protein response is one of the main causes of resistance to chemotherapy and radiotherapy in tumor cells. However, whether the UPR in HCC could regulate the expression levels of MICA/B and affect the sensitivity of HCC cells to NK cell cytolysis has not been established yet. MICA/B expression pattern was evaluated by immunohistochemistry and Kaplan-Meier survival analysis was done to explore the relationship between MICA/B expression level and patient survival. The protein and mRNA expression levels of MICA/B in SMMC7721 and HepG2 cells treated by tunicamycin were evaluated by flow cytometry, Western Blot and RT-PCR. The cytotoxicity analysis was performed with the CytoTox 96 Non-Radioactive LDH Cytotoxicity Assay. MICA/B was highly expressed in human hepatocellular carcinoma and the expression level was significantly and negatively associated with tumor-node metastasis (TNM) stages. Patients with low level of MICA/B expression showed a trend of shorter survival time. The unfolded protein response (UPR) downregulated the expression of MICA/B. This decreased protein expression occurred via post-transcriptional regulation and was associated with proteasomal degradation. Moreover, decreased expression level of MICA/B led to the attenuated sensitivity of human HCC to NK cell cytotoxicity. These new findings of the connection of MICA/B, UPR and NK cells may represent a new concrete theory of NK cell regulation in HCC, and suggest that targeting this novel NK cell-associated immune evasion pathway may be meaningful in treating patients with HCC.

The Role of ARX in Human Pancreatic Endocrine Specification

PubMed Central

Gage, Blair K.; Asadi, Ali; Baker, Robert K.; Webber, Travis D.; Wang, Rennian; Itoh, Masayuki; Hayashi, Masaharu; Miyata, Rie; Akashi, Takumi; Kieffer, Timothy J.

2015-01-01

The in vitro differentiation of human embryonic stem cells (hESCs) offers a model system to explore human development. Humans with mutations in the transcription factor Aristaless Related Homeobox (ARX) often suffer from the syndrome X-linked lissencephaly with ambiguous genitalia (XLAG), affecting many cell types including those of the pancreas. Indeed, XLAG pancreatic islets lack glucagon and pancreatic polypeptide-positive cells but retain somatostatin, insulin, and ghrelin-positive cells. To further examine the role of ARX in human pancreatic endocrine development, we utilized genomic editing in hESCs to generate deletions in ARX. ARX knockout hESCs retained pancreatic differentiation capacity and ARX knockout endocrine cells were biased toward somatostatin-positive cells (94% of endocrine cells) with reduced pancreatic polypeptide (rarely detected), glucagon (90% reduced) and insulin-positive (65% reduced) lineages. ARX knockout somatostatin-positive cells shared expression patterns with human fetal and adult δ-cells. Differentiated ARX knockout cells upregulated PAX4, NKX2.2, ISL1, HHEX, PCSK1, PCSK2 expression while downregulating PAX6 and IRX2. Re-expression of ARX in ARX knockout pancreatic progenitors reduced HHEX and increased PAX6 and insulin expression following differentiation. Taken together these data suggest that ARX plays a key role in pancreatic endocrine fate specification of pancreatic polypeptide, somatostatin, glucagon and insulin positive cells from hESCs. PMID:26633894

The Role of ARX in Human Pancreatic Endocrine Specification.

PubMed

Gage, Blair K; Asadi, Ali; Baker, Robert K; Webber, Travis D; Wang, Rennian; Itoh, Masayuki; Hayashi, Masaharu; Miyata, Rie; Akashi, Takumi; Kieffer, Timothy J

2015-01-01

The in vitro differentiation of human embryonic stem cells (hESCs) offers a model system to explore human development. Humans with mutations in the transcription factor Aristaless Related Homeobox (ARX) often suffer from the syndrome X-linked lissencephaly with ambiguous genitalia (XLAG), affecting many cell types including those of the pancreas. Indeed, XLAG pancreatic islets lack glucagon and pancreatic polypeptide-positive cells but retain somatostatin, insulin, and ghrelin-positive cells. To further examine the role of ARX in human pancreatic endocrine development, we utilized genomic editing in hESCs to generate deletions in ARX. ARX knockout hESCs retained pancreatic differentiation capacity and ARX knockout endocrine cells were biased toward somatostatin-positive cells (94% of endocrine cells) with reduced pancreatic polypeptide (rarely detected), glucagon (90% reduced) and insulin-positive (65% reduced) lineages. ARX knockout somatostatin-positive cells shared expression patterns with human fetal and adult δ-cells. Differentiated ARX knockout cells upregulated PAX4, NKX2.2, ISL1, HHEX, PCSK1, PCSK2 expression while downregulating PAX6 and IRX2. Re-expression of ARX in ARX knockout pancreatic progenitors reduced HHEX and increased PAX6 and insulin expression following differentiation. Taken together these data suggest that ARX plays a key role in pancreatic endocrine fate specification of pancreatic polypeptide, somatostatin, glucagon and insulin positive cells from hESCs.

Prospectively isolated NGN3-expressing progenitors from human embryonic stem cells give rise to pancreatic endocrine cells.

PubMed

Cai, Qing; Bonfanti, Paola; Sambathkumar, Rangarajan; Vanuytsel, Kim; Vanhove, Jolien; Gysemans, Conny; Debiec-Rychter, Maria; Raitano, Susanna; Heimberg, Harry; Ordovas, Laura; Verfaillie, Catherine M

2014-04-01

Pancreatic endocrine progenitors obtained from human embryonic stem cells (hESCs) represent a promising source to develop cell-based therapies for diabetes. Although endocrine pancreas progenitor cells have been isolated from mouse pancreata on the basis of Ngn3 expression, human endocrine progenitors have not been isolated yet. As substantial differences exist between human and murine pancreas biology, we investigated whether it is possible to isolate pancreatic endocrine progenitors from differentiating hESC cultures by lineage tracing of NGN3. We targeted the 3' end of NGN3 using zinc finger nuclease-mediated homologous recombination to allow selection of NGN3eGFP(+) cells without disrupting the coding sequence of the gene. Isolated NGN3eGFP(+) cells express PDX1, NKX6.1, and chromogranin A and differentiate in vivo toward insulin, glucagon, and somatostatin single hormone-expressing cells but not to ductal or exocrine pancreatic cells or other endodermal, mesodermal, or ectodermal lineages. This confirms that NGN3(+) cells represent pancreatic endocrine progenitors in humans. In addition, this hESC reporter line constitutes a unique tool that may aid in gaining insight into the developmental mechanisms underlying fate choices in human pancreas and in developing cell-based therapies.

Influence of antiretroviral therapy on programmed death-1 (CD279) expression on T cells in lymph nodes of human immunodeficiency virus-infected individuals.

PubMed

Ehrhard, Simone; Wernli, Marion; Dürmüller, Ursula; Battegay, Manuel; Gudat, Fred; Erb, Peter

2009-10-01

Human immunodeficiency virus infection leads to T-cell exhaustion and involution of lymphoid tissue. Recently, the programmed death-1 pathway was found to be crucial for virus-specific T-cell exhaustion during human immunodeficiency virus infection. Programmed death-1 expression was elevated on human immunodeficiency virus-specific peripheral blood CD8+ and CD4+ T cells and correlated with disease severity. During human immunodeficiency infection, lymphoid tissue acts as a major viral reservoir and is an important site for viral replication, but it is also essential for regulatory processes important for immune recovery. We compared programmed death-1 expression in 2 consecutive inguinal lymph nodes of 14 patients, excised before antiretroviral therapy (antiretroviral therapy as of 1997-1999) and 16 to 20 months under antiretroviral therapy. In analogy to lymph nodes of human immunodeficiency virus-negative individuals, in all treated patients, the germinal center area decreased, whereas the number of germinal centers did not significantly change. Programmed death-1 expression was mostly found in germinal centers. The absolute extent of programmed death 1 expression per section was not significantly altered after antiretroviral therapy resulting in a significant-relative increase of programmed death 1 per shrunken germinal center. In colocalization studies, CD45R0+ cells that include helper/inducer T cells strongly expressed programmed death-1 before and during therapy, whereas CD8+ T cells, fewer in numbers, showed a weak expression for programmed death-1. Thus, although antiretroviral therapy seems to reduce the number of programmed death-1-positive CD8+ T lymphocytes within germinal centers, it does not down-regulate programmed death-1 expression on the helper/inducer T-cell subset that may remain exhausted and therefore unable to trigger immune recovery.

Dopamine D2 receptor expression in the corticotroph cells of the human normal pituitary gland.

PubMed

Pivonello, Rosario; Waaijers, Marlijn; Kros, Johan M; Pivonello, Claudia; de Angelis, Cristina; Cozzolino, Alessia; Colao, Annamaria; Lamberts, Steven W J; Hofland, Leo J

2017-08-01

The dopamine D 2 receptor is the main dopamine receptor expressed in the human normal pituitary gland. The aim of the current study was to evaluate dopamine D 2 receptor expression in the corticotroph cell populations of the anterior lobe and pars intermedia, as well as posterior lobe of the human normal pituitary gland by immunohistochemistry. Human normal pituitary gland samples obtained from routine autopsies were used for the study. In all cases, histology together with immunostaining for adrenocorticotropic hormone, melanocyte-stimulating hormone, prolactin, and neurofilaments were performed and compared to the immunostaining for D 2 receptor. D 2 receptor was heterogeneously expressed in the majority of the cell populations of the anterior and posterior lobe as well as in the area localized between the anterior and posterior lobe, and arbitrary defined as "intermediate zone". This zone, characterized by the presence of nerve fibers included the residual pars intermedia represented by the colloid-filled cysts lined by the remnant melanotroph cells strongly expressing D 2 receptors, and clusters of corticotroph cells, belonging to the anterior lobe but localized within the cysts and adjacent to the posterior lobe, variably expressing D 2 receptors. D 2 dopamine receptor is expressed in the majority of the cell populations of the human normal pituitary gland, and particularly, in the different corticotroph cell populations localized in the anterior lobe and the intermediate zone of the pituitary gland.

Gene expression analysis uncovers novel Hedgehog interacting protein (HHIP) effects in human bronchial epithelial cells

PubMed Central

Zhou, Xiaobo; Qiu, Weiliang; Sathirapongsasuti, J. Fah.; Cho, Michael H.; Mancini, John D.; Lao, Taotao; Thibault, Derek M.; Litonjua, Gus; Bakke, Per S.; Gulsvik, Amund; Lomas, David A.; Beaty, Terri H.; Hersh, Craig P.; Anderson, Christopher; Geigenmuller, Ute; Raby, Benjamin A.; Rennard, Stephen I.; Perrella, Mark A.; Choi, Augustine M.K.; Quackenbush, John; Silverman, Edwin K.

2013-01-01

Hedgehog Interacting Protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis. PMID:23459001

Therapeutic effect of targeted Fas-expressing adenoviruses method combining γδ T cells in a mouse model of human ovarian carcinoma.

PubMed

Zeng, Dingyuan; Lin, Jiajing; He, Hongying; Tan, Guangping; Lan, Ying; Jiang, Fuyan; Sheng, Shuting

2018-02-01

The present study aimed to investigate the therapeutic effect and safety of targeted use of Fas-expressing adenoviruses combined with γδ T cell-mediated killing to treat human ovarian cancer xenografts in BALB/c mice. Shuttle plasmids containing control elements of human telomerase reverse transcriptase promoter and two-step transcriptional amplification system were constructed and packaged into adenovirus-5 vectors to generate expression of an exogenous Fas gene. A mouse xenograft model of human ovarian carcinoma was constructed. A total of 35 BALB/c mice were randomly divided into five groups, which were injected with PBS, γδ T cells, Fas-expressing adenoviruses, taxol, or Fas-expressing adenovirus and γδ T cells. The weight and volume of tumors in mice in each group was monitored. Tissue sections of the various tissues of mice in the Fas-expressing adenovirus and γδ T cells group was compared with those in the PBS group to evaluate the safety of Fas-expressing adenovirus and γδ T cells in the treatment of human ovarian cancer xenograft tumors. The results of the present study indicated that mice in all treatment groups were alive at the end of the treatment course. Tumor weight and volume was the highest in the PBS group, followed successively by the adenovirus group, the γδ T cell group, the adenovirus and γδ T cell group, and the taxol group. The weight and volume inhibition rate in adenovirus and γδ T cell group were significantly higher compared with in the PBS group (P<0.05). Pathological observation of tissue samples revealed that none of vital organs in the adenovirus and γδ T cell group developed any evident morphological changes during treatment, when compared with healthy controls. In conclusion, the combined therapy with Fas-expressing adenoviruses and γδ T cells is efficient and safe for the treatment of mouse human ovarian carcinoma xenografts.

NFAT5 promotes proliferation and migration of lung adenocarcinoma cells in part through regulating AQP5 expression

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

Guo, Kai, E-mail: gk161@163.com; Department of Respiration, 161th Hospital, PLA, Wuhan 430015; Jin, Faguang, E-mail: jinfag@fmmu.edu.cn

2015-09-25

The osmoregulated transcription factor nuclear factor of activated T-cells 5(NFAT5), has been found to play important roles in the development of many kinds of human cancers, including breast cancer, colon carcinoma, renal cell carcinoma and melanoma. The aim of the present study was to determine whether NFAT5 is involved in the proliferation and migration of lung adenocarcinoma cells. We found that NFAT5 was upregulated in lung adenocarcinoma cells and knockdown of NFAT5 decreased proliferation and migration of the cells, accompanied by a significant reduction in the expression of AQP5. AQP5 was upregulated in lung adenocarcinoma cells and knockdown of AQP5more » also inhibited proliferation and migration of the cells as knockdown of NFAT5 did. Moreover, overexpression of NFAT5 promoted proliferation and migration of lung adenocarcinoma cells, accompanied by a significant increase in the expression of AQP5. These results indicate that NFAT5 plays important roles in proliferation and migration of human lung adenocarcinoma cells through regulating AQP5 expression, providing a new therapeutic option for lung adenocarcinoma therapy. - Highlights: • NFAT5 expression is higher in lung adenocarcinoma cells compared with normal cells. • NFAT5 knockdown decreases proliferation and migration of lung adenocarcinoma cells. • Knockdown of NFAT5 reduces AQP5 expression in human lung adenocarcinoma cells. • Overexpression of NFAT5 promotes proliferation and migration of lung adenocarcinoma cells. • Overexpression of NFAT5 increases AQP5 expression in human lung adenocarcinoma cells.« less

Expression of genes involved in early cell fate decisions in human embryos and their regulation by growth factors.

PubMed

Kimber, S J; Sneddon, S F; Bloor, D J; El-Bareg, A M; Hawkhead, J A; Metcalfe, A D; Houghton, F D; Leese, H J; Rutherford, A; Lieberman, B A; Brison, D R

2008-05-01

Little is understood about the regulation of gene expression in human preimplantation embryos. We set out to examine the expression in human preimplantation embryos of a number of genes known to be critical for early development of the murine embryo. The expression profile of these genes was analysed throughout preimplantation development and in response to growth factor (GF) stimulation. Developmental expression of a number of genes was similar to that seen in murine embryos (OCT3B/4, CDX2, NANOG). However, GATA6 is expressed throughout preimplantation development in the human. Embryos were cultured in IGF-I, leukaemia inhibitory factor (LIF) or heparin-binding EGF-like growth factor (HBEGF), all of which are known to stimulate the development of human embryos. Our data show that culture in HBEGF and LIF appears to facilitate human embryo expression of a number of genes: ERBB4 (LIF) and LIFR and DSC2 (HBEGF) while in the presence of HBEGF no blastocysts expressed EOMES and when cultured with LIF only two out of nine blastocysts expressed TBN. These data improve our knowledge of the similarities between human and murine embryos and the influence of GFs on human embryo gene expression. Results from this study will improve the understanding of cell fate decisions in early human embryos, which has important implications for both IVF treatment and the derivation of human embryonic stem cells.

Altered expression of epithelial mesenchymal transition and pluripotent associated markers by sex steroid hormones in human embryonic stem cells.

PubMed

Jeon, So-Ye; Hwang, Kyung-A; Kim, Cho-Won; Jeung, Eui-Bae; Choi, Kyung-Chul

2017-07-01

Embryonic stem (ES) cells are pluripotent stem cells derived from a developmental stage of pre‑implanted embryos. The present study investigated the effect of female sex steroid hormones on the characteristics of human ES cells by using a feeder‑free culture protocol. In a feeder‑free condition without sex hormones, human ES cells assumed the form of tightly packed cells that grow in a monolayer. The cells had clean and defined edges with no evidence of differentiation and expressed several markers specific for undifferentiated ES cells including POU class 5 homeobox 1 (POU5F1), sex determining region Y‑box 2 (SOX2) and NANOG homeobox (NANOG). It was then investigated if female sex steroid hormones including 17β‑estradiol (E2) and progesterone (P4) altered the protein expression of epithelial-mesenchymal transition (EMT) associated markers in addition to pluripotency markers including POU5F1, SOX2 and NANOG in human ES cells. The protein expression levels of N‑cadherin, Snail and Slug were increased while E‑cadherin expression was decreased by treatment of E2 or P4, and the expression levels of POU5F1, SOX2 and NANOG were decreased by the treatment of E2 or P4. When E2 and P4 were treated in combination with an estrogen receptor inhibitor (ICI 182,780) and progesterone receptor inhibitor (RU486) respectively, their effects on EMT and pluripotency of ES cells were restored to control levels. The results suggested that E2 and P4 may regulate EMT and pluripotency of human ES cells by mediating their receptors. The present study may aid in the understanding of the role of sex steroid hormones in the cellular biology of human ES cells.

STK-1, the human homolog of Flk-2/Flt-3, is selectively expressed in CD34+ human bone marrow cells and is involved in the proliferation of early progenitor/stem cells.

PubMed Central

Small, D; Levenstein, M; Kim, E; Carow, C; Amin, S; Rockwell, P; Witte, L; Burrow, C; Ratajczak, M Z; Gewirtz, A M

1994-01-01

We cloned the cDNA for stem cell tyrosine kinase 1 (STK-1), the human homolog of murine Flk-2/Flt-3, from a CD34+ hematopoietic stem cell-enriched library and investigated its expression in subsets of normal human bone marrow. The cDNA encodes a protein of 993 aa with 85% identity and 92% similarity to Flk-2/Flt-3. STK-1 is a member of the type III receptor tyrosine kinase family that includes KIT (steel factor receptor), FMS (colony-stimulating factor 1R), and platelet-derived growth factor receptor. STK-1 expression in human blood and marrow is restricted to CD34+ cells, a population greatly enriched for stem/progenitor cells. Anti-STK-1 antiserum recognizes polypeptides of 160 and 130 kDa in several STK-1-expressing cell lines and in 3T3 cells transfected with a STK-1 expression vector. Antisense oligonucleotides directed against STK-1 sequences inhibited hematopoietic colony formation, most strongly in long-term bone marrow cultures. These data suggest that STK-1 may function as a growth factor receptor on hematopoietic stem and/or progenitor cells. Images Fig. 2 Fig. 3 Fig. 4 PMID:7507245

Modifying Expression Modes of Human Neurotensin Receptor Type 1 Alters Sensing Capabilities for Agonists in Yeast Signaling Biosensor.

PubMed

Hashi, Hiroki; Nakamura, Yasuyuki; Ishii, Jun; Kondo, Akihiko

2018-04-01

Neurotensin receptor type 1 (NTSR1), a member of the G-protein-coupled receptor (GPCR) family, is naturally activated by binding of a neurotensin peptide, leading to a variety of physiological effects. The budding yeast Saccharomyces cerevisiae is a proven host organism for assaying the agonistic activation of human GPCRs. Previous studies showed that yeast cells can functionally express human NTSR1 receptor, permitting the detection of neurotensin-promoted signaling using a ZsGreen fluorescent reporter gene. However, the fluorescence intensity (sensitivity) of NTSR1-expressing yeast cells is low compared to that of yeast cells expressing other human GPCRs (e.g., human somatostatin receptors). The present study sought to increase the sensitivity of the NTSR1-expressing yeast for use as a fluorescent biosensor, including modification of the expression of human NTSR1 in yeast. Changes in the transcription, translation, and transport of the receptor are attempted by altering the promoter, consensus Kozak-like sequence, and secretion signal sequences of the NTSR1-encoding gene. The resulting yeast cells exhibited increased sensitivity to exogenously added peptide. The cells are further engineered by using cell-surface display technology to ensure that the agonistic peptides are secreted and tethered to the yeast cell wall, yielding cells with enhanced NTSR1 activation. This yeast biosensor holds promise for the identification of agonists to treat NTSR1-related diseases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expression and localisation of aquaporin water channels in human urothelium in situ and in vitro.

PubMed

Rubenwolf, Peter C; Georgopoulos, Nikolaos T; Clements, Lisa A; Feather, Sally; Holland, Philip; Thomas, David F M; Southgate, Jennifer

2009-12-01

Urothelium is generally considered to be impermeable to water and constituents of urine. The possibility that human urothelium expresses aquaporin (AQP) water channels as the basis for water and solute transport has not previously been investigated. To investigate the expression of AQP water channels by human urothelium in situ, in proliferating urothelial cell cultures and in differentiated tissue constructs. AQP expression by human urothelium in situ and cultured urothelial cells was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunolabelling. Expression screening was carried out on samples of freshly isolated urothelia from multiple surgical (bladder and ureteric) specimens and on proliferating and differentiated normal human urothelial (NHU) cells in culture. Urothelial tissue constructs were established and investigated for expression of urothelial differentiation markers and AQPs. Qualitative study. Transcripts for AQP3, AQP4, AQP7, AQP9, and AQP11 were expressed consistently by freshly isolated urothelia as well as by cultured NHU cells. AQP0, AQP1, AQP2, AQP5, AQP6, AQP8, AQP10, and AQP12 were not expressed. Immunochemistry confirmed expression of AQP3, AQP4, AQP7, and AQP9 at the protein level. AQP3 was shown to be intensely expressed at cell borders in the basal and intermediate layers in both urothelium in situ and differentiated tissue constructs in vitro. This is the first study to demonstrate that AQPs are expressed by human urothelium, suggesting a potential role in transurothelial water and solute transport. Our findings challenge the traditional concept of the urinary tract as an impermeable transit and storage unit and provide a versatile platform for further investigations into the biological and clinical relevance of AQPs in human urothelium.

Safeguarding Stem Cell-Based Regenerative Therapy against Iatrogenic Cancerogenesis: Transgenic Expression of DNASE1, DNASE1L3, DNASE2, DFFB Controlled By POLA1 Promoter in Proliferating and Directed Differentiation Resisting Human Autologous Pluripotent Induced Stem Cells Leads to their Death

PubMed Central

Malecki, Marek; LaVanne, Christine; Alhambra, Dominique; Dodivenaka, Chaitanya; Nagel, Sarah; Malecki, Raf

2014-01-01

Introduction The worst possible complication of using stem cells for regenerative therapy is iatrogenic cancerogenesis. The ultimate goal of our work is to develop a self-triggering feedback mechanism aimed at causing death of all stem cells, which resist directed differentiation, keep proliferating, and can grow into tumors. Specific aim The specific aim was threefold: (1) to genetically engineer the DNA constructs for the human, recombinant DNASE1, DNASE1L3, DNASE2, DFFB controlled by POLA promoter; (2) to bioengineer anti-SSEA-4 antibody guided vectors delivering transgenes to human undifferentiated and proliferating pluripotent stem cells; (3) to cause death of proliferating and directed differentiation resisting stem cells by transgenic expression of the human recombinant the DNases (hrDNases). Methods The DNA constructs for the human, recombinant DNASE1, DNASE1L3, DNASE2, DFFB controlled by POLA promoter were genetically engineered. The vectors targeting specifically SSEA-4 expressing stem cells were bioengineered. The healthy volunteers’ bone marrow mononuclear cells (BMMCs) were induced into human, autologous, pluripotent stem cells with non-integrating plasmids. Directed differentiation of the induced stem cells into endothelial cells was accomplished with EGF and BMP. The anti-SSEA 4 antibodies’ guided DNA vectors delivered the transgenes for the human recombinant DNases’ into proliferating stem cells. Results Differentiation of the pluripotent induced stem cells into the endothelial cells was verified by highlighting formation of tight and adherens junctions through transgenic expression of recombinant fluorescent fusion proteins: VE cadherin, claudin, zona occludens 1, and catenin. Proliferation of the stem cells was determined through highlighting transgenic expression of recombinant fluorescent proteins controlled by POLA promoter, while also reporting expression of the transgenes for the hrDNases. Expression of the transgenes for the DNases resulted in complete collapse of the chromatin architecture and degradation of the proliferating cells’ genomic DNA. The proliferating stem cells, but not the differentiating ones, were effectively induced to die. Conclusion Herein, we describe attaining the proof-of-concept for the strategy, whereby transgenic expression of the genetically engineered human recombinant DNases in proliferating and directed differentiation resisting stem cells leads to their death. This novel strategy reduces the risk of iatrogenic neoplasms in stem cell therapy. PMID:25045589

Ovarian Expression and Regulation of the Stromelysins During the Periovulatory Period in the Human and the Rat1

PubMed Central

McCord, Lauren A.; Li, Feixue; Rosewell, Katherine L.; Brännström, Mats; Curry, Thomas E.

2011-01-01

ABSTRACT The matrix metalloproteinases (MMPs) are postulated to facilitate follicular rupture. In the present study, expression of the stromelysins (MMP3, MMP10, MMP11) was analyzed in the periovulatory human and rat ovary. Human granulosa and theca cells were collected from the dominant follicle at various times after human chorionic gonadotropin (hCG). Intact rat ovaries, granulosa cells, and residual tissue (tissue remaining after granulosa cell collection) were isolated from equine CG (eCG)-hCG-primed animals. Mmp10 mRNA was highly induced in human granulosa and theca cells and intact rat ovaries, granulosa cells, and residual tissue. Localization of MMP10 to granulosa and theca cells in both human and rat ovarian follicles was confirmed by immunohistochemistry. Mmp3 mRNA was unchanged in human cells and rat granulosa cells, but increased in intact rat ovaries and residual tissue. Mmp11 mRNA decreased following hCG treatment in human granulosa and theca cells as well as rat granulosa cells. Regulation of Mmp10 in cultured rat granulosa cells revealed that the EGF inhibitor AG1478 and the progesterone receptor antagonist RU486 suppressed the induction of Mmp10 mRNA, whereas the prostaglandin inhibitor NS398 had no effect. Studies on the Mmp10 promoter demonstrated that forskolin plus PMA stimulated promoter activity, which was dependent upon a proximal AP1 site. In conclusion, there are divergent patterns of stromelysin expression associated with ovulation, with a marked induction of Mmp10 mRNA and a decrease in Mmp11 mRNA, yet a species-dependent pattern on Mmp3 mRNA expression. The induction of Mmp10 expression suggests an important role for this MMP in the follicular changes associated with ovulation and subsequent luteinization. PMID:22116802

Ovarian expression and regulation of the stromelysins during the periovulatory period in the human and the rat.

PubMed

McCord, Lauren A; Li, Feixue; Rosewell, Katherine L; Brännström, Mats; Curry, Thomas E

2012-03-01

The matrix metalloproteinases (MMPs) are postulated to facilitate follicular rupture. In the present study, expression of the stromelysins (MMP3, MMP10, MMP11) was analyzed in the periovulatory human and rat ovary. Human granulosa and theca cells were collected from the dominant follicle at various times after human chorionic gonadotropin (hCG). Intact rat ovaries, granulosa cells, and residual tissue (tissue remaining after granulosa cell collection) were isolated from equine CG (eCG)-hCG-primed animals. Mmp10 mRNA was highly induced in human granulosa and theca cells and intact rat ovaries, granulosa cells, and residual tissue. Localization of MMP10 to granulosa and theca cells in both human and rat ovarian follicles was confirmed by immunohistochemistry. Mmp3 mRNA was unchanged in human cells and rat granulosa cells, but increased in intact rat ovaries and residual tissue. Mmp11 mRNA decreased following hCG treatment in human granulosa and theca cells as well as rat granulosa cells. Regulation of Mmp10 in cultured rat granulosa cells revealed that the EGF inhibitor AG1478 and the progesterone receptor antagonist RU486 suppressed the induction of Mmp10 mRNA, whereas the prostaglandin inhibitor NS398 had no effect. Studies on the Mmp10 promoter demonstrated that forskolin plus PMA stimulated promoter activity, which was dependent upon a proximal AP1 site. In conclusion, there are divergent patterns of stromelysin expression associated with ovulation, with a marked induction of Mmp10 mRNA and a decrease in Mmp11 mRNA, yet a species-dependent pattern on Mmp3 mRNA expression. The induction of Mmp10 expression suggests an important role for this MMP in the follicular changes associated with ovulation and subsequent luteinization.

Lent-On-Plus Lentiviral vectors for conditional expression in human stem cells.

PubMed

Benabdellah, Karim; Muñoz, Pilar; Cobo, Marién; Gutierrez-Guerrero, Alejandra; Sánchez-Hernández, Sabina; Garcia-Perez, Angélica; Anderson, Per; Carrillo-Gálvez, Ana Belén; Toscano, Miguel G; Martin, Francisco

2016-11-17

Conditional transgene expression in human stem cells has been difficult to achieve due to the low efficiency of existing delivery methods, the strong silencing of the transgenes and the toxicity of the regulators. Most of the existing technologies are based on stem cells clones expressing appropriate levels of tTA or rtTA transactivators (based on the TetR-VP16 chimeras). In the present study, we aim the generation of Tet-On all-in-one lentiviral vectors (LVs) that tightly regulate transgene expression in human stem cells using the original TetR repressor. By using appropriate promoter combinations and shielding the LVs with the Is2 insulator, we have constructed the Lent-On-Plus Tet-On system that achieved efficient transgene regulation in human multipotent and pluripotent stem cells. The generation of inducible stem cell lines with the Lent-ON-Plus LVs did not require selection or cloning, and transgene regulation was maintained after long-term cultured and upon differentiation toward different lineages. To our knowledge, Lent-On-Plus is the first all-in-one vector system that tightly regulates transgene expression in bulk populations of human pluripotent stem cells and its progeny.

Lent-On-Plus Lentiviral vectors for conditional expression in human stem cells

PubMed Central

Benabdellah, Karim; Muñoz, Pilar; Cobo, Marién; Gutierrez-Guerrero, Alejandra; Sánchez-Hernández, Sabina; Garcia-Perez, Angélica; Anderson, Per; Carrillo-Gálvez, Ana Belén; Toscano, Miguel G.; Martin, Francisco

2016-01-01

Conditional transgene expression in human stem cells has been difficult to achieve due to the low efficiency of existing delivery methods, the strong silencing of the transgenes and the toxicity of the regulators. Most of the existing technologies are based on stem cells clones expressing appropriate levels of tTA or rtTA transactivators (based on the TetR-VP16 chimeras). In the present study, we aim the generation of Tet-On all-in-one lentiviral vectors (LVs) that tightly regulate transgene expression in human stem cells using the original TetR repressor. By using appropriate promoter combinations and shielding the LVs with the Is2 insulator, we have constructed the Lent-On-Plus Tet-On system that achieved efficient transgene regulation in human multipotent and pluripotent stem cells. The generation of inducible stem cell lines with the Lent-ON-Plus LVs did not require selection or cloning, and transgene regulation was maintained after long-term cultured and upon differentiation toward different lineages. To our knowledge, Lent-On-Plus is the first all-in-one vector system that tightly regulates transgene expression in bulk populations of human pluripotent stem cells and its progeny. PMID:27853296

A proteomic chronology of gene expression through the cell cycle in human myeloid leukemia cells.

PubMed

Ly, Tony; Ahmad, Yasmeen; Shlien, Adam; Soroka, Dominique; Mills, Allie; Emanuele, Michael J; Stratton, Michael R; Lamond, Angus I

2014-01-01

Technological advances have enabled the analysis of cellular protein and RNA levels with unprecedented depth and sensitivity, allowing for an unbiased re-evaluation of gene regulation during fundamental biological processes. Here, we have chronicled the dynamics of protein and mRNA expression levels across a minimally perturbed cell cycle in human myeloid leukemia cells using centrifugal elutriation combined with mass spectrometry-based proteomics and RNA-Seq, avoiding artificial synchronization procedures. We identify myeloid-specific gene expression and variations in protein abundance, isoform expression and phosphorylation at different cell cycle stages. We dissect the relationship between protein and mRNA levels for both bulk gene expression and for over ∼6000 genes individually across the cell cycle, revealing complex, gene-specific patterns. This data set, one of the deepest surveys to date of gene expression in human cells, is presented in an online, searchable database, the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/). DOI: http://dx.doi.org/10.7554/eLife.01630.001.

A proteomic chronology of gene expression through the cell cycle in human myeloid leukemia cells

PubMed Central

Ly, Tony; Ahmad, Yasmeen; Shlien, Adam; Soroka, Dominique; Mills, Allie; Emanuele, Michael J; Stratton, Michael R; Lamond, Angus I

2014-01-01

Technological advances have enabled the analysis of cellular protein and RNA levels with unprecedented depth and sensitivity, allowing for an unbiased re-evaluation of gene regulation during fundamental biological processes. Here, we have chronicled the dynamics of protein and mRNA expression levels across a minimally perturbed cell cycle in human myeloid leukemia cells using centrifugal elutriation combined with mass spectrometry-based proteomics and RNA-Seq, avoiding artificial synchronization procedures. We identify myeloid-specific gene expression and variations in protein abundance, isoform expression and phosphorylation at different cell cycle stages. We dissect the relationship between protein and mRNA levels for both bulk gene expression and for over ∼6000 genes individually across the cell cycle, revealing complex, gene-specific patterns. This data set, one of the deepest surveys to date of gene expression in human cells, is presented in an online, searchable database, the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/). DOI: http://dx.doi.org/10.7554/eLife.01630.001 PMID:24596151

[Regulating human interferon-gamma gene expression in marrow stromal cells in mice by Tet-off system].

PubMed

Qin, Xin-Tian; Lu, Yue; Tan, Yin-Duo; Chen, Xiao-Qin; Gen, Qi-Rong

2008-01-01

We have constructed plasmid "pTre-IFN-gamma" and proved that the Tet-off system could regulate the expression of human interferon-gamma (IFN-gamma) gene in murine marrow stromal cells in vitro. This study was to investigate the regulatory reversibility of Tet-off system and its effect on the expression of human IFN-gamma gene in murine marrow stromal cells in mice. Plasmids pTet-off and pTre-IFN-gamma were co-transfected into murine marrow stromal cells. The expression of IFN-gamma in marrow stromal cells was detected with ELISA. The marrow stromal cells were transfused into BABL/c naked mice after co-transfection. The expression of IFN-gamma mRNA in the spleen was detected by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR). IFN-gamma protein was detected in the culture solution of marrow stromal cells after co-transfection. The secretion peak appeared within the first 72 h. The protein level of IFN-gamma was significantly lower in 300 ng/ml tetracycline hydrochloride-treated marrow stroma cells than in untreated cells [(67.11+/-22.14) pg/1 x 10(7) cells vs. (319.96+/-29.04) pg/1 x 10(7) cells, P<0.001]; its expression was increased when removed tetracycline hydrochloride (P=0.032). The expression of human IFN-gamma mRNA was detected in the spleen. The mRNA level of IFN-gamma was significantly higher in untreated group than in continuous tetracycline hydrochloride-treated group [(1.5+/-0.7)x10(5) copies . (100 mg)(-1) vs. (6.9+/-5.3)x10(2) copies . (100 mg)(-1), P<0.001]; its expression in the mice received tetracycline hydrochloride for one single time lay between the above two groups with significant difference. In mice, Tet-off system could rapidly, efficiently and reversibly regulate the expression of human IFN-gamma gene in marrow stromal cells in vitro and in vivo.

Peroxisome proliferator-activated receptor gamma overexpression suppresses proliferation of human colon cancer cells

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

Tsukahara, Tamotsu, E-mail: ttamotsu@shinshu-u.ac.jp; Haniu, Hisao

2012-08-03

Highlights: Black-Right-Pointing-Pointer We examined the correlation between PPAR{gamma} expression and cell proliferation. Black-Right-Pointing-Pointer PPAR{gamma} overexpression reduces cell viability. Black-Right-Pointing-Pointer We show the synergistic effect of cell growth inhibition by a PPAR{gamma} agonist. -- Abstract: Peroxisome proliferator-activated receptor gamma (PPAR{gamma}) plays an important role in the differentiation of intestinal cells and tissues. Our previous reports indicate that PPAR{gamma} is expressed at considerable levels in human colon cancer cells. This suggests that PPAR{gamma} expression may be an important factor for cell growth regulation in colon cancer. In this study, we investigated PPAR{gamma} expression in 4 human colon cancer cell lines, HT-29, LOVO,more » DLD-1, and Caco-2. Real-time polymerase chain reaction (PCR) and Western blot analysis revealed that the relative levels of PPAR{gamma} mRNA and protein in these cells were in the order HT-29 > LOVO > Caco-2 > DLD-1. We also found that PPAR{gamma} overexpression promoted cell growth inhibition in PPAR{gamma} lower-expressing cell lines (Caco-2 and DLD-1), but not in higher-expressing cells (HT-29 and LOVO). We observed a correlation between the level of PPAR{gamma} expression and the cells' sensitivity for proliferation.« less

PDC expressing CD36, CD61 and IL-10 may contribute to propagation of immune tolerance.

PubMed

Parcina, Marijo; Schiller, Martin; Gierschke, Aline; Heeg, Klaus; Bekeredjian-Ding, Isabelle

2009-05-01

Human plasmacytoid dendritic cells (PDC) are blood dendritic cell antigen 2 (BDCA2) and blood dendritic cell antigen 4 (BDCA4) positive leukocytes that do not express common lineage markers. They have been described as proinflammatory innate immune cells and are the major source of alphaIFN in the human body. PDC-derived secretion of type I IFNs upon triggering of nucleic acid-sensing toll-like receptors (TLR) primes immune cells to rapidly respond to microbial stimuli and promotes a Th1 response. Here, we report that human PDC express CD36 and CD61 (beta3 integrin), both involved in uptake of apoptotic cells and in induction of tolerance. Freshly isolated PDC and PDC within human blood leukocytes constitutively express IL-10. Thus, PDC may possess a so far neglected role in propagation of immune tolerance.

Locostatin, a disrupter of Raf kinase inhibitor protein, inhibits extracellular matrix production, proliferation, and migration in human uterine leiomyoma and myometrial cells.

PubMed

Janjusevic, Milijana; Greco, Stefania; Islam, Md Soriful; Castellucci, Clara; Ciavattini, Andrea; Toti, Paolo; Petraglia, Felice; Ciarmela, Pasquapina

2016-11-01

To investigate the presence of Raf kinase inhibitor protein (RKIP) in human myometrium and leiomyoma as well as to determine the effect of locostatin (RKIP inhibitor) on extracellular matrix (ECM) production, proliferation, and migration in human myometrial and leiomyoma cells. Laboratory study. Human myometrium and leiomyoma. Thirty premenopausal women who were admitted to the hospital for myomectomy or hysterectomy. Myometrial and leiomyoma tissues were used to investigate the localization and the expression level of RKIP through immunohistochemistry and Western blotting. Myometrial and leiomyoma cells were treated with locostatin (10 μM) to measure ECM expression by real-time polymerase chain reaction, GSK3β expression by Western blotting, cell migration by wound-healing assay, and cell proliferation by MTT assay and immunocytochemistry. The expression of RKIP in human myometrial and leiomyoma tissue; ECM components and GSK3β expression, migration, and proliferation in myometrial and leiomyoma cells. RKIP is expressed in human myometrial and leiomyoma tissue. Locostatin treatment resulted in the activation of the mitogen-activated protein kinase (MAPK) signal pathway (ERK phosphorylation), providing a powerful validation of our targeting protocol. Further, RKIP inhibition by locostatin reduces ECM components. Moreover, the inhibition of RKIP by locostatin impaired cell proliferation and migration in both leiomyoma and myometrial cells. Finally, locostatin treatment reduced GSK3β expression. Therefore, even if the activation of MAPK pathway should increase proliferation and migration, the destabilization of GSK3β leads to the reduction of proliferation and migration of myometrial and leiomyoma cells. Our results indicate that RKIP may be involved in leiomyoma pathophysiology. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Downregulation of Bit1 expression promotes growth, anoikis resistance, and transformation of immortalized human bronchial epithelial cells via Erk activation-dependent suppression of E-cadherin.

PubMed

Yao, Xin; Gray, Selena; Pham, Tri; Delgardo, Mychael; Nguyen, An; Do, Stephen; Ireland, Shubha Kale; Chen, Renwei; Abdel-Mageed, Asim B; Biliran, Hector

2018-01-01

The mitochondrial Bit1 protein exerts tumor-suppressive function in NSCLC through induction of anoikis and inhibition of EMT. Having this dual tumor suppressive effect, its downregulation in the established human lung adenocarcinoma A549 cell line resulted in potentiation of tumorigenicity and metastasis in vivo. However, the exact role of Bit1 in regulating malignant growth and transformation of human lung epithelial cells, which are origin of most forms of human lung cancers, has not been examined. To this end, we have downregulated the endogenous Bit1 expression in the immortalized non-tumorigenic human bronchial epithelial BEAS-2B cells. Knockdown of Bit1 enhanced the growth and anoikis insensitivity of BEAS-2B cells. In line with their acquired anoikis resistance, the Bit1 knockdown BEAS-2B cells exhibited enhanced anchorage-independent growth in vitro but failed to form tumors in vivo. The loss of Bit1-induced transformed phenotypes was in part attributable to the repression of E-cadherin expression since forced exogenous E-cadherin expression attenuated the malignant phenotypes of the Bit1 knockdown cells. Importantly, we show that the loss of Bit1 expression in BEAS-2B cells resulted in increased Erk activation, which functions upstream to promote TLE1-mediated transcriptional repression of E-cadherin. These collective findings indicate that loss of Bit1 expression contributes to the acquisition of malignant phenotype of human lung epithelial cells via Erk activation-induced suppression of E-cadherin expression. Copyright © 2017 Elsevier Inc. All rights reserved.

Transcription factor EGR-1 suppresses the growth and transformation of human HT-1080 fibrosarcoma cells by induction of transforming growth factor beta 1.

PubMed Central

Liu, C; Adamson, E; Mercola, D

1996-01-01

The early growth response 1 (EGR-1) gene product is a transcription factor with role in differentiation and growth. We have previously shown that expression of exogenous EGR-1 in various human tumor cells unexpectedly and markedly reduces growth and tumorigenicity and, conversely, that suppression of endogenous Egr-1 expression by antisense RNA eliminates protein expression, enhances growth, and promotes phenotypic transformation. However, the mechanism of these effects remained unknown. The promoter of human transforming growth factor beta 1 (TGF-beta 1) contains two GC-rich EGR-1 binding sites. We show that expression of EGR-1 in human HT-1080 fibrosarcoma cells uses increased secretion of biologically active TGF-beta 1 in direct proportion (rPearson = 0.96) to the amount of EGR-1 expressed and addition of recombinant human TGF-beta 1 is strongly growth-suppressive for these cells. Addition of monoclonal anti-TGF-beta 1 antibodies to EGR-1-expressing HT-1080 cells completely reverses the growth inhibitory effects of EGR-1. Reporter constructs bearing the EGR-1 binding segment of the TGF-beta 1 promoter was activated 4- to 6-fold relative to a control reporter in either HT-1080 cells that stably expressed or parental cells cotransfected with an EGR-1 expression vector. Expression of delta EGR-1, a mutant that cannot interact with the corepressors, nerve growth factor-activated factor binding proteins NAB1 and NAB2, due to deletion of the repressor domain, exhibited enhanced transactivation of 2- to 3.5-fold over that of wild-type EGR-1 showing that the reporter construct reflected the appropriate in vivo regulatory context. The EGR-1-stimulated transactivation was inhibited by expression of the Wilms tumor suppressor, a known specific DNA-binding competitor. These results indicate that EGR-1 suppresses growth of human HT-1080 fibrosarcoma cells by induction of TGF-beta 1. Images Fig. 1 Fig. 5 PMID:8876223

Reversal of hyperglycemia in mice by using human expandable insulin-producing cells differentiated from fetal liver progenitor cells

NASA Astrophysics Data System (ADS)

Zalzman, Michal; Gupta, Sanjeev; Giri, Ranjit K.; Berkovich, Irina; Sappal, Baljit S.; Karnieli, Ohad; Zern, Mark A.; Fleischer, Norman; Efrat, Shimon

2003-06-01

Beta-cell replacement is considered to be the most promising approach for treatment of type 1 diabetes. Its application on a large scale is hindered by a shortage of cells for transplantation. Activation of insulin expression, storage, and regulated secretion in stem/progenitor cells offers novel ways to overcome this shortage. We explored whether fetal human progenitor liver cells (FH) could be induced to differentiate into insulin-producing cells after expression of the pancreatic duodenal homeobox 1 (Pdx1) gene, which is a key regulator of pancreatic development and insulin expression in beta cells. FH cells possess a considerable replication capacity, and this was further extended by introduction of the gene for the catalytic subunit of human telomerase. Immortalized FH cells expressing Pdx1 activated multiple beta-cell genes, produced and stored considerable amounts of insulin, and released insulin in a regulated manner in response to glucose. When transplanted into hyperglycemic immunodeficient mice, the cells restored and maintained euglycemia for prolonged periods. Quantitation of human C-peptide in the mouse serum confirmed that the glycemia was normalized by the transplanted human cells. This approach offers the potential of a novel source of cells for transplantation into patients with type 1 diabetes.

CHRFAM7A: a human-specific α7-nicotinic acetylcholine receptor gene shows differential responsiveness of human intestinal epithelial cells to LPS

PubMed Central

Dang, Xitong; Eliceiri, Brian P.; Baird, Andrew; Costantini, Todd W.

2015-01-01

The human genome contains a unique, distinct, and human-specific α7-nicotinic acetylcholine receptor (α7nAChR) gene [CHRNA7 (gene-encoding α7-nicotinic acetylcholine receptor)] called CHRFAM7A (gene-encoding dup-α7-nicotinic acetylcholine receptor) on a locus of chromosome 15 associated with mental illness, including schizophrenia. Located 5′ upstream from the “wild-type” CHRNA7 gene that is found in other vertebrates, we demonstrate CHRFAM7A expression in a broad range of epithelial cells and sequenced the CHRFAM7A transcript found in normal human fetal small intestine epithelial (FHs) cells to prove its identity. We then compared its expression to CHRNA7 in 11 gut epithelial cell lines, showed that there is a differential response to LPS when compared to CHRNA7, and characterized the CHRFAM7A promoter. We report that both CHRFAM7A and CHRNA7 gene expression are widely distributed in human epithelial cell lines but that the levels of CHRFAM7A gene expression vary up to 5000-fold between different gut epithelial cells. A 3-hour treatment of epithelial cells with 100 ng/ml LPS increased CHRFAM7A gene expression by almost 1000-fold but had little effect on CHRNA7 gene expression. Mapping the regulatory elements responsible for CHRFAM7A gene expression identifies a 1 kb sequence in the UTR of the CHRFAM7A gene that is modulated by LPS. Taken together, these data establish the presence, identity, and differential regulation of the human-specific CHRFAM7A gene in human gut epithelial cells. In light of the fact that CHRFAM7A expression is reported to modulate ligand binding to, and alter the activity of, the wild-type α7nAChR ligand-gated pentameric ion channel, the findings point to the existence of a species-specific α7nAChR response that might regulate gut epithelial function in a human-specific fashion.—Dang, X., Eliceiri, B. P., Baird, A., Costantini, T. W. CHRFAM7A: a human-specific α7-nicotinic acetylcholine receptor gene shows differential responsiveness of human intestinal epithelial cells to LPS. PMID:25681457

Molecular cloning, expression and immunolocalization of a novel human cementum-derived protein (CP-23).

PubMed

Alvarez-Pérez, Marco Antonio; Narayanan, Sampath; Zeichner-David, Margarita; Rodríguez Carmona, Bruno; Arzate, Higinio

2006-03-01

Cementum is a unique mineralized connective tissue that covers the root surfaces of the teeth. The cementum is critical for appropriate maturation of the periodontium, both during development as well as that associated with regeneration of periodontal tissues, IU; however, one major impediment to understand the molecular mechanisms that regulate periodontal regeneration is the lack of cementum markers. Here we report on the identification and characterization of one such differentially human expressed gene, termed "cementum protein-23" (CP-23) that appears to be periodontal ligament and cementum-specific. We screened human cementum tumor-derived cDNA libraries by transient expression in COS-7 cells and "panning" with a rabbit polyclonal antibody against a cementoblastoma conditioned media-derived protein (CP). One isolated cDNA, CP-23, was expressed in E. coli and polyclonal antibodies against the recombinant human CP-23 were produced. Expression of CP-23 protein by cells of the periodontium was examined by Northern blot and in situ hybridization. Expression of CP-23 transcripts in human cementoblastoma-derived cells, periodontal ligament cells, human gingival fibroblasts and alveolar bone-derived cells was determined by RT-PCR. Our results show that we have isolated a 1374-bp human cDNA containing an open reading frame that encodes a polypeptide with 247 amino acid residues, with a predicted molecular mass of 25.9 kDa that represents CP species. The recombinant human CP-23 protein cross-reacted with antibodies against CP and type X collagen. Immunoscreening of human periodontal tissues revealed that CP-23 gene product is localized to the cementoid matrix of cementum and cementoblasts throughout the entire surface of the root, cell subpopulations of the periodontal ligament as well as cells located paravascularly to the blood vessels into the periodontal ligament. Furthermore, 98% of putative cementoblasts and 15% of periodontal ligament cells cultured in vitro expressed CP-23 gene product. Cementoblastoma cells and periodontal ligament cells contained a 5.0 kb CP-23 mRNA. In situ hybridization showed strong expression of CP-23 mRNA on cementoblast, cell subpopulations of the periodontal ligament and cells located around blood vessels into the periodontal ligament. Our results demonstrate that CP-23 represents a novel, tissue-specific-gene product being expressed by periodontal ligament subpopulations and cementoblasts. These findings offer the possibility to determine the cellular and molecular events that regulate the cementogenesis process during root development. Furthermore, it might provide new venues for the design of translational studies aimed at achieving predictable new cementogenesis and regeneration of the periodontal tissues.

Silencing of Kv4.1 potassium channels inhibits cell proliferation of tumorigenic human mammary epithelial cells

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

Jang, Soo Hwa; Choi, Changsun; Hong, Seong-Geun

2009-06-26

Potassium channel activity has been shown to facilitate cell proliferation in cancer cells. In the present study, the role of Kv4.1 channels in immortal and tumorigenic human mammary epithelial cells was investigated. Kv4.1 protein expression was positively correlated with tumorigenicity. Moreover, transfection with siRNAs targeting Kv4.1 mRNA suppressed proliferation of tumorigenic mammary epithelial cells. Experiments using mRNA isolated from human breast cancer tissues revealed that the level of Kv4.1 mRNA expression varied depending on the stage of the tumor. Kv4.1 protein expression increased during stages T2 and T3 compared to normal tissue. These results demonstrated that Kv4.1 plays a rolemore » in proliferation of tumorigenic human mammary epithelial cells. In addition, elevated Kv4.1 expression may be useful as a diagnostic marker for staging mammary tumors and selective blockers of Kv4.1 may serve to suppress tumor cell proliferation.« less

PCFT/SLC46A1 promoter methylation and restoration of gene expression in human leukemia cells

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

Gonen, Nitzan; Bram, Eran E.; Assaraf, Yehuda G.

2008-11-28

The proton-coupled folate transporter (PCFT/SLC46A1) displays optimal and prominent folate and antifolate transport activity at acidic pH in human carcinoma cells but poor activity in leukemia cells. Consistently herein, human leukemia cell lines expressed poor PCFT transcript levels, whereas various carcinoma cell lines showed substantial PCFT gene expression. We identified a CpG island with high density at nucleotides -200 through +100 and explored its role in PCFT promoter silencing. Leukemia cells with barely detectable PCFT transcripts consistently harbored 85-100% methylation of this CpG island, whereas no methylation was found in carcinoma cells. Treatment with 5-Aza-2'-deoxycytidine which induced demethylation but notmore » with the histone deacetylase inhibitor trichostatin A, restored 50-fold PCFT expression only in leukemia cells. These findings constitute the first demonstration of the dominant epigenetic silencing of the PCFT gene in leukemia cells. The potential translational implications of the restoration of PCFT expression in chemotherapy of leukemia are discussed.« less

Differentiation of human embryonic stem cells to HOXA+ hemogenic vasculature that resembles the aorta-gonad-mesonephros.

PubMed

Ng, Elizabeth S; Azzola, Lisa; Bruveris, Freya F; Calvanese, Vincenzo; Phipson, Belinda; Vlahos, Katerina; Hirst, Claire; Jokubaitis, Vanta J; Yu, Qing C; Maksimovic, Jovana; Liebscher, Simone; Januar, Vania; Zhang, Zhen; Williams, Brenda; Conscience, Aude; Durnall, Jennifer; Jackson, Steven; Costa, Magdaline; Elliott, David; Haylock, David N; Nilsson, Susan K; Saffery, Richard; Schenke-Layland, Katja; Oshlack, Alicia; Mikkola, Hanna K A; Stanley, Edouard G; Elefanty, Andrew G

2016-11-01

The ability to generate hematopoietic stem cells from human pluripotent cells would enable many biomedical applications. We find that hematopoietic CD34 + cells in spin embryoid bodies derived from human embryonic stem cells (hESCs) lack HOXA expression compared with repopulation-competent human cord blood CD34 + cells, indicating incorrect mesoderm patterning. Using reporter hESC lines to track the endothelial (SOX17) to hematopoietic (RUNX1C) transition that occurs in development, we show that simultaneous modulation of WNT and ACTIVIN signaling yields CD34 + hematopoietic cells with HOXA expression that more closely resembles that of cord blood. The cultures generate a network of aorta-like SOX17 + vessels from which RUNX1C + blood cells emerge, similar to hematopoiesis in the aorta-gonad-mesonephros (AGM). Nascent CD34 + hematopoietic cells and corresponding cells sorted from human AGM show similar expression of cell surface receptors, signaling molecules and transcription factors. Our findings provide an approach to mimic in vitro a key early stage in human hematopoiesis for the generation of AGM-derived hematopoietic lineages from hESCs.

Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment

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

Sustarsic, Elahu G.; Department of Biological Sciences, Ohio University, Athens, OH; Junnila, Riia K.

2013-11-08

Highlights: •Most cancer types of the NCI60 have sub-sets of cell lines with high GHR expression. •GHR is highly expressed in melanoma cell lines. •GHR is elevated in advanced stage IV metastatic tumors vs. stage III. •GH treatment of metastatic melanoma cell lines alters growth and cell signaling. -- Abstract: Accumulating evidence implicates the growth hormone receptor (GHR) in carcinogenesis. While multiple studies show evidence for expression of growth hormone (GH) and GHR mRNA in human cancer tissue, there is a lack of quantification and only a few cancer types have been investigated. The National Cancer Institute’s NCI60 panel includesmore » 60 cancer cell lines from nine types of human cancer: breast, CNS, colon, leukemia, melanoma, non-small cell lung, ovarian, prostate and renal. We utilized this panel to quantify expression of GHR, GH, prolactin receptor (PRLR) and prolactin (PRL) mRNA with real-time RT qPCR. Both GHR and PRLR show a broad range of expression within and among most cancer types. Strikingly, GHR expression is nearly 50-fold higher in melanoma than in the panel as a whole. Analysis of human metastatic melanoma biopsies confirmed GHR gene expression in melanoma tissue. In these human biopsies, the level of GHR mRNA is elevated in advanced stage IV tumor samples compared to stage III. Due to the novel finding of high GHR in melanoma, we examined the effect of GH treatment on three NCI60 melanoma lines (MDA-MB-435, UACC-62 and SK-MEL-5). GH increased proliferation in two out of three cell lines tested. Further analysis revealed GH-induced activation of STAT5 and mTOR in a cell line dependent manner. In conclusion, we have identified cell lines and cancer types that are ideal to study the role of GH and PRL in cancer, yet have been largely overlooked. Furthermore, we found that human metastatic melanoma tumors express GHR and cell lines possess active GHRs that can modulate multiple signaling pathways and alter cell proliferation. Based on this data, GH could be a new therapeutic target in melanoma.« less

Doxorubicin induces ZAKα overexpression with a subsequent enhancement of apoptosis and attenuation of survivability in human osteosarcoma cells.

PubMed

Fu, Chien-Yao; Tseng, Yan-Shen; Chen, Ming-Cheng; Hsu, Hsi-Hsien; Yang, Jaw-Ji; Tu, Chuan-Chou; Lin, Yueh-Min; Viswanadha, Vijaya Padma; Kuo, Wei-Wen; Huang, Chih-Yang

2018-02-01

Human osteosarcoma (OS) is a malignant cancer of the bone. It exhibits a characteristic malignant osteoblastic transformation and produces a diseased osteoid. A previous study demonstrated that doxorubicin (DOX) chemotherapy decreases human OS cell proliferation and might enhance the relative RNA expression of ZAK. However, the impact of ZAKα overexpression on the OS cell proliferation that is inhibited by DOX and the molecular mechanism underlying this effect are not yet known. ZAK is a protein kinase of the MAPKKK family and functions to promote apoptosis. In our study, we found that ZAKα overexpression induced an apoptotic effect in human OS cells. Treatment of human OS cells with DOX enhanced ZAKα expression and decreased cancer cell viability while increasing apoptosis of human OS cells. In the meantime, suppression of ZAKα expression using shRNA and inhibitor D1771 both suppressed the DOX therapeutic effect. These findings reveal a novel molecular mechanism underlying the DOX effect on human OS cells. Taken together, our findings demonstrate that ZAKα enhances the apoptotic effect and decreases cell viability in DOX-treated human OS cells. © 2017 Wiley Periodicals, Inc.

[Distribution diversity of integrins and calcium channels on major human and mouse host cells of Leptospira species].

PubMed

Li, Cheng-xue; Zhao, Xin; Qian, Jing; Yan, Jie

2012-07-01

To determine the distribution of integrins and calcium channels on major human and mouse host cells of Leptospira species. The expression of β1, β2 and β3 integrins was detected with immunofluorescence assay on the surface of human monocyte line THP-1, mouse mononuclear-macrophage-like cell line J774A.1, human vascular endothelial cell line HUVEC, mouse vascular endothelial cell EOMA, human hepatocyte line L-02, mouse hepatocyte line Hepa1-6, human renal tubular epithelial cell line HEK-293, mouse glomerular membrane epithelial cell line SV40-MES13, mouse collagen blast line NIH/3T3, human and mouse platelets. The distribution of voltage gate control calcium channels Cav3.1, Cav3.2, Cav3.3 and Cav2.3, and receptor gate calcium channels P(2)X(1), P(2)2X(2), P(2)X(3), P(2)X(4), P(2)X(5), P(2)X(6) and P(2)X(7) were determined with Western blot assay. β1 integrin proteins were positively expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, L-02, Hepa1-6 and HEK-239 cells as well as human and mouse platelets. β2 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, and NIH/3T3 cells. β3 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, Hepa1-6, HEK-239 and NIH/3T3 cells as well as human and mouse platelets. P(2)X(1) receptor gate calcium channel was expressed on the membrane surface of human and mouse platelets, while P(2)X(5) receptor gate calcium channel was expressed on the membrane surface of J774A.1, THP-1, L-02, Hepa1-6, HEK-239 and HUVEC cells. However, the other calcium channels were not detected on the tested cell lines or platelets. There is a large distribution diversity of integrins and calcium channel proteins on the major human and mouse host cells of Leptospira species, which may be associated with the differences of leptospira-induced injury in different host cells.

SOX2 and nestin expression in human melanoma: an immunohistochemical and experimental study

PubMed Central

Laga, Alvaro C.; Zhan, Qian; Weishaupt, Carsten; Ma, Jie; Frank, Markus H.; Murphy, George F.

2012-01-01

SOX2 is an embryonic neural crest stem-cell transcription factor recently shown to be expressed in human melanoma and to correlate with experimental tumor growth. SOX2 binds to an enhancer region of the gene that encodes for nestin, also a neural progenitor cell biomarker. To define further the potential relationship between SOX2 and nestin, we examined co-expression patterns in 135 melanomas and 37 melanocytic nevi. Immunohistochemical staining in 27 melanoma tissue sections showed an association between SOX2 positivity, spindle cell shape and a peripheral nestin distribution pattern. In contrast, SOX2-negative cells were predominantly epithelioid, and exhibited a cytoplasmic pattern for nestin. In tissue microarrays, co-expression correlated with tumor progression, with only 11% of nevi co-expressing SOX2 and nestin in contrast to 65% of metastatic melanomas, and preliminarily, with clinical outcome. Human melanoma lines that differentially expressed constitutive SOX2 revealed a positive correlation between SOX2 and nestin expression. Experimental melanomas grown from these respective cell lines in murine subcutis and dermis of xenografted human skin maintained the association between SOX2-positivity, spindle cell shape, and peripheral nestin distribution. Moreover, the cytoplasmic pattern of nestin distribution was observed in xenografts generated from SOX2-knockdown A2058 melanoma cells, in contrast to the periperhal nestin pattern seen in tumors grown from A2058 control cells transfected with non-target shRNA. In aggregate, these data further support a biologically significant linkage between SOX2 and nestin expression in human melanoma. PMID:21410764

Novel Immortal Cell Lines Support Cellular Heterogeneity in the Human Annulus Fibrosus

PubMed Central

van den Akker, Guus G. H.; Surtel, Don A. M.; Cremers, Andy; Richardson, Stephen M.; Hoyland, Judith A.; van Rhijn, Lodewijk W.

2016-01-01

Introduction Loss of annulus fibrosus (AF) integrity predisposes to disc herniation and is associated with IVD degeneration. Successful implementation of biomedical intervention therapy requires in-depth knowledge of IVD cell biology. We recently generated unique clonal human nucleus pulposus (NP) cell lines. Recurring functional cellular phenotypes from independent donors provided pivotal evidence for cell heterogeneity in the mature human NP. In this study we aimed to generate and characterize immortal cell lines for the human AF from matched donors. Methods Non-degenerate healthy disc material was obtained as surplus surgical material. AF cells were immortalized by simian virus Large T antigen (SV40LTAg) and human telomerase (hTERT) expression. Early passage cells and immortalized cell clones were characterized based on marker gene expression under standardized culturing and in the presence of Transforming Growth factor β (TGFβ). Results The AF-specific expression signature included COL1A1, COL5A1, COL12A1, SFRP2 and was largely maintained in immortal AF cell lines. Remarkably, TGFβ induced rapid 3D sheet formation in a subgroup of AF clones. This phenotype was associated with inherent differences in Procollagen type I processing and maturation, and correlated with differential mRNA expression of Prolyl 4-hydroxylase alpha polypeptide 1 and 3 (P4HA1,3) and Lysyl oxidase (LOX) between clones and differential P4HA3 protein expression between AF cells in histological sections. Conclusion We report for the first time the generation of representative human AF cell lines. Gene expression profile analysis and functional comparison of AF clones revealed variation between immortalized cells and suggests phenotypic heterogeneity in the human AF. Future characterization of AF cellular (sub-)populations aims to combine identification of additional specific AF marker genes and their biological relevance. Ultimately this knowledge will contribute to clinical application of cell-based technology in IVD repair. PMID:26794306

Novel Immortal Cell Lines Support Cellular Heterogeneity in the Human Annulus Fibrosus.

PubMed

van den Akker, Guus G H; Surtel, Don A M; Cremers, Andy; Richardson, Stephen M; Hoyland, Judith A; van Rhijn, Lodewijk W; Voncken, Jan Willem; Welting, Tim J M

2016-01-01

Loss of annulus fibrosus (AF) integrity predisposes to disc herniation and is associated with IVD degeneration. Successful implementation of biomedical intervention therapy requires in-depth knowledge of IVD cell biology. We recently generated unique clonal human nucleus pulposus (NP) cell lines. Recurring functional cellular phenotypes from independent donors provided pivotal evidence for cell heterogeneity in the mature human NP. In this study we aimed to generate and characterize immortal cell lines for the human AF from matched donors. Non-degenerate healthy disc material was obtained as surplus surgical material. AF cells were immortalized by simian virus Large T antigen (SV40LTAg) and human telomerase (hTERT) expression. Early passage cells and immortalized cell clones were characterized based on marker gene expression under standardized culturing and in the presence of Transforming Growth factor β (TGFβ). The AF-specific expression signature included COL1A1, COL5A1, COL12A1, SFRP2 and was largely maintained in immortal AF cell lines. Remarkably, TGFβ induced rapid 3D sheet formation in a subgroup of AF clones. This phenotype was associated with inherent differences in Procollagen type I processing and maturation, and correlated with differential mRNA expression of Prolyl 4-hydroxylase alpha polypeptide 1 and 3 (P4HA1,3) and Lysyl oxidase (LOX) between clones and differential P4HA3 protein expression between AF cells in histological sections. We report for the first time the generation of representative human AF cell lines. Gene expression profile analysis and functional comparison of AF clones revealed variation between immortalized cells and suggests phenotypic heterogeneity in the human AF. Future characterization of AF cellular (sub-)populations aims to combine identification of additional specific AF marker genes and their biological relevance. Ultimately this knowledge will contribute to clinical application of cell-based technology in IVD repair.

Fv1-like restriction of N-tropic replication-competent murine leukaemia viruses in mCAT-1-expressing human cells.

PubMed

Aagaard, Lars; Mikkelsen, Jacob Giehm; Warming, Søren; Duch, Mogens; Pedersen, Finn Skou

2002-02-01

To study the replication of murine leukaemia viruses in human cells we have used full-length as well as EGFP-tagged ecotropic viruses in combination with mCAT-1-expressing human cells. We present results showing that N-tropic murine leukaemia viruses are restricted in both infection and replication in such cells while B-tropic viruses, modified at capsid position 110, escape restriction. These results support a recently reported Fv1-like restriction in mammalian cells. We extend the analysis of Fv1-like restriction by demonstrating that NB-tropic viruses also escape restriction and human mCAT-1-expressing cells are thus similar to murine Fv1(b) cells with respect to infection though the ecotropic receptor pathway.

Quantitative targeted absolute proteomic analysis of transporters, receptors and junction proteins for validation of human cerebral microvascular endothelial cell line hCMEC/D3 as a human blood-brain barrier model.

PubMed

Ohtsuki, Sumio; Ikeda, Chiemi; Uchida, Yasuo; Sakamoto, Yumi; Miller, Florence; Glacial, Fabienne; Decleves, Xavier; Scherrmann, Jean-Michel; Couraud, Pierre-Olivier; Kubo, Yoshiyuki; Tachikawa, Masanori; Terasaki, Tetsuya

2013-01-07

Human cerebral microvascular endothelial cell line hCMEC/D3 is an established model of the human blood-brain barrier (BBB). The purpose of the present study was to determine, by means of quantitative targeted absolute proteomics, the protein expression levels in hCMEC/D3 cells of multiple transporters, receptors and junction proteins for comparison with our previously reported findings in isolated human brain microvessels. Among 91 target molecules, 12 transporters, 2 receptors, 1 junction protein and 1 membrane marker were present at quantifiable levels in plasma membrane fraction of hCMEC/D3 cells. ABCA2, MDR1, MRP4, BCRP, GLUT1, 4F2hc, MCT1, ENT1, transferrin and insulin receptors and claudin-5 were detected in both hCMEC/D3 cells and human brain microvessels. After normalization based on Na(+)/K(+) ATPase expression, the differences in protein expression levels between hCMEC/D3 cells and human brain microvessels were within 4-fold for these proteins, with the exceptions of ENT1, transferrin receptor and claudin-5. ABCA8, LAT1, LRP1 and γ-GTP were below the limit of quantification in the cells, but were found in human brain microvessels. ABCA3, ABCA6, MRP1 and ATA1 were found only in hCMEC/D3 cells. Furthermore, compared with human umbilical vein endothelial cells (HUVECs) as reference nonbrain endothelial cells, MDR1 was found only in hCMEC/D3 cells, and GLUT1 expression was 15-fold higher in hCMEC/D3 cells than in HUVECs. In conclusion, this is the first study to examine the suitability and limitations of the hCMEC/D3 cell line as a BBB functional model in terms of quantitative expression levels of transporters, receptors and tight junction proteins.

Progesterone Receptor-A and -B Have Opposite Effects on Proinflammatory Gene Expression in Human Myometrial Cells: Implications for Progesterone Actions in Human Pregnancy and Parturition

PubMed Central

Tan, Huiqing; Yi, Lijuan; Rote, Neal S.; Hurd, William W.

2012-01-01

Context: Progesterone promotes uterine relaxation during pregnancy and its withdrawal induces labor. Progesterone withdrawal in human parturition is mediated in part by changes in the relative levels of the nuclear progesterone receptor isoforms, PR-A and PR-B, in myometrial cells. Parturition also involves myometrial inflammation; however, the functional link between nuclear PR-mediated progesterone actions and inflammation in human myometrial cells is unclear. Objective: Our objective was to determine how PR-A and PR-B regulate progesterone action in human myometrial cells and specifically the expression of genes encoding contraction-associated proteins and proinflammatory mediators. Design: Effects of PR-A and PR-B on the capacity for progesterone to modulate gene expression was determined using an immortalized human myometrial cell line stably transfected with inducible PR-A and PR-B expression transgenes and conditioned to express various PR-A and PR-B levels. Gene expression was assessed by genome wide transcriptome analysis, quantitative RT-PCR and immunoblotting. Results: PR-A and PR-B were each transcriptionally active in response to progesterone and affected the expression of distinct gene cohorts. The capacity for progesterone to affect gene expression was dependent on the PR-A to PR-B ratio. This was especially apparent for the expression of proinflammatory genes. Progesterone decreased proinflammatory gene expression when the PR-A to PR-B ratio favored PR-B and increased proinflammatory gene expression when the ratio favored PR-A. Progesterone via PR-B increased expression of inhibitor-κBα, a repressor of the nuclear factor-κB transcription factor, and inhibited basal and lipopolysaccharide-induced proinflammatory gene expression. Both of those PR-B-mediated effects were inhibited by PR-A. Conclusions: Our data suggest that during most of human pregnancy, when myometrial cells are PR-B dominant, progesterone promotes myometrial quiescence through PR-B-mediated antiinflammatory actions. At parturition, the rise in PR-A expression promotes labor by inhibiting the antiinflammatory actions of PR-B and stimulating proinflammatory gene expression in response to progesterone. PMID:22419721

Downregulation of proapoptotic Bim augments IL-2-independent T-cell transformation by human T-cell leukemia virus type-1 Tax

PubMed Central

Higuchi, Masaya; Takahashi, Masahiko; Tanaka, Yuetsu; Fujii, Masahiro

2014-01-01

Human T-cell leukemia virus type 1 (HTLV-1), an etiological agent of adult T-cell leukemia, immortalizes and transforms primary human T cells in vitro in both an interleukin (IL)-2-dependent and IL-2-independent manner. Expression of the HTLV-1 oncoprotein Tax transforms the growth of the mouse T-cell line CTLL-2 from being IL-2-dependent to IL-2-independent. Withdrawal of IL-2 from normal activated T cells induces apoptosis, which is mediated through the inducible expression of several proapoptotic proteins, including Bim. In this study, we found that Tax protects IL-2-depleted T cells against Bim-induced apoptosis. Withdrawal of IL-2 from CTLL-2 cells induced a prominent increase in the level of Bim protein in CTLL-2 cells, but not in Tax-transformed CTLL-2 cells. This inhibition of Bim in Tax-transformed CTLL-2 cells was mediated by two mechanisms: downregulation of Bim mRNA and posttranscriptional reduction of Bim protein. Transient expression of Tax in CTLL-2 cells also inhibited IL-2 depletion–induced expression of Bim, however, this decrease in Bim protein expression was not due to downregulation of Bim mRNA, thus indicating that Bim mRNA downregulation in Tax-transformed CTLL-2 occurs only after long-term expression of Tax. Transient expression of Tax in CTLL-2 cells also induced Erk activation, however, this was not involved in the reduction of Bim protein. Knockdown of Bim expression in CTLL-2 cells augmented Tax-induced IL-2-independent transformation. HTLV-1 infection of human T cells also reduced their levels of Bim protein, and restoring Bim expression in HTLV-1-infected cells reduced their proliferation by inducing apoptosis. Taken together, these results indicate that Tax-induced downregulation of Bim in HTLV-1-infected T cells promotes their IL-2-independent growth, thereby supporting the persistence of HTLV-1 infection in vivo. PMID:25175936

Genetic modification of mesenchymal stem cells to express a single-chain antibody against EGFRvIII on the cell surface.

PubMed

Balyasnikova, Irina V; Franco-Gou, Rosa; Mathis, J Michael; Lesniak, Maciej S

2010-06-01

Human adult mesenchymal stem cells (hMSCs) are under active investigation as cellular carriers for gene therapy. hMSCs possess natural tropism toward tumours; however, the targeting of hMSCs to specific cell populations within tumours is unexplored. In the case of glioblastoma multiforme (GBM), at least half of the tumours express EGFRvIII on the cell surface, an ideal target for antibody-mediated gene/drug delivery. In this study, we investigated the feasibility of genetically modifying hMSCs to express a single-chain antibody (scFv) to EGFRvIII on their surfaces. Nucleofection was used to transfect hMSCs with cDNA encoding scFv EGFRvIII fused with PDGFR or human B7-1 transmembrane domains. The expression of scFv EGFRvIII on the cell surface was assessed by FACS. A stable population of scFv EGFRvIII-expressing hMSCs was selected, based on antibiotic resistance, and enriched using FACS. We found that nucleofection allows the efficient expression of scFv EGFRvIII on the cell surface of hMSCs. hMSCs transfected with the construct encoding scFv EGFRvIII as a fusion with PDGFRtm showed scFv EGFRvIII expression in up to 86% of cells. Most importantly, human MSCs expressing scFv against EGFRvIII demonstrated enhanced binding to U87-EGFRvIII cells in vitro and significantly increased retention in human U87-EGFRvIII-expressing tumours in vivo. In summary, we provide the first conclusive evidence of genetic modification of hMSCs with a single-chain antibody against an antigen expressed on the surface of tumour cells, thereby opening up a new venue for enhanced delivery of gene therapy applications in the context of malignant brain cancer. Copyright 2009 John Wiley & Sons, Ltd.

GDNF and NGF family members and receptors in human fetal and adult spinal cord and dorsal root ganglia.

PubMed

Josephson, A; Widenfalk, J; Trifunovski, A; Widmer, H R; Olson, L; Spenger, C

2001-11-12

We describe the expression of mRNA encoding ligands and receptors of members of the GDNF family and members of the neurotrophin family in the adult human spinal cord and dorsal root ganglia (DRG). Fetal human spinal cord and ganglia were investigated for the presence of ligands and receptors of the neurotrophin family. Tissues were collected from human organ donors and after routine elective abortions. Messenger RNA was found encoding RET, GFR alpha-1, BDNF, trkB, and trkC in the adult human spinal cord and BDNF, NT-3, p75, trkB, and trkC in the fetal human spinal cord. The percentage of adult human DRG cells expressing p75, trkA, trkB, or trkC was 57, 46, 29, and 24%, respectively, and that of DRG cells expressing RET, GFR alpha-1, GFR alpha-2, or GFR alpha-3 was 79, 20, 51, and 32%, respectively. GFR alpha-2 was expressed selectively in small, GFR alpha-3 principally in small and GFR alpha-1 and RET in both large and small adult human DRG neurons. p75 and trkB were expressed by a wide range of DRG neurons while trkA was expressed in most small diameter and trkC primarily in large DRG neurons. Fetal DRG cells were positive for the same probes as adult DRG cells except for NT-3, which was only found in fetal DRG cells. Messenger RNA species only expressed at detectable levels in fetal but not adult spinal cord tissues included GDNF, GFR alpha-2, NT-3, and p75. Notably, GFR alpha-2, which is expressed in the adult rat spinal cord, was not found in the adult human spinal cord. Copyright 2001 Wiley-Liss, Inc.

Glucocorticoid-induced pancreatic-hepatic trans-differentiation in a human cell line in vitro.

PubMed

Fairhall, Emma A; Leitch, Alistair C; Lakey, Anne F; Probert, Philip M E; Richardson, Gabriella; De Santis, Carol; Wright, Matthew C

2018-05-22

The rodent pancreatic AR42J-B13 (B-13) cell line differentiates into non-replicative hepatocyte-like cells in response to glucocorticoid mediated via the glucocorticoid receptor (GR). The aims of this study were to identify a human cell line that responds similarly and investigate the mechanisms underpinning any alteration in differentiation. Exposing the human pancreatic adenocarcinoma (HPAC) cell line to 1-10 µM concentrations of dexamethasone (DEX) resulted an inhibition of proliferation, suppressed carcinoembryonic antigen expression, limited expression of pancreatic acinar and hepatic gene expression and significant induction of the constitutively-expressed hepatic CYP3A5 mRNA transcript. These changes were associated with a pulse of genomic DNA methylation and suppressed notch signalling activity. HPAC cells expressed high levels of GR transcript in contrast to other nuclear receptors - such as the glucocorticoid-activated pregnane X receptor (PXR) - and GR transcriptional function was activated by DEX in HPAC cells. Expression of selected hepatocyte transcripts in response to DEX was blocked by co-treatment with the GR antagonist RU486. These data indicate that the HPAC response to glucocorticoid exposure includes an inhibition in proliferation, alterations in notch signalling and a limited change in the expression of genes associated with an acinar and hepatic phenotype. This is the first demonstration of a human cell responding to similarly to the rodent B-13 cell regarding formation of hepatocyte-like cells in response to glucocorticoid. Identifying and modulating the ablating factor(s) may enhance the hepatocyte-like forming capacity of HPAC cells after exposure to glucocorticoid and generate an unlimited in vitro supply of human hepatocytes for toxicology studies and a variety of clinical applications. Copyright © 2018 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Human Dermal Mast Cells Contain and Release Tumor Necrosis Factor α, which Induces Endothelial Leukocyte Adhesion Molecule 1

NASA Astrophysics Data System (ADS)

Walsh, Laurence J.; Trinchieri, Giorgio; Waldorf, Heidi A.; Whitaker, Diana; Murphy, George F.

1991-05-01

Tumor necrosis factor α (TNF-α) is a proinflammatory cytokine that mediates endothelial leukocyte interactions by inducing expression of adhesion molecules. In this report, we demonstrate that human dermal mast cells contain sizeable stores of immunoreactive and biologically active TNF-α within granules, which can be released rapidly into the extracellular space upon degranulation. Among normal human dermal cells, mast cells are the predominant cell type that expresses both TNF-α protein and TNF-α mRNA. Moreover, induction of endothelial leukocyte adhesion molecule 1 expression is a direct consequence of release of mast cell-derived TNF-α. These findings establish a role for human mast cells as "gatekeepers" of the dermal microvasculature and indicate that mast cell products other than vasoactive amines influence endothelium in a proinflammatory fashion.

Human Nanog pseudogene8 promotes the proliferation of gastrointestinal cancer cells

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

Uchino, Keita, E-mail: uchino13@intmed1.med.kyushu-u.ac.jp; Hirano, Gen; Hirahashi, Minako

2012-09-10

There is emerging evidence that human solid tumor cells originate from cancer stem cells (CSCs). In cancer cell lines, tumor-initiating CSCs are mainly found in the side population (SP) that has the capacity to extrude dyes such as Hoechst 33342. We found that Nanog is expressed specifically in SP cells of human gastrointestinal (GI) cancer cells. Nucleotide sequencing revealed that NanogP8 but not Nanog was expressed in GI cancer cells. Transfection of NanogP8 into GI cancer cell lines promoted cell proliferation, while its inhibition by anti-Nanog siRNA suppressed the proliferation. Immunohistochemical staining of primary GI cancer tissues revealed NanogP8 proteinmore » to be strongly expressed in 3 out of 60 cases. In these cases, NanogP8 was found especially in an infiltrative part of the tumor, in proliferating cells with Ki67 expression. These data suggest that NanogP8 is involved in GI cancer development in a fraction of patients, in whom it presumably acts by supporting CSC proliferation. -- Highlights: Black-Right-Pointing-Pointer Nanog maintains pluripotency by regulating embryonic stem cells differentiation. Black-Right-Pointing-Pointer Nanog is expressed in cancer stem cells of human gastrointestinal cancer cells. Black-Right-Pointing-Pointer Nucleotide sequencing revealed that Nanog pseudogene8 but not Nanog was expressed. Black-Right-Pointing-Pointer Nanog pseudogene8 promotes cancer stem cells proliferation. Black-Right-Pointing-Pointer Nanog pseudogene8 is involved in gastrointestinal cancer development.« less

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression.

PubMed

Trinh, Alice T; Ball, Bret G; Weber, Erin; Gallaher, Timothy K; Gluzman-Poltorak, Zoya; Anderson, French; Basile, Lena A

2009-12-30

Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene. These studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency.

Evaluation of Accessory Lacrimal Gland in Muller's Muscle Conjunctival Resection Specimens for Precursor Cell Markers and Biological Markers of Dry Eye Disease.

PubMed

Ali, Marwan; Shah, Dhara; Pasha, Zeeshan; Jassim, Sarmad H; Jassim Jaboori, Assraa; Setabutr, Pete; Aakalu, Vinay K

2017-04-01

The accessory lacrimal glands (ALGs) are an understudied component of the tear functional unit, even though they are important in the development of dry eye syndrome (DES). To advance our understanding of aging changes, regenerative potential, and histologic correlates to human characteristics, we investigated human ALG tissue from surgical samples to determine the presence or absence of progenitor cell markers and lacrimal epithelial markers and to correlate marker expression to relevant patient characteristics. ALG tissues obtained from Muller's muscle conjunctival resection (MMCR) specimens were created using tissue microarrays (TMAs). Immunofluorescence staining of MMCR sections was performed using primary antibodies specific to cell protein markers. Cell marker localization in TMAs was then assessed by two blinded observers using a standardized scoring system. Patient characteristics including age, race, and status of ocular surface health were then compared against expression of stem cell markers. Human ALG expressed a number of epithelial markers, and in particular, histatin-1 was well correlated with the expression of epithelial markers and was present in most acini. In addition, we noted the presence of precursor cell markers nestin, ABCG2, and CD90 in ALG tissue. There was a decrease in precursor cell marker expression with increasing age. Finally, we noted that a negative association was present between histatin-1 expression and DES. Thus, we report for the first time that human ALG tissues contain precursor marker-positive cells and that this marker expression may decrease with increasing age. Moreover, histatin-1 expression may be decreased in DES. Future studies will be performed to use these cell markers to isolate and culture lacrimal epithelial cells from heterogeneous tissues, determine the relevance of histatin-1 expression to DES, and isolate candidate precursor cells from ALG tissue.

Adult, embryonic and fetal hemoglobin are expressed in human glioblastoma cells.

PubMed

Emara, Marwan; Turner, A Robert; Allalunis-Turner, Joan

2014-02-01

Hemoglobin is a hemoprotein, produced mainly in erythrocytes circulating in the blood. However, non-erythroid hemoglobins have been previously reported in other cell types including human and rodent neurons of embryonic and adult brain, but not astrocytes and oligodendrocytes. Human glioblastoma multiforme (GBM) is the most aggressive tumor among gliomas. However, despite extensive basic and clinical research studies on GBM cells, little is known about glial defence mechanisms that allow these cells to survive and resist various types of treatment. We have shown previously that the newest members of vertebrate globin family, neuroglobin (Ngb) and cytoglobin (Cygb), are expressed in human GBM cells. In this study, we sought to determine whether hemoglobin is also expressed in GBM cells. Conventional RT-PCR, DNA sequencing, western blot analysis, mass spectrometry and fluorescence microscopy were used to investigate globin expression in GBM cell lines (M006x, M059J, M059K, M010b, U87R and U87T) that have unique characteristics in terms of tumor invasion and response to radiotherapy and hypoxia. The data showed that α, β, γ, δ, ζ and ε globins are expressed in all tested GBM cell lines. To our knowledge, we are the first to report expression of fetal, embryonic and adult hemoglobin in GBM cells under normal physiological conditions that may suggest an undefined function of those expressed hemoglobins. Together with our previous reports on globins (Ngb and Cygb) expression in GBM cells, the expression of different hemoglobins may constitute a part of series of active defence mechanisms supporting these cells to resist various types of treatments including chemotherapy and radiotherapy.

Chemotherapeutic Effect of CD147 Antibody-labeled Micelles Encapsulating Doxorubicin Conjugate Targeting CD147-Expressing Carcinoma Cells.

PubMed

Asakura, Tadashi; Yokoyama, Masayuki; Shiraishi, Koichi; Aoki, Katsuhiko; Ohkawa, Kiyoshi

2018-03-01

CD147 (basigin/emmprin) is expressed on the surface of carcinoma cells. For studying the efficacy of CD147-targeting medicine on CD147-expressing cells, we studied the effect of anti-CD147-labeled polymeric micelles (CD147ab micelles) that encapsulated a conjugate of doxorubicin with glutathione (GSH-DXR), with specific accumulation and cytotoxicity against CD147-expressing A431 human epidermoid carcinoma cells, Ishikawa human endometrial adenocarcinoma cells, and PC3 human prostate carcinoma cells. By treatment of each cell type with CD147ab micelles for 1 h, a specific accumulation of CD147ab micelles in CD147-expressing cells was observed. In addition, the cytotoxicity of GSH-DXR-encapsulated micelles against each cell type was measured by treatment of the micelles for 1 h. The cytotoxic effect of CD147ab micelles carrying GSH-DXR was 3- to 10-fold higher for these cells than that of micelles without GSH-DXR. These results suggest that GSH-DXR-encapsulated CD147ab micelles could serve as an effective drug delivery system to CD147-expressing carcinoma cells. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Altered serotonin physiology in human breast cancers favors paradoxical growth and cell survival.

PubMed

Pai, Vaibhav P; Marshall, Aaron M; Hernandez, Laura L; Buckley, Arthur R; Horseman, Nelson D

2009-01-01

The breast microenvironment can either retard or accelerate the events associated with progression of latent cancers. However, the actions of local physiological mediators in the context of breast cancers are poorly understood. Serotonin (5-HT) is a critical local regulator of epithelial homeostasis in the breast and other organs. Herein, we report complex alterations in the intrinsic mammary gland serotonin system of human breast cancers. Serotonin biosynthetic capacity was analyzed in human breast tumor tissue microarrays using immunohistochemistry for tryptophan hydroxylase 1 (TPH1). Serotonin receptors (5-HT1-7) were analyzed in human breast tumors using the Oncomine database. Serotonin receptor expression, signal transduction, and 5-HT effects on breast cancer cell phenotype were compared in non-transformed and transformed human breast cells. In the context of the normal mammary gland, 5-HT acts as a physiological regulator of lactation and involution, in part by favoring growth arrest and cell death. This tightly regulated 5-HT system is subverted in multiple ways in human breast cancers. Specifically, TPH1 expression undergoes a non-linear change during progression, with increased expression during malignant progression. Correspondingly, the tightly regulated pattern of 5-HT receptors becomes dysregulated in human breast cancer cells, resulting in both ectopic expression of some isoforms and suppression of others. The receptor expression change is accompanied by altered downstream signaling of 5-HT receptors in human breast cancer cells, resulting in resistance to 5-HT-induced apoptosis, and stimulated proliferation. Our data constitutes the first report of direct involvement of 5-HT in human breast cancer. Increased 5-HT biosynthetic capacity accompanied by multiple changes in 5-HT receptor expression and signaling favor malignant progression of human breast cancer cells (for example, stimulated proliferation, inappropriate cell survival). This occurs through uncoupling of serotonin from the homeostatic regulatory mechanisms of the normal mammary epithelium. The findings open a new avenue for identification of diagnostic and prognostic markers, and valuable new therapeutic targets for managing breast cancer.

A reevaluation of CD22 expression in human lung cancer.

PubMed

Pop, Laurentiu M; Barman, Stephen; Shao, Chunli; Poe, Jonathan C; Venturi, Guglielmo M; Shelton, John M; Pop, Iliodora V; Gerber, David E; Girard, Luc; Liu, Xiao-yun; Behrens, Carmen; Rodriguez-Canales, Jaime; Liu, Hui; Wistuba, Ignacio I; Richardson, James A; Minna, John D; Tedder, Thomas F; Vitetta, Ellen S

2014-01-01

CD22 is a transmembrane glycoprotein expressed by mature B cells. It inhibits signal transduction by the B-cell receptor and its coreceptor CD19. Recent reports indicate that most human lung cancer cells and cell lines express CD22, making it an important new therapeutic target for lung cancer. The objective of our studies was to independently validate these results with the goal of testing the efficacy of our CD22 immunotoxins on lung cancer cell lines. As determined by quantitative real-time PCR analysis, we found that levels of CD22 mRNA in a panel of human lung cancer cell lines were 200 to 60,000-fold lower than those observed in the human CD22(+) Burkitt lymphoma cells, Daudi. Using flow cytometry with a panel of CD22 monoclonal antibodies and Western blot analyses, we could not detect surface or intracellular expression of CD22 protein in a panel of lung cancer cell lines. In addition, the in vitro proliferation of the lung tumor cell lines was not affected by either CD22 antibodies or our highly potent anti-CD22 immunotoxin. In contrast, CD22(+) Daudi cells expressed high levels of CD22 mRNA and protein, and were sensitive to our CD22 immunotoxin. Importantly, primary non-small cell lung cancers from more than 250 patient specimens did not express detectable levels of CD22 protein as assessed by immunohistochemistry. We conclude that CD22 is not expressed at measurable levels on the surface of lung cancer cells, and that these cells cannot be killed by anti-CD22 immunotoxins.

NADE (p75NTR-associated cell death executor) suppresses cellular growth in vivo.

PubMed

Tong, Xiangjun; Xie, Dong; Roth, Wilfried; Reed, John; Koeffler, H Phillip

2003-06-01

NADE, a p75NTR (low-affinity neurotrophin receptor p75) -associated cell death executor, was initially cloned from a human ovarian granulosa cell cDNA library, as an unknown protein with the name, pHGR74. It was reported to mediate nerve growth factor-induced apoptosis. We independently isolated human NADE (pHGR74) from breast cancer cell lines. Expression of NADE in various human cancer cell lines, and human and murine tissues was examined. NADE was highly expressed in human endocrine-related organs and embryotic murine tissues. Forced expression of NADE in CHO (Chinese hamster ovary) cells and MDA-MB-231 human breast cancer cells had little effect on the growth of the cells in vitro, while it dramatically suppressed cellular growth in vivo. We used the yeast two-hybrid system to search for NADE binding protein. Dynactin was identified as a candidate. The p75NTR was not found in this assay and did not co-immunoprecipitate with human NADE. Furthermore, the cells stably transfected with NADE did not respond to NGF or TNF. Thus, human and murine NADE appear to have different functions.

Low-level expression of human ACAT2 gene in monocytic cells is regulated by the C/EBP transcription factors

PubMed Central

Guo, Dongqing; Lu, Ming; Hu, Xihan; Xu, Jiajia; Hu, Guangjing; Zhu, Ming; Zhang, Xiaowei; Li, Qin; Chang, Catherine C. Y.; Chang, Tayuan; Song, Baoliang; Xiong, Ying; Li, Boliang

2016-01-01

Acyl-coenzyme A:cholesterol acyltransferases (ACATs) are the exclusive intracellular enzymes that catalyze the formation of cholesteryl/steryl esters (CE/SE). In our previous work, we found that the high-level expression of human ACAT2 gene with the CpG hypomethylation of its whole promoter was synergistically regulated by two transcription factors Cdx2 and HNF1α in the intestine and fetal liver. Here, we first observed that the specific CpG-hypomethylated promoter was correlated with the low expression of human ACAT2 gene in monocytic cell line THP-1. Then, two CCAAT/enhancer binding protein (C/EBP) elements within the activation domain in the specific CpG-hypomethylation promoter region were identified, and the expression of ACAT2 in THP-1 cells was evidently decreased when the C/EBP transcription factors were knock-downed using RNAi technology. Furthermore, ChIP assay confirmed that C/EBPs directly bind to their elements for low-level expression of human ACAT2 gene in THP-1 cells. Significantly, the increased expressions of ACAT2 and C/EBPs were also found in macrophages differentiated from both ATRA-treated THP-1 cells and cultured human blood monocytes. These results demonstrate that the low-level expression of human ACAT2 gene with specific CpG-hypomethylated promoter is regulated by the C/EBP transcription factors in monocytic cells, and imply that the lowly expressed ACAT2 catalyzes the synthesis of certain CE/SE that are assembled into lipoproteins for the secretion. PMID:27688151

Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line.

PubMed Central

Malik, P; McQuiston, S A; Yu, X J; Pepper, K A; Krall, W J; Podsakoff, G M; Kurtzman, G J; Kohn, D B

1997-01-01

We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate for gene therapy of nondividing cells, a very high MOI or improvements in basic aspects of AAV-based vectors may be necessary to improve integration frequency in the rapidly dividing hematopoietic cell population. PMID:9032306

Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line.

PubMed

Malik, P; McQuiston, S A; Yu, X J; Pepper, K A; Krall, W J; Podsakoff, G M; Kurtzman, G J; Kohn, D B

1997-03-01

We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate for gene therapy of nondividing cells, a very high MOI or improvements in basic aspects of AAV-based vectors may be necessary to improve integration frequency in the rapidly dividing hematopoietic cell population.

Peroxisome proliferator-activated receptor (PPAR)-gamma expression in human vascular smooth muscle cells: inhibition of growth, migration, and c-fos expression by the peroxisome proliferator-activated receptor (PPAR)-gamma activator troglitazone.

PubMed

Benson, S; Wu, J; Padmanabhan, S; Kurtz, T W; Pershadsingh, H A

2000-01-01

This study was conducted to determine whether cultured human coronary artery and aorta vascular smooth muscle (VSM) cells express the nuclear transcription factor peroxisome proliferator-activated receptor-gamma (PPARgamma); whether the thiazolidinedione troglitazone, a ligand for PPARgamma, would inhibit c-fos expression by these cells; and whether troglitazone would inhibit proliferation and migration induced in these cells by mitogenic growth factors. Using immunoblotting and reverse-transcriptase polymerase chain reaction (RT-PCR) techniques, we show that both human aorta and coronary artery VSM cell lines expressed PPARgamma protein and mRNA for both PPARgamma isoforms, PPARgamma1 and PPARgamma2. Immunocytochemical staining localized the PPARgamma protein primarily within the nucleus. Troglitazone inhibited basic fibroblast growth factor and platelet-derived growth factor-BB induced DNA synthesis in a dose-dependent manner and downregulated the growth-factor-induced expression of c-fos. Troglitazone also inhibited the migration of coronary artery VSM cells along a platelet-derived growth factor-BB concentration gradient. These findings demonstrate for the first time the expression and nuclear localization of PPARgamma in human coronary artery and aorta VSM cells. The data also suggest that the downregulation of c-fos expression, growth-factor-induced proliferation, and migration by VSM may, in part, be mediated by activation of the PPARgamma receptor.

Buprenorphine-elicited alteration of adenylate cyclase activity in human embryonic kidney 293 cells coexpressing κ-, μ-opioid and nociceptin receptors

PubMed Central

Wang, Pei-Chen; Ho, Ing-Kang; Lee, Cynthia Wei-Sheng

2015-01-01

Buprenorphine, a maintenance drug for heroin addicts, exerts its pharmacological function via κ- (KOP), μ-opioid (MOP) and nociceptin/opioid receptor-like 1 (NOP) receptors. Previously, we investigated its effects in an in vitro model expressing human MOP and NOP receptors individually or simultaneously (MOP, NOP, and MOP+NOP) in human embryonic kidney 293 cells. Here, we expanded this cell model by expressing human KOP, MOP and NOP receptors individually or simultaneously (KOP, KOP+MOP, KOP+NOP and KOP+MOP+NOP). Radioligand binding with tritium-labelled diprenorphine confirmed the expression of KOP receptors. Immunoblotting and immunocytochemistry indicated that the expressed KOP, MOP and NOP receptors are N-linked glycoproteins and colocalized in cytoplasmic compartments. Acute application of the opioid receptor agonists— U-69593, DAMGO and nociceptin— inhibited adenylate cyclase (AC) activity in cells expressing KOP, MOP and NOP receptors respectively. Buprenorphine, when applied acutely, inhibited AC activity to ~90% in cells expressing KOP+MOP+NOP receptors. Chronic exposure to buprenorphine induced concentration-dependent AC superactivation in cells expressing KOP+NOP receptors, and the level of this superactivation was even higher in KOP+MOP+NOP-expressing cells. Our study demonstrated that MOP receptor could enhance AC regulation in the presence of coexpressed KOP and NOP receptors, and NOP receptor is essential for concentration-dependent AC superactivation elicited by chronic buprenorphine exposure. PMID:26153065

Expression of lnterleukin-8 Correlates with Angiogenesis, Tumorigenicity, and Metastasis of Human Prostate Cancer Cells Implanted Orthotopically in Nude Mice1

PubMed Central

Sun, Jin Kim; Uehara, Hisanori; Karashima, Takashi; Mccarty, Marya; Shih, Nancy; Fidler, Isaiah J

2001-01-01

Abstract We determined whether the expression of interleukin-8 (IL-8) by human prostate cancer cells correlates with induction of angiogenesis, tumorigenicity, and production of metastasis. Low and high IL-8-producing clones were isolated from the heterogeneous PC-3 human prostate cancer cell line. The secretion of IL-8 protein correlated with transcriptional activity and levels of IL-8 mRNA. All PC-3 cells expressed both IL-8 receptors, CXCR1 and CXCR2. The low and high IL-8-producing clones were injected into the prostate of nude mice. Titration studies indicated that PC-3 cells expressing high levels of IL-8 were highly tumorigenic, producing rapidly growing, highly vascularized prostate tumors with and a 100% incidence of lymph node metastasis. Low IL-8-expressing PC-3 cells were less tumorigenic, producing slower growing and less vascularized primary tumors and a significantly lower incidence of metastasis. In situ hybridization (ISH) analysis of the tumors for expression of genes that regulate angiogenesis and metastasis showed that the expression level of IL-8, matrix metalloproteinases, vascular endothelial growth factor (VEGF), and E-cadherin corresponded with microvascular density and biological behavior of the prostate cancers in nude mice. Collectively, the data show that the expression level of IL-8 in human prostate cancer cells is associated with angiogenesis, tumorigenicity, and metastasis. PMID:11326314

Phenotype, effector function, and tissue localization of PD-1-expressing human follicular helper T cell subsets

PubMed Central

2011-01-01

Background It is well established that PD-1 is expressed by follicular T cells but its function in regulation of human T helper cells has been unclear. We investigated the expression modality and function of PD-1 expressed by human T cells specialized in helping B cells. Results We found that PD-1-expressing T cells are heterogeneous in PD-1 expression. We identified three different PD-1-expressing memory T cell subsets (i.e. PD-1low (+), PD-1medium (++), and PD-1high (+++) cells). PD-1+++ T cells expressed CXCR5 and CXCR4 and were localized in the rim of germinal centers. PD-1+ or PD-1++ cells expressed CCR7 and were present mainly in the T cell area or other parts of the B cell follicles. Utilizing a novel antigen density-dependent magnetic sorting (ADD-MS) method, we isolated the three T cell subsets for functional characterization. The germinal center-located PD-1+++ T cells were most efficient in helping B cells and in producing IL-21 and CXCL13. Other PD-1-expressing T cells, enriched with Th1 and Th17 cells, were less efficient than PD-1+++ T cells in these capacities. PD-1+++ T cells highly expressed Ki-67 and therefore appear active in cell activation and proliferation in vivo. IL-2 is a cytokine important for proliferation and survival of the PD-1+++ T cells. In contrast, IL-21, while a major effector cytokine produced by the PD-1-expressing T helper cells, had no function in generation, survival, or proliferation of the PD-1-expressing helper T cells at least in vitro. PD-1 triggering has a suppressive effect on the proliferation and B cell-helping function of PD-1+++ germinal center T cells. Conclusion Our results revealed the phenotype and effector function of PD-1-expressing T helper cell subsets and indicate that PD-1 restrains the B cell-helping function of germinal center-localized T cells to prevent excessive antibody response. PMID:21914188

The role of the obestatin/GPR39 system in human gastric adenocarcinomas.

PubMed

Alén, Begoña O; Leal-López, Saúl; Alén, María Otero; Viaño, Patricia; García-Castro, Victoria; Mosteiro, Carlos S; Beiras, Andrés; Casanueva, Felipe F; Gallego, Rosalía; García-Caballero, Tomás; Camiña, Jesús P; Pazos, Yolanda

2016-02-02

Obestatin, a 23-amino acid peptide encoded by the ghrelin gene, and the GPR39 receptor were reported to be involved in the control of mitogenesis of gastric cancer cell lines; however, the relationship between the obestatin/GPR39 system and gastric cancer progression remains unknown. In the present study, we determined the expression levels of the obestatin/GPR39 system in human gastric adenocarcinomas and explored their potential functional roles. Twenty-eight patients with gastric adenocarcinomas were retrospectively studied, and clinical data were obtained. The role of obestatin/GPR39 in gastric cancer progression was studied in vitro using the human gastric adenocarcinoma AGS cell line. Obestatin exogenous administration in these GPR39-bearing cells deregulated the expression of several hallmarks of the epithelial-mesenchymal transition (EMT) and angiogenesis. Moreover, obestatin signaling promoted phenotypic changes via GPR39, increasingly impacting on the cell morphology, proliferation, migration and invasion of these cells. In healthy human stomachs, obestatin expression was observed in the neuroendocrine cells and GPR39 expression was localized mainly in the chief cells of the oxyntic glands. In human gastric adenocarcinomas, no obestatin expression was found; however, an aberrant pattern of GPR39 expression was discovered, correlating to the dedifferentiation of the tumor. Altogether, our data strongly suggest the involvement of the obestatin/GPR39 system in the pathogenesis and/or clinical outcome of human gastric adenocarcinomas and highlight the potential usefulness of GPR39 as a prognostic marker in gastric cancer.

The role of the obestatin/GPR39 system in human gastric adenocarcinomas

PubMed Central

Alén, Begoña O.; Leal-López, Saúl; Alén, María Otero; Viaño, Patricia; García-Castro, Victoria; Mosteiro, Carlos S.; Beiras, Andrés; Casanueva, Felipe F.; Gallego, Rosalía; García-Caballero, Tomás; Camiña, Jesús P.; Pazos, Yolanda

2016-01-01

Obestatin, a 23-amino acid peptide encoded by the ghrelin gene, and the GPR39 receptor were reported to be involved in the control of mitogenesis of gastric cancer cell lines; however, the relationship between the obestatin/GPR39 system and gastric cancer progression remains unknown. In the present study, we determined the expression levels of the obestatin/GPR39 system in human gastric adenocarcinomas and explored their potential functional roles. Twenty-eight patients with gastric adenocarcinomas were retrospectively studied, and clinical data were obtained. The role of obestatin/GPR39 in gastric cancer progression was studied in vitro using the human gastric adenocarcinoma AGS cell line. Obestatin exogenous administration in these GPR39-bearing cells deregulated the expression of several hallmarks of the epithelial-mesenchymal transition (EMT) and angiogenesis. Moreover, obestatin signaling promoted phenotypic changes via GPR39, increasingly impacting on the cell morphology, proliferation, migration and invasion of these cells. In healthy human stomachs, obestatin expression was observed in the neuroendocrine cells and GPR39 expression was localized mainly in the chief cells of the oxyntic glands. In human gastric adenocarcinomas, no obestatin expression was found; however, an aberrant pattern of GPR39 expression was discovered, correlating to the dedifferentiation of the tumor. Altogether, our data strongly suggest the involvement of the obestatin/GPR39 system in the pathogenesis and/or clinical outcome of human gastric adenocarcinomas and highlight the potential usefulness of GPR39 as a prognostic marker in gastric cancer. PMID:26716511

Susceptibility of human liver cells to porcine endogenous retrovirus.

PubMed

Lin, Xinzi; Qi, Lin; Li, Zhiguo; Chi, Hao; Lin, Wanjun; Wang, Yan; Jiang, Zesheng; Pan, Mingxin; Gao, Yi

2013-12-01

The risk of porcine endogenous retrovirus infection is a major barrier for pig-to-human xenotransplant. Porcine endogenous retrovirus, present in porcine cells, can infect many human and nonhuman primate cells in vitro, but there is no evidence available about in vitro infection of human liver cells. We investigated the susceptibility of different human liver cells to porcine endogenous retrovirus. The supernatant from a porcine kidney cell line was added to human liver cells, including a normal hepatocyte cell line (HL-7702 cells), primary hepatocytes (Phh cells), and a liver stellate cell line (Lx-2 cells), and to human embryonic kidney cells as a reference control. Expression of the porcine endogenous retrovirus antigen p15E in the human cells was evaluated with polymerase chain reaction, reverse transcription-polymerase chain reaction, and Western blot. The porcine endogenous retrovirus antigen p15E was not expressed in any human liver cells (HL-7702, Phh, or Lx-2 cells) that had been exposed to supernatants from porcine kidney cell lines. Porcine endogenous retrovirus-specific fragments were amplified in human kidney cells. Human liver cells tested were not susceptible to infection by porcine endogenous retrovirus. Therefore, not all human cells are susceptible to porcine endogenous retrovirus.

Localization of migraine susceptibility genes in human brain by single-cell RNA sequencing.

PubMed

Renthal, William

2018-01-01

Background Migraine is a debilitating disorder characterized by severe headaches and associated neurological symptoms. A key challenge to understanding migraine has been the cellular complexity of the human brain and the multiple cell types implicated in its pathophysiology. The present study leverages recent advances in single-cell transcriptomics to localize the specific human brain cell types in which putative migraine susceptibility genes are expressed. Methods The cell-type specific expression of both familial and common migraine-associated genes was determined bioinformatically using data from 2,039 individual human brain cells across two published single-cell RNA sequencing datasets. Enrichment of migraine-associated genes was determined for each brain cell type. Results Analysis of single-brain cell RNA sequencing data from five major subtypes of cells in the human cortex (neurons, oligodendrocytes, astrocytes, microglia, and endothelial cells) indicates that over 40% of known migraine-associated genes are enriched in the expression profiles of a specific brain cell type. Further analysis of neuronal migraine-associated genes demonstrated that approximately 70% were significantly enriched in inhibitory neurons and 30% in excitatory neurons. Conclusions This study takes the next step in understanding the human brain cell types in which putative migraine susceptibility genes are expressed. Both familial and common migraine may arise from dysfunction of discrete cell types within the neurovascular unit, and localization of the affected cell type(s) in an individual patient may provide insight into to their susceptibility to migraine.

[Effect of EMP-1 gene on human esophageal cancer cell line].

PubMed

Wang, Hai-tao; Liu, Zhi-hua; Wang, Xiu-qin; Wu, Min

2002-03-01

EMP-1 was selected from a series of differential expressed genes obtained from cDNA microarray in the authors' lab. Epithelial membrane pnteiu-1 gene (EMP-1) was expressed 6 fold lower in esophageal cancer than in normal tissue. The authors further designed the experiment to study the effect of human EMP-1 gene on human esophageal cancer cell line in order to explain the function of this gene on the carcinogensis and progression esophageal cancer. EMP-1 gene was cloned into eukaryotic vector and transfected into the human esophageal cancer cell line. The transfection effect was qualified by Western blot and RT-PCR method. The cell growth curve was observed and the cell cycle was checked by FACS method. EMP-1 was transfected into EC9706 cell line and its expression was up-regulated. The cell growth is accelerated and expression of EMP-1 is linked to induction of S phase arrest. EMP-1 gene has some relationship with carcinogenesis of esophagus.

Expansion of mesenchymal stem cells from human pancreatic ductal epithelium.

PubMed

Seeberger, Karen L; Dufour, Jannette M; Shapiro, Andrew M James; Lakey, Jonathan R T; Rajotte, Ray V; Korbutt, Gregory S

2006-02-01

Fibroblast-like cells emerging from cultured human pancreatic endocrine and exocrine tissue have been reported. Although a thorough phenotypic characterization of these cells has not yet been carried out, these cells have been hypothesized to be contaminating fibroblasts, mesenchyme and/or possibly beta-cell progenitors. In this study, we expanded fibroblast-like cells from adult human exocrine pancreas following islet isolation and characterized these cells as mesenchymal stem cells (MSCs) based on their cell surface antigen expression and ability to differentiate into mesoderm. Analysis by flow cytometry demonstrated that pancreatic MSCs express cell surface antigens used to define MSCs isolated from bone marrow such as CD13, CD29, CD44, CD49b, CD54, CD90 and CD105. In addition, utilizing protocols used to differentiate MSCs isolated from other somatic tissues, we successfully differentiated pancreatic MSCs into: (1) osteocytes that stained positive for alkaline phosphatase, collagen, mineralization (calcification) and expressed osteocalcin, (2) adipocytes that contained lipid inclusions and expressed fatty acid binding protein 4 and (3) chondrocytes that expressed aggrecan. We also demonstrated that pancreatic MSCs are multipotent and capable of deriving cells of endodermal origin. Pancreatic MSCs were differentiated into hepatocytes that stained positive for human serum albumin and expressed endoderm and liver-specific genes such as GATA 4 and tyrosine aminotransferase. In addition, preliminary protocols used to differentiate these cells into insulin-producing cells resulted in the expression of genes necessary for islet and beta-cell development such as Pax4 and neurogenin 3. Therefore, multipotent MSCs residing within the adult exocrine pancreas could represent a progenitor cell, which when further manipulated could result in the production of functional islet beta-cells.

Harnessing Gene Conversion in Chicken B Cells to Create a Human Antibody Sequence Repertoire

PubMed Central

Schusser, Benjamin; Yi, Henry; Collarini, Ellen J.; Izquierdo, Shelley Mettler; Harriman, William D.; Etches, Robert J.; Leighton, Philip A.

2013-01-01

Transgenic chickens expressing human sequence antibodies would be a powerful tool to access human targets and epitopes that have been intractable in mammalian hosts because of tolerance to conserved proteins. To foster the development of the chicken platform, it is beneficial to validate transgene constructs using a rapid, cell culture-based method prior to generating fully transgenic birds. We describe a method for the expression of human immunoglobulin variable regions in the chicken DT40 B cell line and the further diversification of these genes by gene conversion. Chicken VL and VH loci were knocked out in DT40 cells and replaced with human VK and VH genes. To achieve gene conversion of human genes in chicken B cells, synthetic human pseudogene arrays were inserted upstream of the functional human VK and VH regions. Proper expression of chimeric IgM comprised of human variable regions and chicken constant regions is shown. Most importantly, sequencing of DT40 genetic variants confirmed that the human pseudogene arrays contributed to the generation of diversity through gene conversion at both the Igl and Igh loci. These data show that engineered pseudogene arrays produce a diverse pool of human antibody sequences in chicken B cells, and suggest that these constructs will express a functional repertoire of chimeric antibodies in transgenic chickens. PMID:24278246

Human CD22 cannot fully substitute murine CD22 functions in vivo, as shown in a new knockin mouse model.

PubMed

Wöhner, Miriam; Born, Stefanie; Nitschke, Lars

2012-11-01

CD22, an inhibitory co-receptor of the B-cell receptor, shows a B-cell-specific expression pattern and is expressed on most B-cell lymphomas. The anti-CD22 antibody Epratuzumab is in clinical trials for B-cell non-Hodgkin lymphoma and systemic lupus erythematosus, but shows a mostly unknown mode of action. We generated a new mouse model that expresses human CD22 instead of murine CD22 (Huki CD22 mice), in which human CD22 can be targeted. Expression of human CD22 on the B cells of Huki CD22 mice does not generally interfere with B-cell development. However, Huki CD22 mice show a reduction of the population of mature recirculating B cells in the bone marrow and reduced transitional and marginal zone B cells in the spleen, phenotypes resembling that of CD22-deficient mice. Similarly, enhanced BCR-induced Ca(2+) signalling is observed in Huki CD22 mice, which also mount normal immune responses toward different classes of antigens. Huki CD22 B cells show a normal anti-hCD22 antibody-mediated endocytosis. In conclusion, human CD22 cannot fully substitute for murine CD22 functions, possibly due to the changed intracellular tail of the protein or due to lower expression levels. Huki CD22 mice are a valuable new model for both antibody- and immunotoxin-mediated targeting of human CD22. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Differential expression of connexin 43 in human autoimmune thyroid disease.

PubMed

Jiang, Xiao-Yan; Feng, Xiao-Hong; Li, Guo-Yan; Zhao, Qian; Yin, Hui-Qing

2010-05-01

Gap junctions provide a pathway for cell-to-cell communication. Reduced thyroid epithelial cell-cell communication has been reported in some animal models of autoimmune thyroid disease. In order to assess whether this change was similar to human autoimmune thyroid disease, we identified some connexin proteins and their corresponding mRNA in human thyroid gland. The aim of our study was to explore the expression of connexin 43 (Cx43) in the thyroid gland from normal and diseased human thyroid tissue by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). The expression levels of Cx43 in Grave's disease were significantly increased in comparison with those of normal thyroid tissue. There was a significant decrease in expression of Cx43 in Hashimoto's thyroiditis, compared with normal thyroid tissue. These data indicate that changes of Cx43 expression in human autoimmune thyroid disease were associated with variations in thyroid function and hormone secretion. 2009 Elsevier GmbH. All rights reserved.

Kindlin-2 Expression in Arsenite and Cadmium Transformed Bladder Cancer Cell Lines and in Archival Specimens of Human Bladder Cancer

PubMed Central

Talaat, Sherine; Somji, Seema; Toni, Conrad; Garrett, Scott H.; Zhou, Xu Dong; Sens, Mary Ann; Sens, Donald A.

2011-01-01

Objective The goal of this study was to confirm a microarray study that suggested that Kindlin-2 might play a role in the development and progression of bladder cancer. There has been no previous examination of Kindlin-2 expression in human bladder cancer. Methods A combination of real time PCR, western analysis and immunohistochemistry was used to characterize Kindlin-2 expression in arsenite (As+3) and cadmium (Cd+2) transformed human cell lines, their tumor transplants in immune-compromised mice, and in archival specimens of human bladder and bladder cancer. Results The results show that the Kindlin-2 expression patterns in the cell lines were not duplicated in the tumor tissues. However, it was shown that Kindlin-2 was expressed in the stromal element of all the transplanted tumors and archival specimens of human bladder cancer. It was also shown that a small number of high grade invasive urothelial cancers have focal expression of Kindlin-2 in the tumor cells. Conclusion Kindlin-2 is expressed in the stromal component of most, if not all, human bladder cancers. Kindlin-2 is not expressed in normal urothelium. Kindlin-2 is expressed in a small subset of high grade invasive bladder cancers and may have potential as a prognostic marker for tumor progression. PMID:21624607

Mena invasive (MenaINV) and Mena11a isoforms play distinct roles in breast cancer cell cohesion and association with TMEM

PubMed Central

Roussos, Evanthia T.; Goswami, Sumanta; Balsamo, Michele; Wang, Yarong; Stobezki, Robert; Adler, Esther; Robinson, Brian D.; Jones, Joan G.; Gertler, Frank B.; Condeelis, John S.; Oktay, Maja H.

2012-01-01

Mena, an actin regulatory protein, functions at the convergence of motility pathways that drive breast cancer cell invasion and migration in vivo. The tumor microenvironment spontaneously induces both increased expression of the MenaINV and decreased expression of Mena11a isoforms in invasive and migratory tumor cells. Tumor cells with this Mena expression pattern participate with macrophages in migration and intravasation in mouse mammary tumors in vivo. Consistent with these findings, anatomical sites containing tumor cells with high levels of Mena expression associated with perivascular macrophages were identified in human invasive ductal breast carcinomas and called TMEM. The number of TMEM sites positively correlated with the development of distant metastasis in humans. Here we demonstrate that mouse mammary tumors generated from EGFP-MenaINV expressing tumor cells are significantly less cohesive and have discontinuous cell-cell contacts compared to Mena11a xenografts. Using the mouse PyMT model we show that metastatic mammary tumors express 8.7 fold more total Mena and 7.5 fold more MenaINV mRNA than early non-metastatic ones. Furthermore, MenaINV expression in fine needle aspiration biopsy (FNA) samples of human invasive ductal carcinomas correlate with TMEM score while Mena11a does not. These results suggest that MenaINV is the isoform associated with breast cancer cell discohesion, invasion and intravasation in mice and in humans. They also imply that MenaINV expression and TMEM score measure related aspects of a common tumor cell dissemination mechanism and provide new insight into metastatic risk. PMID:21484349

Mena invasive (Mena(INV)) and Mena11a isoforms play distinct roles in breast cancer cell cohesion and association with TMEM.

PubMed

Roussos, Evanthia T; Goswami, Sumanta; Balsamo, Michele; Wang, Yarong; Stobezki, Robert; Adler, Esther; Robinson, Brian D; Jones, Joan G; Gertler, Frank B; Condeelis, John S; Oktay, Maja H

2011-08-01

Mena, an actin regulatory protein, functions at the convergence of motility pathways that drive breast cancer cell invasion and migration in vivo. The tumor microenvironment spontaneously induces both increased expression of the Mena invasive (Mena(INV)) and decreased expression of Mena11a isoforms in invasive and migratory tumor cells. Tumor cells with this Mena expression pattern participate with macrophages in migration and intravasation in mouse mammary tumors in vivo. Consistent with these findings, anatomical sites containing tumor cells with high levels of Mena expression associated with perivascular macrophages were identified in human invasive ductal breast carcinomas and called TMEM. The number of TMEM sites positively correlated with the development of distant metastasis in humans. Here we demonstrate that mouse mammary tumors generated from EGFP-Mena(INV) expressing tumor cells are significantly less cohesive and have discontinuous cell-cell contacts compared to Mena11a xenografts. Using the mouse PyMT model we show that metastatic mammary tumors express 8.7 fold more total Mena and 7.5 fold more Mena(INV) mRNA than early non-metastatic ones. Furthermore, Mena(INV) expression in fine needle aspiration biopsy (FNA) samples of human invasive ductal carcinomas correlate with TMEM score while Mena11a does not. These results suggest that Mena(INV) is the isoform associated with breast cancer cell discohesion, invasion and intravasation in mice and in humans. They also imply that Mena(INV) expression and TMEM score measure related aspects of a common tumor cell dissemination mechanism and provide new insight into metastatic risk.

Lysophosphatidic Acid Acyltransferase β (LPAATβ) Promotes the Tumor Growth of Human Osteosarcoma

PubMed Central

Rastegar, Farbod; Gao, Jian-Li; Shenaq, Deana; Luo, Qing; Shi, Qiong; Kim, Stephanie H.; Jiang, Wei; Wagner, Eric R.; Huang, Enyi; Gao, Yanhong; Shen, Jikun; Yang, Ke; He, Bai-Cheng; Chen, Liang; Zuo, Guo-Wei; Luo, Jinyong; Luo, Xiaoji; Bi, Yang; Liu, Xing; Li, Mi; Hu, Ning; Wang, Linyuan; Luther, Gaurav; Luu, Hue H.; Haydon, Rex C.; He, Tong-Chuan

2010-01-01

Background Osteosarcoma is the most common primary malignancy of bone with poorly characterized molecular pathways important in its pathogenesis. Increasing evidence indicates that elevated lipid biosynthesis is a characteristic feature of cancer. We sought to investigate the role of lysophosphatidic acid acyltransferase β (LPAATβ, aka, AGPAT2) in regulating the proliferation and growth of human osteosarcoma cells. LPAATβ can generate phosphatidic acid, which plays a key role in lipid biosynthesis as well as in cell proliferation and survival. Although elevated expression of LPAATβ has been reported in several types of human tumors, the role of LPAATβ in osteosarcoma progression has yet to be elucidated. Methodology/Principal Findings Endogenous expression of LPAATβ in osteosarcoma cell lines is analyzed by using semi-quantitative PCR and immunohistochemical staining. Adenovirus-mediated overexpression of LPAATβ and silencing LPAATβ expression is employed to determine the effect of LPAATβ on osteosarcoma cell proliferation and migration in vitro and osteosarcoma tumor growth in vivo. We have found that expression of LPAATβ is readily detected in 8 of the 10 analyzed human osteosarcoma lines. Exogenous expression of LPAATβ promotes osteosarcoma cell proliferation and migration, while silencing LPAATβ expression inhibits these cellular characteristics. We further demonstrate that exogenous expression of LPAATβ effectively promotes tumor growth, while knockdown of LPAATβ expression inhibits tumor growth in an orthotopic xenograft model of human osteosarcoma. Conclusions/Significance Our results strongly suggest that LPAATβ expression may be associated with the aggressive phenotypes of human osteosarcoma and that LPAATβ may play an important role in regulating osteosarcoma cell proliferation and tumor growth. Thus, targeting LPAATβ may be exploited as a novel therapeutic strategy for the clinical management of osteosarcoma. This is especially attractive given the availability of selective pharmacological inhibitors. PMID:21152068

Mind-controlled transgene expression by a wireless-powered optogenetic designer cell implant.

PubMed

Folcher, Marc; Oesterle, Sabine; Zwicky, Katharina; Thekkottil, Thushara; Heymoz, Julie; Hohmann, Muriel; Christen, Matthias; Daoud El-Baba, Marie; Buchmann, Peter; Fussenegger, Martin

2014-11-11

Synthetic devices for traceless remote control of gene expression may provide new treatment opportunities in future gene- and cell-based therapies. Here we report the design of a synthetic mind-controlled gene switch that enables human brain activities and mental states to wirelessly programme the transgene expression in human cells. An electroencephalography (EEG)-based brain-computer interface (BCI) processing mental state-specific brain waves programs an inductively linked wireless-powered optogenetic implant containing designer cells engineered for near-infrared (NIR) light-adjustable expression of the human glycoprotein SEAP (secreted alkaline phosphatase). The synthetic optogenetic signalling pathway interfacing the BCI with target gene expression consists of an engineered NIR light-activated bacterial diguanylate cyclase (DGCL) producing the orthogonal second messenger cyclic diguanosine monophosphate (c-di-GMP), which triggers the stimulator of interferon genes (STING)-dependent induction of synthetic interferon-β promoters. Humans generating different mental states (biofeedback control, concentration, meditation) can differentially control SEAP production of the designer cells in culture and of subcutaneous wireless-powered optogenetic implants in mice.

Protein expression of preferred human codon-optimized Gaussia luciferase genes with an artificial open-reading frame in mammalian and bacterial cells.

PubMed

Inouye, Satoshi; Suzuki, Takahiro

2016-12-01

The protein expressions of three preferred human codon-optimized Gaussia luciferase genes (pGLuc, EpGLuc, and KpGLuc) were characterized in mammalian and bacterial cells by comparing them with those of wild-type Gaussia luciferase gene (wGLuc) and human codon-optimized Gaussia luciferase gene (hGLuc). Two synthetic genes of EpGLuc and KpGLuc containing the complete preferred human codons have an artificial open-reading frame; however, they had the similar protein expression levels to those of pGLuc and hGLuc in mammalian cells. In bacterial cells, the protein expressions of pGLuc, EpGLuc, and KpGLuc with approximately 65% GC content were the same and showed approximately 60% activities of wGLuc and hGLuc. The artificial open-reading frame in EpGLuc and KpGLuc did not affect the protein expression in mammalian and bacterial cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Cysteinyl leukotrienes C4 and D4 downregulate human mast cell expression of toll-like receptors 1 through 7.

PubMed

Karpov, V; Ilarraza, R; Catalli, A; Kulka, M

2018-01-01

Cysteinyl leukotrienes (CysLT) are potent inflammatory lipid molecules that mediate some of the pathophysiological responses associated with asthma such as bronchoconstriction, vasodilation and increased microvascular permeability. As a result, CysLT receptor antagonists (LRA), such as montelukast, have been used to effectively treat patients with asthma. We have recently shown that mast cells are necessary modulators of innate immune responses to bacterial infection and an important component of this innate immune response may involve the production of CysLT. However, the effect of LRA on innate immune receptors, particularly on allergic effector cells, is unknown. This study determined the effect of CysLT on toll-like receptor (TLR) expression by the human mast cell line LAD2. Real-time PCR analysis determined that LTC4, LTD4 and LTE4 downregulated mRNA expression of several TLR. Specifically in human CD34+-derived human mast cells (HuMC), LTC4 inhibited expression of TLR1, 2, 4, 5, 6 and 7 while LTD4 inhibited expression of TLR1-7. Montelukast blocked LTC4-mediated downregulation of all TLR, suggesting that these effects were mediated by activation of the CysLT1 receptor (CysLT1R). Flow cytometry analysis confirmed that LTC4 downregulated surface expression of TLR2 which was blocked by montelukast. These data show that CysLT can modulate human mast cell expression of TLR and that montelukast may be beneficial for innate immune responses mediated by mast cells.

The expression patterns of pro-apoptotic and anti-apoptotic factors in human fetal and adult ovary.

PubMed

Poljicanin, Ana; Vukusic Pusic, Tanja; Vukojevic, Katarina; Caric, Ana; Vilovic, Katarina; Tomic, Snjezana; Soljic, Violeta; Saraga-Babic, Mirna

2013-07-01

The influence of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins on the cell death (caspase-3, TUNEL) of different ovarian cell lineages was immunohistochemically analyzed in six fetal and five adult human ovaries in order to disclose possible mechanisms of cell number control. Mild to moderate expression of Bcl-2 characterized ovarian surface epithelium, follicular cells and oocytes of 15 and 22 week human ovaries, while expression of Bax and caspase-3 gradually increased in all ovarian cell populations, except caspase-3 in the ovarian surface epithelium. Different levels of Bax and Bcl-2 proteins co-expression characterized fetal ovarian cells, while TUNEL and caspase-3 co-expression was found only in some of them. In adult ovaries, Bcl-2 was moderately and Bax strongly expressed in the surface ovarian epithelium and stroma. Bcl-2 and Bax expression in granulosa and theca interna cells varied depending on the stage of follicular atresia. Caspase-3 apoptotic cells characterized granulosa cells of adult atretic follicles. Our results indicate that intracellular levels of Bcl-2 and Bax protein might regulate the final destiny of developing germ cells. Caspase-3 dependent apoptosis seems to be the most important, but not the only cell death pathway in ovaries. In adult ovaries, caspase-dependent cell death characterized granulosa cells, but not the germ cells. Copyright © 2012 Elsevier GmbH. All rights reserved.

Human CD22 Inhibits Murine B Cell Receptor Activation in a Human CD22 Transgenic Mouse Model.

PubMed

Bednar, Kyle J; Shanina, Elena; Ballet, Romain; Connors, Edward P; Duan, Shiteng; Juan, Joana; Arlian, Britni M; Kulis, Michael D; Butcher, Eugene C; Fung-Leung, Wai-Ping; Rao, Tadimeti S; Paulson, James C; Macauley, Matthew S

2017-11-01

CD22, a sialic acid-binding Ig-type lectin (Siglec) family member, is an inhibitory coreceptor of the BCR with established roles in health and disease. The restricted expression pattern of CD22 on B cells and most B cell lymphomas has made CD22 a therapeutic target for B cell-mediated diseases. Models to better understand how in vivo targeting of CD22 translates to human disease are needed. In this article, we report the development of a transgenic mouse expressing human CD22 (hCD22) in B cells and assess its ability to functionally substitute for murine CD22 (mCD22) for regulation of BCR signaling, Ab responses, homing, and tolerance. Expression of hCD22 on transgenic murine B cells is comparable to expression on human primary B cells, and it colocalizes with mCD22 on the cell surface. Murine B cells expressing only hCD22 have identical calcium (Ca 2+ ) flux responses to anti-IgM as mCD22-expressing wild-type B cells. Furthermore, hCD22 transgenic mice on an mCD22 -/- background have restored levels of marginal zone B cells and Ab responses compared with deficiencies observed in CD22 -/- mice. Consistent with these observations, hCD22 transgenic mice develop normal humoral responses in a peanut allergy oral sensitization model. Homing of B cells to Peyer's patches was partially rescued by expression of hCD22 compared with CD22 -/- B cells, although not to wild-type levels. Notably, Siglec-engaging antigenic liposomes formulated with an hCD22 ligand were shown to prevent B cell activation, increase cell death, and induce tolerance in vivo. This hCD22 transgenic mouse will be a valuable model for investigating the function of hCD22 and preclinical studies targeting hCD22. Copyright © 2017 by The American Association of Immunologists, Inc.

Induction of chemokine receptor CXCR4 expression by transforming growth factor-β1 in human basal cell carcinoma cells.

PubMed

Chu, Chia-Yu; Sheen, Yi-Shuan; Cha, Shih-Ting; Hu, Yeh-Fang; Tan, Ching-Ting; Chiu, Hsien-Ching; Chang, Cheng-Chi; Chen, Min-Wei; Kuo, Min-Liang; Jee, Shiou-Hwa

2013-11-01

Higher CXCR4 expression enhances basal cell carcinoma (BCC) invasion and angiogenesis. The underlying mechanism of increased CXCR4 expression in invasive BCC is still not well understood. To investigate the mechanisms involved in the regulation of CXCR4 expression in invasive BCC. We used qRT-PCR, RT-PCR, Western blot, and flow cytometric analyses to examine different CXCR4 levels among the clinical samples, co-cultured BCC cells and BCC cells treated with recombinant transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF). Immunohistochemical studies were used to demonstrate the correlation between TGF-β1 and CXCR4 expressions. The signal transduction pathway and transcriptional regulation were confirmed by treatments with chemical inhibitors, neutralizing antibodies, or short interfering RNAs, as well as luciferase reporter activity. Invasive BCC has higher TGF-β1 and CTGF levels compared to non-invasive BCC. Non-contact dermal fibroblasts co-culture with human BCC cells also increases the expression of CXCR4 in BCC cells. Treatment with recombinant human TGF-β1, but not CTGF, enhanced the CXCR4 levels in time- and dose-dependent manners. The protein level and surface expression of CXCR4 in human BCC cells was increased by TGF-β1 treatment. TGF-β1 was intensely expressed in the surrounding fibroblasts of invasive BCC and was positively correlated with the CXCR4 expression of BCC cells. The transcriptional regulation of CXCR4 by TGF-β1 is mediated by its binding to the TGF-β receptor II and phosphorylation of the extracellular signal-related kinase 1/2 (ERK1/2)-ETS-1 pathway. TGF-β1 induces upregulation of CXCR4 in human BCC cells by phosphorylation of ERK1/2-ETS-1 pathway. Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies.

PubMed

Tsukahara, T; Iwase, N; Kawakami, K; Iwasaki, M; Yamamoto, C; Ohmine, K; Uchibori, R; Teruya, T; Ido, H; Saga, Y; Urabe, M; Mizukami, H; Kume, A; Nakamura, M; Brentjens, R; Ozawa, K

2015-02-01

Engineered T-cell therapy using a CD19-specific chimeric antigen receptor (CD19-CAR) is a promising strategy for the treatment of advanced B-cell malignancies. Gene transfer of CARs to T-cells has widely relied on retroviral vectors, but transposon-based gene transfer has recently emerged as a suitable nonviral method to mediate stable transgene expression. The advantages of transposon vectors compared with viral vectors include their simplicity and cost-effectiveness. We used the Tol2 transposon system to stably transfer CD19-CAR into human T-cells. Normal human peripheral blood lymphocytes were co-nucleofected with the Tol2 transposon donor plasmid carrying CD19-CAR and the transposase expression plasmid and were selectively propagated on NIH3T3 cells expressing human CD19. Expanded CD3(+) T-cells with stable and high-level transgene expression (~95%) produced interferon-γ upon stimulation with CD19 and specifically lysed Raji cells, a CD19(+) human B-cell lymphoma cell line. Adoptive transfer of these T-cells suppressed tumor progression in Raji tumor-bearing Rag2(-/-)γc(-/-) immunodeficient mice compared with control mice. These results demonstrate that the Tol2 transposon system could be used to express CD19-CAR in genetically engineered T-cells for the treatment of refractory B-cell malignancies.

Iodine-131 induces apoptosis in human cardiac muscle cells through the p53/Bax/caspase-3 and PIDD/caspase-2/ t‑BID/cytochrome c/caspase-3 signaling pathway.

PubMed

Wang, Yansheng; Liu, Changqing; Wang, Jianchun; Zhang, Yang; Chen, Linlin

2017-09-01

The aim of this study was to elucidate the effects of iodine-131 on the induction of apoptosis in human cardiac muscle cells and the underlying molecular mechanisms. We found that iodine-131 reduced cell proliferation, induced apoptosis, induced p53, PIDD, t-BID (mitochondria) protein expression, suppressed cytochrome c (mitochondria) protein expression, and increased Bax protein expression, and promoted caspase-2, -3 and -9 expression levels in human cardiac muscle cells. Meanwhile, si-p53 inhibited the effects of iodine-131 on the reduction in cell proliferation and induction of apoptosis in human cardiac muscle cells through regulation of Bax/cytochrome c/caspase-3 and PIDD/caspase‑2/t-BID/cytochrome c/caspase-3 signaling pathway. After si-Bax reduced the effects of iodine-131, it reduced cell proliferation and induced apoptosis in human cardiac muscle cells through the cytochrome c/caspase-3 signaling pathway. However, si-caspase-2 also reduced the effects of iodine-131 on the reduction of cell proliferation and induction of apoptosis in human cardiac muscle cells through the t-BID/cytochrome c/caspase-3 signaling pathway. These findings demonstrated that iodine-131 induces apoptosis in human cardiac muscle cells through the p53/Bax/caspase-3 and PIDD/caspase-2/t-BID/cytochrome c/caspase-3 signaling pathway.

Matched rabbit monoclonal antibodies against αv-series integrins reveal a novel αvβ3-LIBS epitope, and permit routine staining of archival paraffin samples of human tumors

PubMed Central

Goodman, Simon L.; Grote, Hans Juergen; Wilm, Claudia

2012-01-01

Summary The relationship between integrin expression and function in pathologies is often contentious as comparisons between human pathological expression and expression in cell lines is difficult. In addition, the expression of even integrins αvβ6 and αvβ8 in tumor cell lines is not comprehensively documented. Here, we describe rabbit monoclonal antibodies (RabMabs) against the extracellular domains of αv integrins that react with both native integrins and formalin fixed, paraffin embedded (FFPE) human tissues. These RabMabs, against αvβ3 (EM22703), αvβ5 (EM09902), αvβ6 (EM05201), αvβ8 (EM13309), and pan-αv (EM01309), recognize individual integrin chains in Western blots and in flow cytometry. EM22703 detected a ligand-induced binding site (LIBS), reporting an epitope enhanced by the binding of an RGD-peptide to αvβ3. αvβ8 was rarely expressed in human tumor specimens, and weakly expressed in non-small-cell lung carcinoma (NSCLC). However, ovarian carcinoma cell lines expressed αvβ8, as did some melanoma cells, whereas U87MG glioma lacked αvβ8 expression. We observed an unexpected strong expression of αvβ6 in tumor samples of invasive ductal breast adenoma, colorectal carcinoma (CRC), and NSCLC. αvβ3 was strongly expressed in some invasive NSCLC cohorts. Interestingly, PC3 prostate cell and human prostate tumors did not express αvβ3. The RabMabs stained plasma membranes in FFPE-immunohistochemistry (IHC) samples of tumor cell lines from lung, ovary, colon, prostate, squamous cell carcinoma of head and neck (SCCHN), breast, and pancreas carcinomas. The RabMabs are unique tools for probing αv integrin biology, and suggest that especially αvβ6 and αvβ8 biologies still have much to reveal. PMID:23213423

Human Induced Pluripotent Stem Cell-Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation.

PubMed

Sharmin, Sazia; Taguchi, Atsuhiro; Kaku, Yusuke; Yoshimura, Yasuhiro; Ohmori, Tomoko; Sakuma, Tetsushi; Mukoyama, Masashi; Yamamoto, Takashi; Kurihara, Hidetake; Nishinakamura, Ryuichi

2016-06-01

Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator-like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in vitro These induced human podocytes exhibited apicobasal polarity, with nephrin proteins accumulated close to the basal domain, and possessed primary processes that were connected with slit diaphragm-like structures. Microarray analysis of sorted iPS cell-derived podocytes identified well conserved marker gene expression previously shown in mouse and human podocytes in vivo Furthermore, we developed a novel transplantation method using spacers that release the tension of host kidney capsules, thereby allowing the effective formation of glomeruli from human iPS cell-derived nephron progenitors. The human glomeruli were vascularized with the host mouse endothelial cells, and iPS cell-derived podocytes with numerous cell processes accumulated around the fenestrated endothelial cells. Therefore, the podocytes generated from iPS cells retain the podocyte-specific molecular and structural features, which will be useful for dissecting human glomerular development and diseases. Copyright © 2016 by the American Society of Nephrology.

Differential expression of extracellular-signal-regulated kinase 5 (ERK5) in normal and degenerated human nucleus pulposus tissues and cells

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

Liang, Weiguo, E-mail: liangweiguo@tom.com; Fang, Dejian; Ye, Dongping

2014-07-11

Highlights: • ERK5 involved in NP cells. • ERK5 involved in NP tissue. • It was important modulator. - Abstract: Extracellular-signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family and regulates a wide variety of cellular processes such as proliferation, differentiation, necrosis, apoptosis and degeneration. However, the expression of ERK5 and its role in degenerated human nucleus pulposus (NP) is hitherto unknown. In this study, we observed the differential expression of ERK5 in normal and degenerated human nucleus pulposus tissues by using immunohistochemical staining and Western blot. Treatment of NP cells with Pro-inflammatory cytokine, TNF-αmore » decreased ERK5 gene expression as well as NP marker gene expression; including the type II collagen and aggrecan. Suppression of ERK5 gene expression in NP cells by ERK5 siRNA resulted in decreased gene expression of type II collagen and aggrecan. Furthermore, inhibition of ERK5 activation by BIX02188 (5 μM) decreased the gene expression of type II collagen and aggrecan in NP cells. Our results document the expression of ERK5 in degenerated nucleus pulposus tissues, and suggest a potential involvement of ERK5 in human degenerated nucleus pulposus.« less

HIF-1α activates hypoxia-induced BCL-9 expression in human colorectal cancer cells

PubMed Central

Chen, Tian-Rui; Wei, Hai-feng; Song, Dian-Wen; Liu, Tie-Long; Yang, Xing-Hai; Fu, Chuan-Gang; Hu, Zhi-qian; Zhou, Wang; Yan, Wang-Jun; Xiao, Jian-Ru

2017-01-01

B-cell CLL/lymphoma 9 protein (BCL-9), a multi-functional co-factor in Wnt signaling, induced carcinogenesis as well as promoting tumor progression, metastasis and chemo-resistance in colorectal cancer (CRC). However, the mechanisms for increased BCL-9 expression in CRC were not well understood. Here, we report that hypoxia, a hallmark of solid tumors, induced BCL-9 mRNA expression in human CRC cells. Analysis of BCL-9 promoter revealed two functional hypoxia-responsive elements (HRE-B and HRE-C) that can be specifically bound with and be transactivated by hypoxia inducible factors (HIF) -1α but not HIF-2α. Consistently, ectopic expression of HIF-1α but not HIF-2α transcriptionally induced BCL-9 expression levels in cells. Knockdown of endogenous HIF-1α but not HIF-2α by siRNA largely abolished the induction of HIF by hypoxia. Furthermore, there was a strong association of HIF-1α expression with BCL-9 expression in human CRC specimens. In summary, results from this study demonstrated that hypoxia induced BCL-9 expression in human CRC cells mainly through HIF-1α, which could be an important underlying mechanism for increased BCL-9 expression in CRC. PMID:27121066

Knockdown of the schizophrenia susceptibility gene TCF4 alters gene expression and proliferation of progenitor cells from the developing human neocortex.

PubMed

Hill, Matthew J; Killick, Richard; Navarrete, Katherinne; Maruszak, Aleksandra; McLaughlin, Gemma M; Williams, Brenda P; Bray, Nicholas J

2017-05-01

Common variants in the TCF4 gene are among the most robustly supported genetic risk factors for schizophrenia. Rare TCF4 deletions and loss-of-function point mutations cause Pitt-Hopkins syndrome, a developmental disorder associated with severe intellectual disability. To explore molecular and cellular mechanisms by which TCF4 perturbation could interfere with human cortical development, we experimentally reduced the endogenous expression of TCF4 in a neural progenitor cell line derived from the developing human cerebral cortex using RNA interference. Effects on genome-wide gene expression were assessed by microarray, followed by Gene Ontology and pathway analysis of differentially expressed genes. We tested for genetic association between the set of differentially expressed genes and schizophrenia using genome-wide association study data from the Psychiatric Genomics Consortium and competitive gene set analysis (MAGMA). Effects on cell proliferation were assessed using high content imaging. Genes that were differentially expressed following TCF4 knockdown were highly enriched for involvement in the cell cycle. There was a nonsignificant trend for genetic association between the differentially expressed gene set and schizophrenia. Consistent with the gene expression data, TCF4 knockdown was associated with reduced proliferation of cortical progenitor cells in vitro. A detailed mechanistic explanation of how TCF4 knockdown alters human neural progenitor cell proliferation is not provided by this study. Our data indicate effects of TCF4 perturbation on human cortical progenitor cell proliferation, a process that could contribute to cognitive deficits in individuals with Pitt-Hopkins syndrome and risk for schizophrenia.

Identification of prostaglandin receptors in human ureters.

PubMed

Oll, Matthias; Baumann, Claudia; Behbahani, Turang E; von Ruecker, Alexander; Müller, Stefan C; Ellinger, Jörg

2012-12-10

Prostaglandins play an important role in ureteral obstruction, but the detailed expression profiles of the prostaglandin receptors (PTGER1, PTGER2, PTGER3, PTGER4, PTGFR) remain unknown in the different parts of the human ureter. The expression pattern of PTGER1, PTGER2, PTGER3, PTGER4 and PTGFR was determined in human distal, mid and proximal ureter and renal pelvis samples using immunohistochemistry (protein levels) and quantitative real-time PCR (mRNA). PTGER1 was highly expressed in most samples irrespective of the ureteral localization; however, urothelial cells had higher levels of PTGER1 than smooth muscle cells. PTGFR was also moderately to strongly expressed in urothelial and smooth muscle cells. In comparison, PTGER2-4 expression was mostly unexpressed or weakly expressed in urothelial and smooth cells in all regions. Our data indicate high levels of PTGER1 in ureters.

Downregulation of the c-Fes protein-tyrosine kinase inhibits the proliferation of human renal carcinoma cells

PubMed Central

Kanda, Shigeru; Miyata, Yasuyoshi; Kanetake, Hiroshi; Smithgall, Thomas E.

2009-01-01

The c-Fes protein-tyrosine kinase is associated with growth and differentiation of hematopoietic, neuronal, vascular endothelial and epithelial cell types. In this study, we investigated whether small interfering RNA (siRNA)-mediated knockdown of c-Fes expression affected proliferation of the human renal carcinoma cell lines, ACHN and VMRC-RCW. Immunofluorescence microscopy showed that c-Fes was expressed in both the cytosol and nuclei of these cells, and siRNA treatment preferentially downregulated c-Fes expression in the cytosol. Knock-down of c-Fes inhibited cellular proliferation in a dose-dependent manner with minimal increase in cell death. c-Fes siRNA treatment also downregulated the phosphorylation of Akt1 on S473 and IKKα on T23, and cyclin D1 expression, enhanced the expression of IκBα, and prevented the nuclear localization of NFκB. Treatment with an NFκB inhibitory peptide (SN50) also blocked the proliferation and nuclear localization of NFκB in these cells. The effect of SN50 treatment was not enhanced by c-Fes siRNA, suggesting that downregulation of c-Fes expression inhibited cell cycle progression through the Akt1/NFκB pathway. In contrast to siRNA-mediated knockdown, ectopic expression of either wild-type or kinase-inactive c-Fes in renal carcinoma cells failed to alter their proliferation in vitro and in vivo. Thus, suppression of proliferation resulting from siRNA-mediated knockdown may depend upon an expression of c-Fes protein rather than its kinase activity. Taken together, our results indicate that downregulation of c-Fes expression may be a potential therapeutic strategy for advanced human renal cell carcinoma and inhibition of its kinase activity as an antiangiogenic therapy does not seem to induce the growth of human renal carcinoma cells. PMID:19082481

Aqueous Humor Rapidly Stimulates Myocilin Secretion from Human Trabecular Meshwork Cells

PubMed Central

Resch, Zachary T.; Hann, Cheryl R.; Cook, Kimberly A.; Fautsch, Michael P.

2010-01-01

Myocilin, a protein associated with the development of glaucoma, is expressed in most eye tissues with highest expression observed in trabecular meshwork cells. In culture, primary human trabecular meshwork cells incubated in 10% fetal bovine serum have reduced myocilin expression compared to in vivo, but incubation in human aqueous humor, their normal in vivo nutrient source, restores myocilin expression to near in vivo levels. To investigate the mechanism by which human aqueous humor stimulates myocilin accumulation in conditioned media from normal human trabecular meshwork cells, three independent trabecular meshwork cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing various supplements: fetal bovine serum (10%), human serum (0.2%), porcine aqueous humor (50%), bovine serum albumin (0.1%), dexamethasone (10−7 M), human aqueous humor (50%) or heat-inactivated human aqueous humor (50%). Conditioned media from cultured primary trabecular meshwork cells following incubation in human aqueous humor showed significant accumulation of myocilin in a time- (15 minutes) and dose-dependent manner (half maximal effective concentration ~ 30%) while intracellular myocilin levels decreased. Minimal myocilin accumulation was observed in conditioned media isolated from trabecular meshwork cells cultured in DMEM containing fetal bovine or human serum, bovine serum albumin, porcine aqueous humor, dexamethasone or DMEM alone. Heat inactivation of human aqueous humor nearly eliminated human aqueous humor-stimulated myocilin secretion. Inhibitors of new protein synthesis, gene transcription, the endoplasmic reticulum/Golgi system and endocytic/exocytic secretory pathways failed to inhibit human aqueous humor-stimulated myocilin secretion. Using immunolabeling and transmission electron microscopy, myocilin was found associated with 70–90 nm vesicle-like structures within the cytoplasm of human aqueous humor treated trabecular meshwork cells. These studies suggest that myocilin secretion from trabecular meshwork cells occurs in a Golgi-independent manner following human aqueous humor treatment. Heat-labile factors in human aqueous humor are responsible for the time- and dose-dependent release of myocilin from vesicle-like structures within the cytoplasm of trabecular meshwork cells. PMID:20932969

Functional importance of GLP-1 receptor species and expression levels in cell lines.

PubMed

Knudsen, Lotte Bjerre; Hastrup, Sven; Underwood, Christina Rye; Wulff, Birgitte Schjellerup; Fleckner, Jan

2012-04-10

Of the mammalian species, only the GLP-1 receptors of rat and human origin have been described and characterized. Here, we report the cloning of the homologous GLP-1 receptors from mouse, rabbit, pig, cynomolgus monkey and chimp. The GLP-1 receptor is highly conserved across species, thus underlining the physiological importance of the peptide hormone and its receptor across a wide range of mammals. We expressed the receptors by stable transfection of BHK cells, both in cell lines with high expression levels of the cloned receptors, as well as in cell lines with lower expression levels, more comparable to endogenous expression of these receptors. High expression levels of cloned GLP-1 receptors markedly increased the potency of GLP-1 and other high affinity ligands, whereas the K(d) values were not affected. For a low affinity ligand like the ago-allosteric modulator Compound 2, expression levels of the human GLP-1 receptor were important for maximal efficacy as well as potency. The two natural metabolites of GLP-1, GLP-1(9-37) and GLP-1(9-36)amide were agonists when tested on a cell line with high expression of the recombinant human GLP-1 receptor, whereas they behaved as (low potent) antagonists on a cell line that expressed the receptor endogenously, as well as cells expressing a moderate level of the recombinant human GLP-1 receptor. The amide form was a more potent agonist than the free acid from. In conclusion, receptor expression level is an important parametre for selecting cell lines with cloned GLP-1 receptors for functional characterization of physiological and pharmaceutical ligands. Copyright © 2011 Elsevier B.V. All rights reserved.

METABOLISM AND TOXICITY OF AS IN HUMAN UROTHELIAL CELLS EXPRESSING RAT ARSENIC (+3 OXIDATION STATE)-METHYLTRANSFERASE

EPA Science Inventory

The enzymatic methylation of inorganic As (iAs) is catalyzed by As(+3 oxidation state)-methyltransferase (AS3MT). AS3MT is expressed in rat liver and in human hepatocytes However, AS3MT is not expressed in UROtsa, human urothelial cells that do not methylate iAs. Thus, UROtsa ce...

TGF-β induces surface LAP expression on murine CD4 T cells independent of Foxp3 induction.

PubMed

Oida, Takatoku; Weiner, Howard L

2010-11-24

It has been reported that human FOXP3(+) CD4 Tregs express GARP-anchored surface latency-associated peptide (LAP) after activation, based on the use of an anti-human LAP mAb. Murine CD4 Foxp3(+) Tregs have also been reported to express surface LAP, but these studies have been hampered by the lack of suitable anti-mouse LAP mAbs. We generated anti-mouse LAP mAbs by immunizing TGF-β(-/-) animals with a mouse Tgfb1-transduced P3U1 cell line. Using these antibodies, we demonstrated that murine Foxp3(+) CD4 Tregs express LAP on their surface. In addition, retroviral transduction of Foxp3 into mouse CD4(+)CD25(-) T cells induced surface LAP expression. We then examined surface LAP expression after treating CD4(+)CD25(-) T cells with TGF-β and found that TGF-β induced surface LAP not only on T cells that became Foxp3(+) but also on T cells that remained Foxp3(-) after TGF-β treatment. GARP expression correlated with the surface LAP expression, suggesting that surface LAP is GARP-anchored also in murine T cells. Unlike human CD4 T cells, surface LAP expression on mouse CD4 T cells is controlled by Foxp3 and TGF-β. Our newly described anti-mouse LAP mAbs will provide a useful tool for the investigation and functional analysis of T cells that express LAP on their surface.

Human cells: new platform for recombinant therapeutic protein production.

PubMed

Swiech, Kamilla; Picanço-Castro, Virgínia; Covas, Dimas Tadeu

2012-07-01

The demand for recombinant therapeutic proteins is significantly increasing. There is a constant need to improve the existing expression systems, and also developing novel approaches to face the therapeutic proteins demands. Human cell lines have emerged as a new and powerful alternative for the production of human therapeutic proteins because this expression system is expected to produce recombinant proteins with post translation modifications more similar to their natural counterpart and reduce the potential immunogenic reactions against nonhuman epitopes. Currently, little information about the cultivation of human cells for the production of biopharmaceuticals is available. These cells have shown efficient production in laboratory scale and represent an important tool for the pharmaceutical industry. This review presents the cell lines available for large-scale recombinant proteins production and evaluates critically the advantages of this expression system in comparison with other expression systems for recombinant therapeutic protein production. Copyright © 2012 Elsevier Inc. All rights reserved.

Characterization of the human oncogene SCL/TAL1 interrupting locus (Stil) mediated Sonic hedgehog (Shh) signaling transduction in proliferating mammalian dopaminergic neurons

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

Sun, Lei; Department of Physiology, Nankai University School of Medicine, Tianjin 300071; Carr, Aprell L.

2014-07-11

Highlights: • Stil is a human oncogene that is conserved in vertebrate species. • Stil functions in the Shh pathway in mammalian cells. • The expression of Stil is required for mammalian dopaminergic cell proliferation. - Abstract: The human oncogene SCL/TAL1 interrupting locus (Stil) is highly conserved in all vertebrate species. In humans, the expression of Stil is involved in cancer cell survival, apoptosis and proliferation. In this research, we investigated the roles of Stil expression in cell proliferation of mammalian dopaminergic (DA) PC12 cells. Stil functions through the Sonic hedgehog (Shh) signal transduction pathway. Co-immunoprecipitation tests revealed that STILmore » interacts with Shh downstream components, which include SUFU and GLI1. By examining the expression of Stil, Gli1, CyclinD2 (cell-cycle marker) and PCNA (proliferating cell nuclear antigen), we found that up-regulation of Stil expression (transfection with overexpression plasmids) increased Shh signaling transduction and PC12 cell proliferation, whereas down-regulation of Stil expression (by shRNA) inhibited Shh signaling transduction, and thereby decreased PC12 cell proliferation. Transient transfection of PC12 cells with Stil knockdown or overexpression plasmids did not affect PC12 cell neural differentiation, further indicating the specific roles of Stil in cell proliferation. The results from this research suggest that Stil may serve as a bio-marker for neurological diseases involved in DA neurons, such as Parkinson’s disease.« less

Human endogenous retrovirus rec interferes with germ cell development in mice and may cause carcinoma in situ, the predecessor lesion of germ cell tumors.

PubMed

Galli, Uwe M; Sauter, Marlies; Lecher, Bernd; Maurer, Simone; Herbst, Hermann; Roemer, Klaus; Mueller-Lantzsch, Nikolaus

2005-04-28

Germ cell tumors (GCTs) are among the most common malignancies in young men. We have previously documented that patients with GCT frequently produce serum antibodies directed against proteins encoded by human endogenous retrovirus (HERV) type K sequences. Transcripts originating from the env gene of HERV-K, including the rec-relative of human immunodeficiency virus rev, are highly expressed in GCTs. We report here that mice that inducibly express HERV-K rec show a disturbed germ cell development and may exhibit, by 19 months of age, changes reminiscent of carcinoma in situ, the predecessor lesion of classic seminoma in humans. This provides the first direct evidence that the expression of a human endogenous retroviral gene previously established as a marker in human germ cell tumors may contribute to organ-specific tumorigenesis in a transgenic mouse model.

HIF-2α mediates hypoxia-induced LIF expression in human colorectal cancer cells

PubMed Central

Zhao, Yuhan; Zhang, Cen; Wang, Jiabei; Yue, Xuetian; Yang, Qifeng; Hu, Wenwei

2015-01-01

Leukemia inhibitory factor (LIF), a multi-functional cytokine, has a complex role in cancer. While LIF induces the differentiation of several myeloid leukemia cells and inhibits their growth, it also promotes tumor progression, metastasis and chemoresistance in many solid tumors. LIF is frequently overexpressed in a variety of human tumors and its overexpression is often associated with poor prognosis of patients. Currently, the mechanism for LIF overexpression in tumor cells is not well-understood. Here, we report that hypoxia, a hallmark of solid tumors, induced LIF mRNA expression in human colorectal cancer cells. Analysis of LIF promoter revealed several hypoxia-responsive elements (HREs) that can specifically interact with and be transactivated by HIF-2α but not HIF-1α. Consistently, ectopic expression of HIF-2α but not HIF-1α transcriptionally induced LIF expression levels in cells. Knockdown of endogenous HIF-2α but not HIF-1α by siRNA largely abolished the induction of LIF by hypoxia in cells. Furthermore, there is a strong association of HIF-2α overexpression with LIF overexpression in human colorectal cancer specimens. In summary, results from this study demonstrate that hypoxia induces LIF expression in human cancer cells mainly through HIF-2α, which could be an important underlying mechanism for LIF overexpression in human cancers. PMID:25726527

Expression of Eph receptor tyrosine kinases and their ligands in human Granulosa lutein cells and human umbilical vein endothelial cells.

PubMed

Xu, Y; Zagoura, D; Keck, C; Pietrowski, D

2006-11-01

Corpus luteum development is regulated by gonadotropins and accompanied by extremely rapid vascularization of the avascular granulosa cell compartiment by endothelial cells (EC). The proliferation of Granulosa cells (GC) and EC is a complex interplay and takes place in a spatially and temporarily coordinated manner. The erythropoietin-producing hepatoma amplified sequence (Eph) receptors and their ligands-the ephrins- are a recently detected family of membrane located protein tyrosine kinases which play a crucial role in the growth and development of nerve and blood vessel network. We report about the mRNA expression pattern of Ephs and their ligands in human GC, in human EC, and in carcinoma cell lines OvCar-3 and Hela. The mRNA of EphA4, EphA7, ephrinA4, ephrinB1 and ephrinB2 was detected in GC and EC, while EphA2 was expressed only in GC. The expression of various Ephs and ephrins did not change in GC after stimulation with human chorion gonadotropin. Our study analyzes for the first time the expression of the complete human Eph/ephriny-system in GC and in EC. The remarkable similarity between these two cell types supports the theory of a functional relationship of EC and GC. In addition, it was shown that hCG is not a major determinant of Eph/ephrin regulation in GC.

Expression of Inflammation-related Intercellular Adhesion Molecules in Cardiomyocytes In Vitro and Modulation by Pro-inflammatory Agents.

PubMed

El-Battrawy, Ibrahim; Tülümen, Erol; Lang, Siegfried; Akin, Ibrahim; Behnes, Michael; Zhou, Xiabo; Mavany, Martin; Bugert, Peter; Bieback, Karen; Borggrefe, Martin; Elmas, Elif

2016-01-01

Cell-surface adhesion molecules regulate multiple intercellular and intracellular processes and play important roles in inflammation by facilitating leukocyte endothelial transmigration. Whether cardiomyocytes express surface-adhesion molecules related to inflammation and the effect of pro-inflammatory mediators remain unknown. In the present study, the expression of different cell-adhesion molecules (CD11a, CD11b, CD31, CD62P, CD162, F11 receptor and mucosal vascular addressin cell adhesion molecule 1 (MADCAM1)) and the effect of pro-inflammatory mediators were investigated in an in vitro model of human cardiomyocytes. Cells were supplied as a primary culture of cardiac alpha actin-positive cells from human heart tissue. The cells were incubated for 24 h with 1 U/ml thrombin or 700 ng/ml lipopolysaccharide (LPS) or with a combination of both. The expression of the cell adhesion molecules was measured by flow cytometry. In cultured human cardiomyocytes, 22.8% of cells expressed CD31, 7.1% MADCAM1 and 2.6% F11R. CD11a, CD11b, CD62P and CD162 were expressed by fewer than 2% of the cells at baseline. CD31 expression increased on incubation of cardiomyocytes with thrombin by 26% (p<0.05) and with LPS by 26% (p=0.06). The combination of thrombin and LPS did not result in increased levels of CD31 (p>0.10). The pro-inflammatory agents LPS and thrombin had no effect on the expression of MADCAM1 and F11R. Inflammation-related cell-adhesion molecules CD31, MADCAM1 and F11R were shown to be expressed on the surface of human cardiomyocytes in an in vitro model. Incubation with LPS or thrombin resulted in increased expression of CD31, however, it did not modify the expression of the cell adhesion molecules MADCAM1 and F11R. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Malignant pericytes expressing GT198 give rise to tumor cells through angiogenesis.

PubMed

Zhang, Liyong; Wang, Yan; Rashid, Mohammad H; Liu, Min; Angara, Kartik; Mivechi, Nahid F; Maihle, Nita J; Arbab, Ali S; Ko, Lan

2017-08-01

Angiogenesis promotes tumor development. Understanding the crucial factors regulating tumor angiogenesis may reveal new therapeutic targets. Human GT198 ( PSMC3IP or Hop2) is an oncoprotein encoded by a DNA repair gene that is overexpressed in tumor stromal vasculature to stimulate the expression of angiogenic factors. Here we show that pericytes expressing GT198 give rise to tumor cells through angiogenesis. GT198 + pericytes and perivascular cells are commonly present in the stromal compartment of various human solid tumors and rodent xenograft tumor models. In human oral cancer, GT198 + pericytes proliferate into GT198 + tumor cells, which migrate into lymph nodes. Increased GT198 expression is associated with increased lymph node metastasis and decreased progression-free survival in oral cancer patients. In rat brain U-251 glioblastoma xenografts, GT198 + pericytes of human tumor origin encase endothelial cells of rat origin to form mosaic angiogenic blood vessels, and differentiate into pericyte-derived tumor cells. The net effect is continued production of glioblastoma tumor cells from malignant pericytes via angiogenesis. In addition, activation of GT198 induces the expression of VEGF and promotes tube formation in cultured U251 cells. Furthermore, vaccination using GT198 protein as an antigen in mouse xenograft of GL261 glioma delayed tumor growth and prolonged mouse survival. Together, these findings suggest that GT198-expressing malignant pericytes can give rise to tumor cells through angiogenesis, and serve as a potential source of cells for distant metastasis. Hence, the oncoprotein GT198 has the potential to be a new target in anti-angiogenic therapies in human cancer.

The role of human chorionic gonadotropin in regulation of naïve and memory T cells activity in vitro.

PubMed

Zamorina, S A; Litvinova, L S; Yurova, K A; Khaziakhmatova, O G; Timganova, V P; Bochkova, M S; Khramtsov, P V; Rayev, M B

2018-01-01

The role of human chorionic gonadotropin (hCG) in the regulation of molecular genetics factors determining the functional activity of human naïve and memory T cells in vitro was studied. It was found that hCG (10 and 100IU/ml) inhibited CD28 and CD25 expression on the naïve T cells (CD45RA+) and CD25 expression on the memory T cells (CD45R0+). hCG didn't affect the CD71 proliferation marker expression in total. Nevertheless, hCG reduced the percentage of proliferating memory T cells with simultaneous suppression of CD71 expression on proliferating CD45R0+cells. In parallel, expression of U2af1l4, Gfi1, and hnRNPLL genes, which are Ptprc gene alternative splicing regulators was evaluated. It was established that hCG stimulated the expression of U2af1l4 and hnRNPLL genes, responsible for the assembly of CD45R0 in memory T cells, but reduced the expression of Gfi1 in these cells. In general, hCG promotes the differentiation of memory T cells by increasing of CD45R0 expression, but inhibits proliferation and CD25 expression which reflects their functional activity. Copyright © 2017 Elsevier B.V. All rights reserved.

Production of heterozygous alpha 1,3-galactosyltransferase (GGTA1) knock-out transgenic miniature pigs expressing human CD39.

PubMed

Choi, Kimyung; Shim, Joohyun; Ko, Nayoung; Eom, Heejong; Kim, Jiho; Lee, Jeong-Woong; Jin, Dong-Il; Kim, Hyunil

2017-04-01

Production of transgenic pigs for use as xenotransplant donors is a solution to the severe shortage of human organs for transplantation. The first barrier to successful xenotransplantation is hyperacute rejection, a rapid, massive humoral immune response directed against the pig carbohydrate GGTA1 epitope. Platelet activation, adherence, and clumping, all major features of thrombotic microangiopathy, are inevitable results of immune-mediated transplant rejection. Human CD39 rapidly hydrolyzes ATP and ADP to AMP; AMP is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine, an anti-thrombotic and cardiovascular protective mediator. In this study, we developed a vector-based strategy for ablation of GGTA1 function and concurrent expression of human CD39 (hCD39). An hCD39 expression cassette was constructed to target exon 4 of GGTA1. We established heterozygous GGTA1 knock-out cell lines expressing hCD39 from pig ear fibroblasts for somatic cell nuclear transfer (SCNT). We also described production of heterozygous GGTA1 knock-out piglets expressing hCD39 and analyzed expression and function of the transgene. Human CD39 was expressed in heart, kidney and aorta. Human CD39 knock-in heterozygous ear fibroblast from transgenic cloned pigs, but not in non-transgenic pig's cells. Expression of GGTA1 gene was lower in the knock-in heterozygous ear fibroblast from transgenic pigs compared to the non-transgenic pig's cell. The peripheral blood mononuclear cells (PBMC) from the transgenic pigs were more resistant to lysis by pooled complement-preserved normal human serum than that from wild type (WT) pig. Accordingly, GGTA1 mutated piglets expressing hCD39 will provide a new organ source for xenotransplantation research.

Detection of the human endogenous retrovirus ERV3-encoded Env-protein in human tissues using antibody-based proteomics.

PubMed

Fei, Chen; Atterby, Christina; Edqvist, Per-Henrik; Pontén, Fredrik; Zhang, Wei Wei; Larsson, Erik; Ryan, Frank P

2014-01-01

There is growing evidence to suggest that human endogenous retroviruses (HERVs) have contributed to human evolution, being expressed in development, normal physiology and disease. A key difficulty in the scientific evaluation of this potential viral contribution is the accurate demonstration of virally expressed protein in specific human cells and tissues. In this study, we have adopted the endogenous retrovirus, ERV3, as our test model in developing a reliable high-capacity methodology for the expression of such endogenous retrovirus-coded protein. Two affinity-purified polyclonal antibodies to ERV3 Env-encoded protein were generated to detect the corresponding protein expression pattern in specific human cells, tissues and organs. Sampling included normal tissues from 144 individuals ranging from childhood to old age. This included more than forty different tissues and organs and some 216 different cancer tissues representing the twenty commonest forms of human cancer. The Rudbeck Laboratory, Uppsala University and Uppsala University Hospital, Uppsala, Sweden. The potential expression at likely physiological level of the ERV3Env encoded protein in a wide range of human cells, tissues and organs. We found that ERV3 encoded Env protein is expressed at substantive levels in placenta, testis, adrenal gland, corpus luteum, Fallopian tubes, sebaceous glands, astrocytes, bronchial epithelium and the ducts of the salivary glands. Substantive expression was also seen in a variety of epithelial cells as well as cells known to undergo fusion in inflammation and in normal physiology, including fused macrophages, myocardium and striated muscle. This contrasted strongly with the low levels expressed in other tissues types. These findings suggest that this virus plays a significant role in human physiology and may also play a possible role in disease. This technique can now be extended to the study of other HERV genomes within the human chromosomes that may have contributed to human evolution, physiology and disease.

Hematopoietic Colony Formation from Human Growth Factor-Dependent TF1 Cells and Human Cord Blood Myeloid Progenitor Cells Depends on SHP2 Phosphatase Function

PubMed Central

Etienne-Julan, Maryse; Gotoh, Akihiko; Braun, Stephen E.; Lu, Li; Cooper, Scott; Feng, Gen-Sheng; Li, Xing Jun

2013-01-01

The protein tyrosine phosphatase, SHP2, is widely expressed; however, previous studies demonstrated that hematopoietic cell development more stringently requires Shp2 expression compared to other tissues. Furthermore, somatic gain-of-function SHP2 mutants are commonly found in human myeloid leukemias. Given that pharmacologic inhibitors to SHP2 phosphatase activity are currently in development as putative antileukemic agents, we conducted a series of experiments examining the necessity of SHP2 phosphatase activity for human hematopoiesis. Anti-sense oligonucleotides to human SHP2 coding sequences reduced human cord blood- and human cell line, TF1-derived colony formation. Expression of truncated SHP2 bearing its Src homology 2 (SH2) domains, but lacking the phosphatase domain similarly reduced human cord blood- and TF1-derived colony formation. Mechanistically, expression of truncated SHP2 reduced the interaction between endogenous, full-length SHP2 with the adapter protein, Grb2. To verify the role of SHP2 phosphatase function in human hematopoietic cell development, human cord blood CD34+ cells were transduced with a leukemia-associated phosphatase gain-of-function SHP2 mutant or with a phosphatase dead SHP2 mutant, which indicated that increased phosphatase function enhanced, while decreased SHP2 phosphatase function reduced, human cord blood-derived colonies. Collectively, these findings indicate that SHP2 phosphatase function regulates human hematopoietic cell development and imply that the phosphatase component of SHP2 may serve as a pharmacologic target in human leukemias bearing increased SHP2 phosphatase activity. PMID:23082805

Hematopoietic colony formation from human growth factor-dependent TF1 cells and human cord blood myeloid progenitor cells depends on SHP2 phosphatase function.

PubMed

Broxmeyer, Hal E; Etienne-Julan, Maryse; Gotoh, Akihiko; Braun, Stephen E; Lu, Li; Cooper, Scott; Feng, Gen-Sheng; Li, Xing Jun; Chan, Rebecca J

2013-03-15

The protein tyrosine phosphatase, SHP2, is widely expressed; however, previous studies demonstrated that hematopoietic cell development more stringently requires Shp2 expression compared to other tissues. Furthermore, somatic gain-of-function SHP2 mutants are commonly found in human myeloid leukemias. Given that pharmacologic inhibitors to SHP2 phosphatase activity are currently in development as putative antileukemic agents, we conducted a series of experiments examining the necessity of SHP2 phosphatase activity for human hematopoiesis. Anti-sense oligonucleotides to human SHP2 coding sequences reduced human cord blood- and human cell line, TF1-derived colony formation. Expression of truncated SHP2 bearing its Src homology 2 (SH2) domains, but lacking the phosphatase domain similarly reduced human cord blood- and TF1-derived colony formation. Mechanistically, expression of truncated SHP2 reduced the interaction between endogenous, full-length SHP2 with the adapter protein, Grb2. To verify the role of SHP2 phosphatase function in human hematopoietic cell development, human cord blood CD34+ cells were transduced with a leukemia-associated phosphatase gain-of-function SHP2 mutant or with a phosphatase dead SHP2 mutant, which indicated that increased phosphatase function enhanced, while decreased SHP2 phosphatase function reduced, human cord blood-derived colonies. Collectively, these findings indicate that SHP2 phosphatase function regulates human hematopoietic cell development and imply that the phosphatase component of SHP2 may serve as a pharmacologic target in human leukemias bearing increased SHP2 phosphatase activity.

4-Nitroquinoline-1-oxide effects human lung adenocarcinoma A549 cells by regulating the expression of POLD4

PubMed Central

HUANG, QIN-MIAO; ZENG, YI-MING; ZHANG, HUA-PING; LV, LIANG-CHAO; YANG, DONG-YONG; LIN, HUI-HUANG

2016-01-01

The aim of the present study was to explore the expression of POLD4 in human lung adenocarcinoma A549 cells under 4-nitroquinoline-1-oxide (4NQO) stimulation to investigate the role of POLD4 in smoking-induced lung cancer. The lung cancer A549 cell line was treated with 4NQO, with or without MG132 (an inhibitor of proteasome activity), and subsequently the POLD4 level was determined by western blot analysis. Secondly, the cell sensitivity to 4NQO and Taxol was determined when the POLD4 expression level was downregulated by siRNA. The POLD4 protein levels in the A549 cells decreased following treatment with 4NQO; however, MG132 could reverse this phenotype. Downregulation of the POLD4 expression by siRNA enhanced A549 cell sensitivity to 4NQO, but not to Taxol. In conclusion, 4NQO affects human lung adenocarcinoma A549 cells by regulating the expression of POLD4. PMID:26998273

Phenotypical and Pharmacological Characterization of Stem-Like Cells in Human Pituitary Adenomas.

PubMed

Würth, Roberto; Barbieri, Federica; Pattarozzi, Alessandra; Gaudenzi, Germano; Gatto, Federico; Fiaschi, Pietro; Ravetti, Jean-Louis; Zona, Gianluigi; Daga, Antonio; Persani, Luca; Ferone, Diego; Vitale, Giovanni; Florio, Tullio

2017-09-01

The presence and functional role of tumor stem cells in benign tumors, and in human pituitary adenomas in particular, is a debated issue that still lacks a definitive formal demonstration. Fifty-six surgical specimens of human pituitary adenomas were processed to establish tumor stem-like cultures by selection and expansion in stem cell-permissive medium or isolating CD133-expressing cells. Phenotypic and functional characterization of these cells was performed (1) ex vivo, by immunohistochemistry analysis on paraffin-embedded tissues; (2) in vitro, attesting marker expression, proliferation, self-renewal, differentiation, and drug sensitivity; and (3) in vivo, using a zebrafish model. Within pituitary adenomas, we identified rare cell populations expressing stem cell markers but not pituitary hormones; we isolated and expanded in vitro these cells, obtaining fibroblast-free, stem-like cultures from 38 pituitary adenoma samples. These cells grow as spheroids, express stem cell markers (Oct4, Sox2, CD133, and nestin), show sustained in vitro proliferation as compared to primary cultures of differentiated pituitary adenoma cells, and are able to differentiate in hormone-expressing pituitary cells. Besides, pituisphere cells, apparently not tumorigenic in mice, engrafted in zebrafish embryos, inducing pro-angiogenic and invasive responses. Finally, pituitary adenoma stem-like cells express regulatory pituitary receptors (D2R, SSTR2, and SSTR5), whose activation by a dopamine/somatostatin chimeric agonist exerts antiproliferative effects. In conclusion, we provide evidence that human pituitary adenomas contain a subpopulation fulfilling biological and phenotypical signatures of tumor stem cells that may represent novel therapeutic targets for therapy-resistant tumors.

Curcumin Significantly Enhances Dual PI3K/Akt and mTOR Inhibitor NVP-BEZ235-Induced Apoptosis in Human Renal Carcinoma Caki Cells through Down-Regulation of p53-Dependent Bcl-2 Expression and Inhibition of Mcl-1 Protein Stability

PubMed Central

Cho, Il Je; Kim, Sang Chan; Kwon, Taeg Kyu

2014-01-01

The PI3K/Akt and mTOR signaling pathways are important for cell survival and growth, and they are highly activated in cancer cells compared with normal cells. Therefore, these signaling pathways are targets for inducing cancer cell death. The dual PI3K/Akt and mTOR inhibitor NVP-BEZ235 completely inhibited both signaling pathways. However, NVP-BEZ235 had no effect on cell death in human renal carcinoma Caki cells. We tested whether combined treatment with natural compounds and NVP-BEZ235 could induce cell death. Among several chemopreventive agents, curcumin, a natural biologically active compound that is extracted from the rhizomes of Curcuma species, markedly induced apoptosis in NVP-BEZ235-treated cells. Co-treatment with curcumin and NVP-BEZ235 led to the down-regulation of Mcl-1 protein expression but not mRNA expression. Ectopic expression of Mcl-1 completely inhibited curcumin plus NVP-NEZ235-induced apoptosis. Furthermore, the down-regulation of Bcl-2 was involved in curcumin plus NVP-BEZ235-induced apoptosis. Curcumin or NVP-BEZ235 alone did not change Bcl-2 mRNA or protein expression, but co-treatment reduced Bcl-2 mRNA and protein expression. Combined treatment with NVP-BEZ235 and curcumin reduced Bcl-2 expression in wild-type p53 HCT116 human colon carcinoma cells but not p53-null HCT116 cells. Moreover, Bcl-2 expression was completely reversed by treatment with pifithrin-α, a p53-specific inhibitor. Ectopic expression of Bcl-2 also inhibited apoptosis in NVP-BE235 plus curcumin-treated cells. In contrast, NVP-BEZ235 combined with curcumin did not have a synergistic effect on normal human skin fibroblasts and normal human mesangial cells. Taken together, combined treatment with NVP-BEZ235 and curcumin induces apoptosis through p53-dependent Bcl-2 mRNA down-regulation at the transcriptional level and Mcl-1 protein down-regulation at the post-transcriptional level. PMID:24743574

c-Myc-Dependent Cell Competition in Human Cancer Cells.

PubMed

Patel, Manish S; Shah, Heta S; Shrivastava, Neeta

2017-07-01

Cell Competition is an interaction between cells for existence in heterogeneous cell populations of multicellular organisms. This phenomenon is involved in initiation and progression of cancer where heterogeneous cell populations compete directly or indirectly for the survival of the fittest based on differential gene expression. In Drosophila, cells having lower dMyc expression are eliminated by cell competition through apoptosis when present in the milieu of cells having higher dMyc expression. Thus, we designed a study to develop c-Myc (human homolog) dependent in vitro cell competition model of human cancer cells. Cells with higher c-Myc were transfected with c-myc shRNA to prepare cells with lower c-Myc and then co-cultured with the same type of cells having a higher c-Myc in equal ratio. Cells with lower c-Myc showed a significant decrease in numbers when compared with higher c-Myc cells, suggesting "loser" and "winner" status of cells, respectively. During microscopy, engulfment of loser cells by winner cells was observed with higher expression of JNK in loser cells. Furthermore, elimination of loser cells was prevented significantly, when co-cultured cells were treated with the JNK (apoptosis) inhibitor. Above results indicate elimination of loser cells in the presence of winner cells by c-Myc-dependent mechanisms of cell competition in human cancer cells. This could be an important mechanism in human tumors where normal cells are eliminated by c-Myc-overexpressed tumor cells. J. Cell. Biochem. 118: 1782-1791, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Expression pattern of G protein-coupled receptor 30 in human seminiferous tubular cells.

PubMed

Oliveira, Pedro F; Alves, Marco G; Martins, Ana D; Correia, Sara; Bernardino, Raquel L; Silva, Joaquina; Barros, Alberto; Sousa, Mário; Cavaco, José E; Socorro, Sílvia

2014-05-15

The role of estrogens in male reproductive physiology has been intensively studied over the last few years. Yet, the involvement of their specific receptors has long been a matter of debate. The selective testicular expression of the classic nuclear estrogen receptors (ERα and ERβ) argues in favor of ER-specific functions in the spermatogenic event. Recently, the existence of a G protein-coupled estrogen receptor (GPR30) mediating non-genomic effects of estrogens has also been described. However, little is known about the specific testicular expression pattern of GPR30, as well as on its participation in the control of male reproductive function. Herein, by means of immunohistochemical and molecular biology techniques (RT-PCR and Western blot), we aimed to present the first exhaustive evaluation of GPR30 expression in non-neoplastic human testicular cells. Indeed, we were able to demonstrate that GPR30 was expressed in human testicular tissue and that the staining pattern was consistent with its cytoplasmic localization. Additionally, by using cultured human Sertoli cells (SCs) and isolated haploid and diploid germ cells fractions, we confirmed that GPR30 is expressed in SCs and diploid germ cells but not in haploid germ cells. This specific expression pattern suggests a role for GPR30 in spermatogenesis. Copyright © 2014 Elsevier Inc. All rights reserved.

Human hematopoietic progenitors express erythropoietin.

PubMed

Stopka, T; Zivny, J H; Stopkova, P; Prchal, J F; Prchal, J T

1998-05-15

Erythropoietin (EPO) is a factor essential for erythroid cell proliferation, differentiation, and survival. The production of EPO by the kidneys in response to hypoxia and anemia is well documented. To determine whether EPO is also produced by hematopoietic cells, we analyzed the expression of EPO in normal human hematopoietic progenitors and in their progeny. Undifferentiated CD34(+)lin- hematopoietic progenitors do not have detectable EPO mRNA. Differentiating CD34(+) cells that are stimulated with recombinant human EPO in serum-free liquid cultures express both EPO and EPO receptor (EPOR). Because CD34(+) cells represent a heterogeneous cell population, we analyzed individual burst-forming units-erythroid (BFU-E) and nonerythroid colony-forming unit-granulocyte-macrophage colonies for EPO mRNA. Only BFU-E colonies were positive for EPO mRNA. Lysates from pooled BFU-E colonies stained positively for EPO by immunoblotting. To further confirm the intrinsic nature of erythroid EPO, we replaced extrinsic EPO in erythroid colony cultures with EPO-mimicking peptide (EMP). We show EPO expression in the EMP-stimulated BFU-Es at both mRNA and protein levels. Stimulation of bone marrow mononuclear cells (BMMCs) with EMP upregulated EPO expression. Furthermore, we found EPO and EPOR mRNAs as well as EPO protein in K562 cells, a human erythroleukemia cell line. Stimulation of K562 cells with EMP upregulated EPO expression. We suggest that EPO of erythroid origin may have a role in the regulation of erythropoiesis.

Expression and distribution of endocan in human tissues.

PubMed

Zhang, S M; Zuo, L; Zhou, Q; Gui, S Y; Shi, R; Wu, Q; Wei, W; Wang, Y

2012-04-01

Endocan is a novel human endothelial cell specific molecule. Its expression is regulated by cytokines and vascular endothelial growth factor (VEGF). The distribution of endocan in normal human tissues, however, remains unclear. We examined the expression of endocan in normal human tissue using immunohistochemical stains. Endocan was expressed in actively proliferative or neogeneic tissues and cells such as glandular tissues, endothelium of neovasculature, bronchial epithelium, germinal centers of lymph nodes etc. Endocan was not present in silent or resting tissues or cells such as endothelium of great arteries and spleen etc. Our findings suggest that endocan may act as a marker for angiogenesis or oncogenesis and could be regarded as a candidate gene for inflammatory tissue, neoplasia, tumor development and metastasis. The expression level of endocan may assist early diagnosis and prognosis of some tumors.

Gossypol inhibition of mitosis, cyclin D1 and Rb protein in human mammary cancer cells and cyclin-D1 transfected human fibrosarcoma cells.

PubMed Central

Ligueros, M.; Jeoung, D.; Tang, B.; Hochhauser, D.; Reidenberg, M. M.; Sonenberg, M.

1997-01-01

The antiproliferative effects of gossypol on human MCF-7 mammary cancer cells and cyclin D1-transfected HT-1060 human fibrosarcoma cells were investigated by cell cycle analysis and effects on the cell cycle regulatory proteins Rb and cyclin D1. Flow cytometry of MCF-7 cells at 24 h indicated that 10 microM gossypol inhibited DNA synthesis by producing a G1/S block. Western blot analysis using anti-human Rb antibodies and anti-human cyclin D1 antibodies in MCF-7 cells and high- and low-expression cyclin D1-transfected fibrosarcoma cells indicated that, after 6 h exposure, gossypol decreased the expression levels of these proteins in a dose-dependent manner. Gossypol also decreased the ratio of phosphorylated to unphosphorylated Rb protein in human mammary cancer and fibrosarcoma cell lines. Gossypol (10 microM) treated also decreased cyclin D1-associated kinase activity on histone H1 used as a substrate in MCF-7 cells. These results suggest that gossypol might suppress growth by modulating the expression of cell cycle regulatory proteins Rb and cyclin D1 and the phosphorylation of Rb protein. Images Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:9218727

Beclin-1 Expression in Normal Bladder and in Cd+2 and As+3 Exposed and Transformed Human Urothelial Cells (UROtsa)

PubMed Central

Larson, Jennifer L.; Somji, Seema; Zhou, Xu Dong; Sens, Mary Ann; Garrett, Scott H.; Sens, Donald A.; Dunlevy, Jane R.

2010-01-01

The expression of beclin-1 in normal human bladder and in Cd+2 and As+3 exposed and transformed urothelial cells (UROtsa) was examined in this study. It was shown using a combination of real time PCR, western analysis and immunohistochemistry that beclin-1 was expressed in the urothelial cells of the normal bladder. It was also demonstrated that the parental UROtsa cell line expressed beclin-1 mRNA and protein at levels similar to that of the in situ urothelium. The level of beclin-1 expression underwent only modest alterations when the UROtsa cells were malignantly transformed by Cd+2 or As+3 or when the parental cells were exposed acutely to Cd+2 or As+3. While there were instances of significant alterations at individual time points and within cell line-to-cell line comparisons there was no evidence of a dose response relationship or correlations to the phenotypic properties of the cell lines. Similar results were obtained for the expression of the Atg-5, Atg-7, Atg-12 and LC3B autophagy-related proteins. The findings provide initial evidence for beclin-1 expression in normal bladder and that large alterations in the expression of beclin-1 and associated proteins do not occur when human urothelial cells are malignantly transformed with, or exposed to, either Cd+2 or As+3. PMID:20206246

Epithelial to mesenchymal transition in human endocrine islet cells

PubMed Central

Moreno-Amador, José Luis; Téllez, Noèlia; Marin, Sandra; Aloy-Reverté, Caterina; Semino, Carlos; Nacher, Montserrat

2018-01-01

Background β-cells undergo an epithelial to mesenchymal transition (EMT) when expanded in monolayer culture and give rise to highly proliferative mesenchymal cells that retain the potential to re-differentiate into insulin-producing cells. Objective To investigate whether EMT takes place in the endocrine non-β cells of human islets. Methodology Human islets isolated from 12 multiorgan donors were dissociated into single cells, purified by magnetic cell sorting, and cultured in monolayer. Results Co-expression of insulin and the mesenchymal marker vimentin was identified within the first passage (p1) and increased subsequently (insulin+vimentin+ 7.2±6% at p1; 43±15% at p4). The endocrine non-β-cells did also co-express vimentin (glucagon+vimentin+ 59±1.5% and 93±6%, somatostatin+vimentin+ 16±9.4% and 90±10% at p1 and p4 respectively; PP+vimentin+ 74±14% at p1; 88±12% at p2). The percentage of cells expressing only endocrine markers was progressively reduced (0.6±0.2% insulin+, 0.2±0.1% glucagon+, and 0.3±0.2% somatostatin+ cells at p4, and 0.7±0.3% PP+ cells at p2. Changes in gene expression were also indicated of EMT, with reduced expression of endocrine markers and the epithelial marker CDH-1 (p<0.01), and increased expression of mesenchymal markers (CDH-2, SNAI2, ZEB1, ZEB2, VIM, NT5E and ACTA2; p<0.05). Treatment with the EMT inhibitor A83-01 significantly reduced the percentage of co-expressing cells and preserved the expression of endocrine markers. Conclusions In adult human islets, all four endocrine islet cell types undergo EMT when islet cells are expanded in monolayer conditions. The presence of EMT in all islet endocrine cells could be relevant to design of strategies aiming to re-differentiate the expanded islet cells towards a β-cell phenotype. PMID:29360826

Productive Infection of Human Embryonic Stem Cell-Derived NKX2.1+ Respiratory Progenitors with Human Rhinovirus.

PubMed

Jenny, Robert A; Hirst, Claire; Lim, Sue Mei; Goulburn, Adam L; Micallef, Suzanne J; Labonne, Tanya; Kicic, Anthony; Ling, Kak-Ming; Stick, Stephen M; Ng, Elizabeth S; Trounson, Alan; Giudice, Antonietta; Elefanty, Andrew G; Stanley, Edouard G

2015-06-01

Airway epithelial cells generated from pluripotent stem cells (PSCs) represent a resource for research into a variety of human respiratory conditions, including those resulting from infection with common human pathogens. Using an NKX2.1-GFP reporter human embryonic stem cell line, we developed a serum-free protocol for the generation of NKX2.1(+) endoderm that, when transplanted into immunodeficient mice, matured into respiratory cell types identified by expression of CC10, MUC5AC, and surfactant proteins. Gene profiling experiments indicated that day 10 NKX2.1(+) endoderm expressed markers indicative of early foregut but lacked genes associated with later stages of respiratory epithelial cell differentiation. Nevertheless, NKX2.1(+) endoderm supported the infection and replication of the common respiratory pathogen human rhinovirus HRV1b. Moreover, NKX2.1(+) endoderm upregulated expression of IL-6, IL-8, and IL-1B in response to infection, a characteristic of human airway epithelial cells. Our experiments provide proof of principle for the use of PSC-derived respiratory epithelial cells in the study of cell-virus interactions. This report provides proof-of-principle experiments demonstrating, for the first time, that human respiratory progenitor cells derived from stem cells in the laboratory can be productively infected with human rhinovirus, the predominant cause of the common cold. ©AlphaMed Press.

Effect of human vascular endothelial growth factor gene transfer on endogenous vascular endothelial growth factor mRNA expression in a rat fibroblast and osteoblast culture model.

PubMed

Li, Ru; Li, Claire H; Nauth, Aaron; McKee, Michael D; Schemitsch, Emil H

2010-09-01

Vascular endothelial growth factor (VEGF) plays an important role in promoting angiogenesis and osteogenesis during fracture repair. Our previous studies have shown that cell-based VEGF gene therapy enhances bone healing of a rabbit tibia segmental bone defect in vivo. The aim of this project was to examine the effect of exogenous human VEGF on the endogenous rat VEGF messenger RNA (mRNA) expression in a cell-based gene transfer model. Rat fibroblasts and osteoblasts were harvested from the dermal tissue and periosteum, respectively, of Fisher 344 rats. The cells were then cultured and transfected with pcDNA-human VEGF using Superfect reagent (Qiagen). Four experimental groups were created: 1) fibroblast-VEGF; 2) osteoblast-VEGF; 3) nontransfected fibroblast controls; and 4) nontransfected osteoblast controls. The cultured cells were harvested at 1, 3, and 7 days after the gene transfection. The total mRNA was extracted (Trizol; Invitrogen); both human VEGF and rat VEGF mRNA were measured by reverse transcriptase-polymerase chain reaction and quantified by VisionWorksLS. The human VEGF165 mRNA was detected by reverse transcriptase-polymerase chain reaction from transfected fibroblasts and osteoblasts at 1, 3, and 7 days after gene transfection. The human VEGF165 levels peaked at Day 1 and then gradually reduced expression in both transfected fibroblasts and osteoblasts. Two endogenous rat VEGF isoforms were detected in this cell culture model: rat VEGF120 and rat VEGF164. We compared the rat VEGF120 and rat VEGF164 expression level of the fibroblasts or osteoblasts that were transfected with human VEGF165, with nontransfected control cells. Both the transfected fibroblasts and osteoblasts showed greater expression of rat VEGF164 than nontransfected controls at Day 1 (peak level) and Day 3, but not at Day 7. The expression of rat VEGF120 was lower in transfected fibroblasts, but higher in transfected osteoblasts, than the relevant control groups at any time point after transfection. In addition, human VEGF gene transfection increased osteoblast cell proliferation after 3 days. These in vitro results suggest that cell-based human VEGF gene therapy is not only effective at causing human VEGF expression, but also enhances endogenous rat VEGF mRNA expression in both fibroblasts and osteoblasts, particularly the rat VEGF164 isoform.

Parallels between Global Transcriptional Programs of Polarizing Caco-2 Intestinal Epithelial Cells In Vitro and Gene Expression Programs in Normal Colon and Colon Cancer

PubMed Central

Sääf, Annika M.; Halbleib, Jennifer M.; Chen, Xin; Yuen, Siu Tsan; Leung, Suet Yi

2007-01-01

Posttranslational mechanisms are implicated in the development of epithelial cell polarity, but little is known about the patterns of gene expression and transcriptional regulation during this process. We characterized temporal patterns of gene expression during cell–cell adhesion-initiated polarization of cultured human Caco-2 cells, which develop structural and functional polarity resembling enterocytes in vivo. A distinctive switch in gene expression patterns occurred upon formation of cell–cell contacts. Comparison to gene expression patterns in normal human colon and colon tumors revealed that the pattern in proliferating, nonpolarized Caco-2 cells paralleled patterns seen in human colon cancer in vivo, including expression of genes involved in cell proliferation. The pattern switched in polarized Caco-2 cells to one more closely resembling that in normal colon tissue, indicating that regulation of transcription underlying Caco-2 cell polarization is similar to that during enterocyte differentiation in vivo. Surprisingly, the temporal program of gene expression in polarizing Caco-2 cells involved changes in signaling pathways (e.g., Wnt, Hh, BMP, FGF) in patterns similar to those during migration and differentiation of intestinal epithelial cells in vivo, despite the absence of morphogen gradients and interactions with stromal cells characteristic of enterocyte differentiation in situ. The full data set is available at http://microarray-pubs.stanford.edu/CACO2. PMID:17699589

Redox-mediated enrichment of self-renewing adult human pancreatic cells that possess endocrine differentiation potential.

PubMed

Linning, Katrina D; Tai, Mei-Hui; Madhukar, Burra V; Chang, C C; Reed, Donald N; Ferber, Sarah; Trosko, James E; Olson, L Karl

2004-10-01

The limited availability of transplantable human islets has stimulated the development of methods needed to isolate adult pancreatic stem/progenitor cells capable of self-renewal and endocrine differentiation. The objective of this study was to determine whether modulation of intracellular redox state with N-acetyl-L-cysteine (NAC) would allow for the propagation of pancreatic stem/progenitor cells from adult human pancreatic tissue. Cells were propagated from human pancreatic tissue using a serum-free, low-calcium medium supplemented with NAC and tested for their ability to differentiate when cultured under different growth conditions. Human pancreatic cell (HPC) cultures coexpressed alpha-amylase, albumin, vimentin, and nestin. The HPC cultures, however, did not express other genes associated with differentiated pancreatic exocrine, duct, or endocrine cells. A number of transcription factors involved in endocrine cell development including Beta 2, Islet-1, Nkx6.1, Pax4, and Pax6 were expressed at variable levels in HPC cultures. In contrast, pancreatic duodenal homeobox factor 1 (Pdx-1) expression was extremely low and at times undetectable. Overexpression of Pdx-1 in HPC cultures stimulated somatostatin, glucagon, and carbonic anhydrase expression but had no effect on insulin gene expression. HPC cultures could form 3-dimensional islet-like cell aggregates, and this was associated with expression of somatostatin and glucagon but not insulin. Cultivation of HPCs in a differentiation medium supplemented with nicotinamide, exendin-4, and/or LY294002, an inhibitor of phosphatidylinositol-3 kinase, stimulated expression of insulin mRNA and protein. These data support the use of intracellular redox modulation for the enrichment of pancreatic stem/progenitor cells capable of self-renewal and endocrine differentiation.

Notch-Induced Expression of FZD7 Requires Noncanonical NOTCH3 Signaling in Human Breast Epithelial Cells.

PubMed

Bhat, Vasudeva; Sun, Yu Jia; Weger, Steve; Raouf, Afshin

2016-04-01

The evolutionarily conserved Notch and Wnt signaling pathways have demonstrated roles in normal mammary gland development and in breast carcinogenesis. We previously reported that in human mammary gland, signaling through NOTCH3 alone regulates the commitment of the undifferentiated bipotential progenitors to the luminal cell fate, indicating that NOTCH3 may regulate the expression of unique genes apart from the other Notch receptors. In this study, we used gain of function and loss of function experiments and found that a Wnt signaling receptor, Frizzled7 (FZD7), is a unique and nonredundant target of NOTCH3 in human breast epithelial cells. Interestingly, neither the constitutively active forms of NOTCH1-2, 4 nor loss of expression of these receptors were able to alter expression of FZD7 in human breast epithelial cells. We further show that FZD7-expressing cells are found more frequently in the luminal progenitor-enriched subpopulation of cells obtained from breast reduction samples compared with the undifferentiated bipotent progenitors. Also, we show that NOTCH3-induced expression of FZD7 occurs in the absence of CSL (CBF1-Suppressor of Hairless-Lag-1). Our data suggest that noncanonical Notch signaling through NOTCH3 could modulate Wnt signaling via FZD7 and in this way, might be involved in luminal cell differentiation.

Dasatinib is preclinically active against Src-overexpressing human transitional cell carcinoma of the urothelium with activated Src signaling.

PubMed

Levitt, Jonathan M; Yamashita, Hideyuki; Jian, Weiguo; Lerner, Seth P; Sonpavde, Guru

2010-05-01

Dasatinib is an orally administered multitargeted kinase inhibitor that targets Src family tyrosine kinases, Abl, c-Kit, and PDGFR. A preclinical study was conducted to evaluate dasatinib alone or combined with cisplatin for human transitional cell carcinoma (TCC). Expression of Src in a human TCC tissue microarray was evaluated by immunohistochemistry. The activity of dasatinib and/or cisplatin was evaluated in six human TCC cell lines. Western blot was done to assess Src and phosphorylated-Src (p-Src) expression. The activity of dasatinib alone and in combination with cisplatin was determined in murine subcutaneous xenografts. Sixty-two percent to 75% of human TCC expressed Src. Dasatinib displayed significant antiproliferative activity at nanomolar concentrations against two human TCC cell lines (RT4 and Hu456) that exhibited high Src and p-Src expression and were cisplatin-resistant. RT4 cells were the most sensitive and displayed the highest level of Src pathway activation (p-Src/Src ratio). Dasatinib downregulated p-Src in either sensitive or resistant cells. TCC cells that were sensitive to cisplatin (5637 and TCC-SUP) were highly resistant to dasatinib and exhibited low Src expression. Dasatinib showed antitumor activity in RT4 murine xenografts, and the combination of dasatinib and cisplatin was significantly more active than placebo. Combination dasatinib plus cisplatin significantly inhibited proliferation and promoted apoptosis in vivo. In conclusion, dasatinib displayed significant preclinical antitumor activity against Src-overexpressing human TCC with active Src signaling and was highly active in combination with cisplatin in vivo. Further clinical development might be warranted in selected human subjects.

A Novel In Vitro Model for Studying Quiescence and Activation of Primary Isolated Human Myoblasts

PubMed Central

Sellathurai, Jeeva; Cheedipudi, Sirisha; Dhawan, Jyotsna; Schrøder, Henrik Daa

2013-01-01

Skeletal muscle stem cells, satellite cells, are normally quiescent but become activated upon muscle injury. Recruitment of resident satellite cells may be a useful strategy for treatment of muscle disorders, but little is known about gene expression in quiescent human satellite cells or the mechanisms involved in their early activation. We have developed a method to induce quiescence in purified primary human myoblasts isolated from healthy individuals. Analysis of the resting state showed absence of BrdU incorporation and lack of KI67 expression, as well as the extended kinetics during synchronous reactivation into the cell cycle, confirming arrest in the G0 phase. Reactivation studies showed that the majority (>95%) of the G0 arrested cells were able to re-enter the cell cycle, confirming reversibility of arrest. Furthermore, a panel of important myogenic factors showed expression patterns similar to those reported for mouse satellite cells in G0, reactivated and differentiated cultures, supporting the applicability of the human model. In addition, gene expression profiling showed that a large number of genes (4598) were differentially expressed in cells activated from G0 compared to long term exponentially proliferating cultures normally used for in vitro studies. Human myoblasts cultured through many passages inevitably consist of a mixture of proliferating and non-proliferating cells, while cells activated from G0 are in a synchronously proliferating phase, and therefore may be a better model for in vivo proliferating satellite cells. Furthermore, the temporal propagation of proliferation in these synchronized cultures resembles the pattern seen in vivo during regeneration. We therefore present this culture model as a useful and novel condition for molecular analysis of quiescence and reactivation of human myoblasts. PMID:23717533

Identification of a subset of human natural killer cells expressing high levels of programmed death 1: A phenotypic and functional characterization.

PubMed

Pesce, Silvia; Greppi, Marco; Tabellini, Giovanna; Rampinelli, Fabio; Parolini, Silvia; Olive, Daniel; Moretta, Lorenzo; Moretta, Alessandro; Marcenaro, Emanuela

2017-01-01

Programmed death 1 (PD-1) is an immunologic checkpoint that limits immune responses by delivering potent inhibitory signals to T cells on interaction with specific ligands expressed on tumor/virus-infected cells, thus contributing to immune escape mechanisms. Therapeutic PD-1 blockade has been shown to mediate tumor eradication with impressive clinical results. Little is known about the expression/function of PD-1 on human natural killer (NK) cells. We sought to clarify whether human NK cells can express PD-1 and analyze their phenotypic/functional features. We performed multiparametric cytofluorimetric analysis of PD-1 + NK cells and their functional characterization using degranulation, cytokine production, and proliferation assays. We provide unequivocal evidence that PD-1 is highly expressed (PD-1 bright ) on an NK cell subset detectable in the peripheral blood of approximately one fourth of healthy subjects. These donors are always serologically positive for human cytomegalovirus. PD-1 is expressed by CD56 dim but not CD56 bright NK cells and is confined to fully mature NK cells characterized by the NKG2A - KIR + CD57 + phenotype. Proportions of PD-1 bright NK cells were higher in the ascites of a cohort of patients with ovarian carcinoma, suggesting their possible induction/expansion in tumor environments. Functional analysis revealed a reduced proliferative capability in response to cytokines, low degranulation, and impaired cytokine production on interaction with tumor targets. We have identified and characterized a novel subpopulation of human NK cells expressing high levels of PD-1. These cells have the phenotypic characteristics of fully mature NK cells and are increased in patients with ovarian carcinoma. They display low proliferative responses and impaired antitumor activity that can be partially restored by antibody-mediated disruption of PD-1/programmed death ligand interaction. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Autocrine stimulation of VEGFR-2 activates human leukemic cell growth and migration

PubMed Central

Dias, Sergio; Hattori, Koichi; Zhu, Zhenping; Heissig, Beate; Choy, Margaret; Lane, William; Wu, Yan; Chadburn, Amy; Hyjek, Elizabeth; Gill, Muhammad; Hicklin, Daniel J.; Witte, Larry; Moore, M.A.S.; Rafii, Shahin

2000-01-01

Emerging data suggest that VEGF receptors are expressed by endothelial cells as well as hematopoietic stem cells. Therefore, we hypothesized that functional VEGF receptors may also be expressed in malignant counterparts of hematopoietic stem cells such as leukemias. We demonstrate that certain leukemias not only produce VEGF but also express functional VEGFR-2 in vivo and in vitro, resulting in the generation of an autocrine loop that may support leukemic cell survival and proliferation. Approximately 50% of freshly isolated leukemias expressed mRNA and protein for VEGFR-2. VEGF165 induced phosphorylation of VEGFR-2 and increased proliferation of leukemic cells, demonstrating these receptors were functional. VEGF165 also induced the expression of MMP-9 by leukemic cells and promoted their migration through reconstituted basement membrane. The neutralizing mAb IMC-1C11, specific to human VEGFR-2, inhibited leukemic cell survival in vitro and blocked VEGF165-mediated proliferation of leukemic cells and VEGF-induced leukemic cell migration. Xenotransplantation of primary leukemias and leukemic cell lines into immunocompromised nonobese diabetic mice resulted in significant elevation of human, but not murine, VEGF in plasma and death of inoculated mice within 3 weeks. Injection of IMC-1C11 inhibited proliferation of xenotransplanted human leukemias and significantly increased the survival of inoculated mice. Interruption of signaling by VEGFRs, particularly VEGFR-2, may provide a novel strategy for inhibiting leukemic cell proliferation. PMID:10953026

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.

Role of ID Proteins in BMP4 Inhibition of Profibrotic Effects of TGF-β2 in Human TM Cells.

PubMed

Mody, Avani A; Wordinger, Robert J; Clark, Abbot F

2017-02-01

Increased expression of TGF-β2 in primary open-angle glaucoma (POAG) aqueous humor (AH) and trabecular meshwork (TM) causes deposition of extracellular matrix (ECM) in the TM and elevated IOP. Bone morphogenetic proteins (BMPs) regulate TGF-β2-induced ECM production. The underlying mechanism for BMP4 inhibition of TGF-β2-induced fibrosis remains undetermined. Bone morphogenic protein 4 induces inhibitor of DNA binding proteins (ID1, ID3), which suppress transcription factor activities to regulate gene expression. Our study will determine whether ID1and ID3 proteins are downstream targets of BMP4, which attenuates TGF-β2 induction of ECM proteins in TM cells. Primary human TM cells were treated with BMP4, and ID1 and ID3 mRNA, and protein expression was determined by quantitative PCR (Q-PCR) and Western immunoblotting. Intracellular ID1 and ID3 protein localization was studied by immunocytochemistry. Transformed human TM cells (GTM3 cells) were transfected with ID1 or ID3 expression vectors to determine their potential inhibitory effects on TGF-β2-induced fibronectin and plasminogen activator inhibitor-I (PAI-1) protein expression. Basal expression of ID1-3 was detected in primary human TM cells. Bone morphogenic protein 4 significantly induced early expression of ID1 and ID3 mRNA (P < 0.05) and protein in primary TM cells, and a BMP receptor inhibitor blocked this induction. Overexpression of ID1 and ID3 significantly inhibited TGF-β2-induced expression of fibronectin and PAI-1 in TM cells (P < 0.01). Bone morphogenic protein 4 induced ID1 and ID3 expression suppresses TGF-β2 profibrotic activity in human TM cells. In the future, targeting specific regulators may control the TGF-β2 profibrotic effects on the TM, leading to disease modifying IOP lowering therapies.

A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase

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

Tratschin, J.D.; West, M.H.P.; Sandbank, T.

1984-10-01

The authors have used the defective human parvovirus adeno-associated virus (AAV) as a novel eurocaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p/sub 40/ (pAVHiCAT) and p/sub 19/more » (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product. The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p/sub 19/ is increased by E1A, whereas p/sub 40/ yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus.« less

Expression of 11beta-hydroxysteroid-dehydrogenase type 2 in human thymus.

PubMed

Almanzar, Giovanni; Mayerl, Christina; Seitz, Jan-Christoph; Höfner, Kerstin; Brunner, Andrea; Wild, Vanessa; Jahn, Daniel; Geier, Andreas; Fassnacht, Martin; Prelog, Martina

2016-06-01

11beta-hydroxysteroid-dehydrogenase type 2 (11β-HSD2) is a high affinity dehydrogenase which rapidly inactivates physiologically-active glucocorticoids to protect key tissues. 11β-HSD2 expression has been described in peripheral cells of the innate and the adaptive immune system as well as in murine thymus. In absence of knowledge of 11β-HSD2 expression in human thymus, the study aimed to localize 11β-HSD2 in human thymic tissue. Thymic tissue was taken of six healthy, non-immunologically impaired male infants below 12months of age with congenital heart defects who had to undergo correction surgery. 11β-HSD2 protein expression was analyzed by immunohistochemistry and Western blot. Kidney tissue, peripheral blood mononuclear cells (PBMCs) and human umbilical vein endothelial cells (HUVEC) were taken as positive controls. Significant expression of 11β-HSD2 protein was found at single cell level in thymus parenchyma, at perivascular sites of capillaries and small vessels penetrating the thymus lobuli and within Hassall's bodies. The present study demonstrates that 11β-HSD2 is expressed in human thymus with predominant perivascular expression and also within Hassall's bodies. To our knowledge, this is the first report confirming 11β-HSD2 expression at the protein level in human thymic tissue underlining a potential role of this enzyme in regulating glucocorticoid function at the thymic level. Copyright © 2016 Elsevier Inc. All rights reserved.

Neuron-derived orphan receptor 1 promoted human pulmonary artery smooth muscle cells proliferation.

PubMed

Wang, Chang-Guo; Lei, Wei; Li, Chang; Zeng, Da-Xiong; Huang, Jian-An

2015-05-01

As a transcription factor of the nuclear receptor superfamily, neuron-derived orphan receptor 1 (NOR1) is induced rapidly in response to various extracellular stimuli. But, it is still unclear its role in pulmonary artery smooth muscle cells proliferation. Human PASMCs were cultured in vitro and stimulated by serum. The special antisense oligodeoxynucleotides (AS-ODNs) were used to knockdown human NOR1 gene expression. Real-time PCR and Western-blot were used to evaluate the gene expression and protein levels. Fetal bovine serum (FBS) induced human PASMCs proliferation in a dose dependent manner. Furthermore, FBS promoted NOR1 gene expression in a dose dependent manner and a time dependent manner. 10% FBS induced a maximal NOR1 mRNA levels at 2 h. FBS also induced a significant higher NOR1 protein levels as compared with control. The NOR1 over-expressed plasmid significantly promoted DNA synthesis and cells proliferation. Moreover, the special AS-ODNs against human NOR1 not only prevented NOR1 expression but also inhibited DNA synthesis and cells proliferation significantly. The NOR1 over-expression plasmid could up-regulate cyclin D1 expression markedly, but the AS-ODNs inhibited cyclin D1 expression significantly. So, we concluded that NOR1 could promote human PASMCs proliferation. Cyclin D1 might be involved in this process.

JAG1-Mediated Notch Signaling Regulates Secretory Cell Differentiation of the Human Airway Epithelium.

PubMed

Gomi, Kazunori; Staudt, Michelle R; Salit, Jacqueline; Kaner, Robert J; Heldrich, Jonna; Rogalski, Allison M; Arbelaez, Vanessa; Crystal, Ronald G; Walters, Matthew S

2016-08-01

Basal cells (BC) are the stem/progenitor cells of the human airway epithelium capable of differentiating into secretory and ciliated cells. Notch signaling activation increases BC differentiation into secretory cells, but the role of individual Notch ligands in regulating this process in the human airway epithelium is largely unknown. The objective of this study was to define the role of the Notch ligand JAG1 in regulating human BC differentiation. JAG1 over-expression in BC increased secretory cell differentiation, with no effect on ciliated cell differentiation. Conversely, knockdown of JAG1 decreased expression of secretory cell genes. These data demonstrate JAG1-mediated Notch signaling regulates differentiation of BC into secretory cells.

A High-Dimensional Atlas of Human T Cell Diversity Reveals Tissue-Specific Trafficking and Cytokine Signatures.

PubMed

Wong, Michael Thomas; Ong, David Eng Hui; Lim, Frances Sheau Huei; Teng, Karen Wei Weng; McGovern, Naomi; Narayanan, Sriram; Ho, Wen Qi; Cerny, Daniela; Tan, Henry Kun Kiaang; Anicete, Rosslyn; Tan, Bien Keem; Lim, Tony Kiat Hon; Chan, Chung Yip; Cheow, Peng Chung; Lee, Ser Yee; Takano, Angela; Tan, Eng-Huat; Tam, John Kit Chung; Tan, Ern Yu; Chan, Jerry Kok Yen; Fink, Katja; Bertoletti, Antonio; Ginhoux, Florent; Curotto de Lafaille, Maria Alicia; Newell, Evan William

2016-08-16

Depending on the tissue microenvironment, T cells can differentiate into highly diverse subsets expressing unique trafficking receptors and cytokines. Studies of human lymphocytes have primarily focused on a limited number of parameters in blood, representing an incomplete view of the human immune system. Here, we have utilized mass cytometry to simultaneously analyze T cell trafficking and functional markers across eight different human tissues, including blood, lymphoid, and non-lymphoid tissues. These data have revealed that combinatorial expression of trafficking receptors and cytokines better defines tissue specificity. Notably, we identified numerous T helper cell subsets with overlapping cytokine expression, but only specific cytokine combinations are secreted regardless of tissue type. This indicates that T cell lineages defined in mouse models cannot be clearly distinguished in humans. Overall, our data uncover a plethora of tissue immune signatures and provide a systemic map of how T cell phenotypes are altered throughout the human body. Copyright © 2016 Elsevier Inc. All rights reserved.

Expression profile of senescence-associated beta-galactosidase and activation of telomerase in human ovarian surface epithelial cells undergoing immortalization.

PubMed

Litaker, J R; Pan, J; Cheung, Y; Zhang, D K; Liu, Y; Wong, S C; Wan, T S; Tsao, S W

1998-11-01

Senescence is a specific physiological stage of cells characterized by long population doubling time. It accounts for the inability of normal somatic cells to undergo indefinite cell division. As the number of population doublings increase, cell cycle regulatory mechanisms come into play and signal cells to exit the cell cycle and become senescent. Senescence has been implicated in the aging process and may function as a tumor suppressor mechanism in human cells. The ability to measure the degree of cellular senescence is important in understanding the biological processes regulating cell aging and immortalization. Senescent cells exhibit an enzyme termed senescence-associated histochemical staining. Cells immortalized by viral oncogenes often enter a stage of crisis at the early phase of immortalization. The cells at crisis have a long population doubling time. Cells at the crisis stage resemble senescent cells and the expression of SA- beta-Gal may be used to monitor the process of immortalization. In this study the expression profile of SA-beta-Gal was examined in human ovarian surface epithelial cells (HOSE 6-3) undergoing immortalization by the human papilloma viral oncogene E6 and E7 (HPV E6 and E7). Our results showed a low percentage (12.0%) of HOSE 6-3 cells expressing SA-beta-Gal activity at the pre-crisis stage. The percentage of HOSE 6-3 cells expressing SA-beta-Gal activity was highest (39.2%) at the crisis stage. When HOSE 6-3 cells achieved immortalized status there was a sharp decrease in cells (1. 3%) expressing SA-beta-Gal activity. In addition, an inverse relationship between the expression of SA-beta-Gal activity and telomerase activity was noted in cells undergoing immortalization. The results confirm that the SA-beta-Gal enzyme is a good marker for monitoring the population of cells undergoing senescence at different stages of immortalization and that telomerase activation is a characteristic feature of post-crisis cells.

Humanization of the mouse mammary gland by replacement of the luminal layer with genetically engineered preneoplastic human cells.

PubMed

Verbeke, Stephanie; Richard, Elodie; Monceau, Elodie; Schmidt, Xenia; Rousseau, Benoit; Velasco, Valerie; Bernard, David; Bonnefoi, Herve; MacGrogan, Gaetan; Iggo, Richard D

2014-12-20

The cell of origin for estrogen receptor α-positive (ERα+) breast cancer is probably a luminal stem cell in the terminal duct lobular units. To model these cells, we have used the murine myoepithelial layer in the mouse mammary ducts as a scaffold upon which to build a human luminal layer. To prevent squamous metaplasia, a common artifact in genetically-engineered breast cancer models, we sought to limit activation of the epidermal growth factor receptor (EGFR) during in vitro cell culture before grafting the cells. Human reduction mammoplasty cells were grown in vitro in WIT medium. Epidermal growth factor in the medium was replaced with amphiregulin and neuregulin to decrease activation of EGFR and increase activation of EGFR homologs 3 and 4 (ERBB3 and ERBB4). Lentiviral vectors were used to express oncogenic transgenes and fluorescent proteins. Human mammary epithelial cells were mixed with irradiated mouse fibroblasts and Matrigel, then injected through the nipple into the mammary ducts of immunodeficient mice. Engrafted cells were visualized by stereomicroscopy for fluorescent proteins and characterized by histology and immunohistochemistry. Growth of normal mammary epithelial cells in conditions favoring ERBB3/4 signaling prevented squamous metaplasia in vitro. Normal human cells were quickly lost after intraductal injection, but cells infected with lentiviruses expressing CCND1, MYC, TERT, BMI1 and a short-hairpin RNA targeting TP53 were able to engraft and progressively replace the luminal layer in the mouse mammary ducts, resulting in the formation of an extensive network of humanized ducts. Despite expressing multiple oncogenes, the human cells formed a morphologically normal luminal layer. Expression of a single additional oncogene, PIK3CA-H1047R, converted the cells into invasive cancer cells. The resulting tumors were ERα+, Ki67+ luminal B adenocarcinomas that were resistant to treatment with fulvestrant. Injection of preneoplastic human mammary epithelial cells into the mammary ducts of immunodeficient mice leads to replacement of the murine luminal layer with morphologically normal human cells. Genetic manipulation of the injected cells makes it possible to study defined steps in the transformation of human mammary epithelial cells in a more physiological environment than has hitherto been possible.

Induction of interleukin-8 by Naegleria fowleri lysates requires activation of extracellular signal-regulated kinase in human astroglial cells.

PubMed

Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Sang-Hee; Kwon, Daeho; Shin, Ho-Joon

2012-08-01

Naegleria fowleri is a pathogenic free-living amoeba which causes primary amoebic meningoencephalitis in humans and experimental animals. To investigate the mechanisms of such inflammatory diseases, potential chemokine gene activation in human astroglial cells was investigated following treatment with N. fowleri lysates. We demonstrated that N. fowleri are potent inducers for the expression of interleukin-8 (IL-8) genes in human astroglial cells which was preceded by activation of extracellular signal-regulated kinase (ERK). In addition, N. fowleri lysates induces the DNA binding activity of activator protein-1 (AP-1), an important transcription factor for IL-8 induction. The specific mitogen-activated protein kinase kinase/ERK inhibitor, U0126, blocks N. fowleri-mediated AP-1 activation and subsequent IL-8 induction. N. fowleri-induced IL-8 expression requires activation of ERK in human astroglial cells. These findings indicate that treatment of N. fowleri on human astroglial cells leads to the activation of AP-1 and subsequent expression of IL-8 which are dependent on ERK activation. These results may help understand the N. fowleri-mediated upregulation of chemokine and cytokine expression in the astroglial cells.

Transcriptome Dynamics of Developing Photoreceptors in Three‐Dimensional Retina Cultures Recapitulates Temporal Sequence of Human Cone and Rod Differentiation Revealing Cell Surface Markers and Gene Networks

PubMed Central

Kaewkhaw, Rossukon; Kaya, Koray Dogan; Brooks, Matthew; Homma, Kohei; Zou, Jizhong; Chaitankar, Vijender; Rao, Mahendra

2015-01-01

Abstract The derivation of three‐dimensional (3D) stratified neural retina from pluripotent stem cells has permitted investigations of human photoreceptors. We have generated a H9 human embryonic stem cell subclone that carries a green fluorescent protein (GFP) reporter under the control of the promoter of cone‐rod homeobox (CRX), an established marker of postmitotic photoreceptor precursors. The CRXp‐GFP reporter replicates endogenous CRX expression in vitro when the H9 subclone is induced to form self‐organizing 3D retina‐like tissue. At day 37, CRX+ photoreceptors appear in the basal or middle part of neural retina and migrate to apical side by day 67. Temporal and spatial patterns of retinal cell type markers recapitulate the predicted sequence of development. Cone gene expression is concomitant with CRX, whereas rod differentiation factor neural retina leucine zipper protein (NRL) is first observed at day 67. At day 90, robust expression of NRL and its target nuclear receptor NR2E3 is evident in many CRX+ cells, while minimal S‐opsin and no rhodopsin or L/M‐opsin is present. The transcriptome profile, by RNA‐seq, of developing human photoreceptors is remarkably concordant with mRNA and immunohistochemistry data available for human fetal retina although many targets of CRX, including phototransduction genes, exhibit a significant delay in expression. We report on temporal changes in gene signatures, including expression of cell surface markers and transcription factors; these expression changes should assist in isolation of photoreceptors at distinct stages of differentiation and in delineating coexpression networks. Our studies establish the first global expression database of developing human photoreceptors, providing a reference map for functional studies in retinal cultures. Stem Cells 2015;33:3504–3518 PMID:26235913

A Re-evaluation of CD22 Expression by Human Lung Cancer

PubMed Central

Pop, Laurentiu M.; Barman, Stephen; Shao, Chunli; Poe, Jonathan C.; Venturi, Guglielmo M.; Shelton, John M.; Pop, Iliodora V.; Gerber, David E.; Girard, Luc; Liu, Xiao-yun; Behrens, Carmen; Rodriguez-Canales, Jaime; Liu, Hui; Wistuba, Ignacio I.; Richardson, James A.; Minna, John D.; Tedder, Thomas F.; Vitetta, Ellen S.

2014-01-01

CD22 is a transmembrane glycoprotein expressed by mature B cells. It inhibits signal transduction by the B cell receptor and its co-receptor CD19. Recently it was reported that most human lung cancer cells and cell lines express CD22 making it an important new lung cancer therapeutic target (Can Res 72:5556, 2012). The objective of our studies was to independently validate these results with the goal of testing the efficacy of our CD22 immunotoxins on lung cancer cell lines. As determined by qRT-PCR analysis, we found that levels of CD22 mRNA in a panel of human lung cancer cell lines were 200–60,000- fold lower than those observed in the human CD22+ Burkitt’s lymphoma cells, Daudi. Using flow cytometry with a panel of CD22 monoclonal antibodies and Western blot analyses, we could not detect surface or intracellular expression of CD22 protein in a panel of lung cancer cell lines. In addition, the in vitro proliferation of the lung tumor cell lines was not affected by CD22 antibodies or our highly potent anti-CD22 immunotoxin. By contrast, CD22+ Daudi cells expressed high levels of CD22 mRNA and protein and were sensitive to our CD22 immunotoxin. Importantly, primary non-small cell lung cancers from over 250 patient specimens did not express detectable levels of CD22 protein as assessed by immunohistochemistry. We conclude that CD22 is not expressed at measurable levels on the surface of lung cancer cells and that these cells can not be killed by anti-CD22 immunotoxins. PMID:24395821

Expression of beta-dystroglycan is reduced or absent in many human carcinomas.

PubMed

Cross, S S; Lippitt, J; Mitchell, A; Hollingsbury, F; Balasubramanian, S P; Reed, M W R; Eaton, C; Catto, J W; Hamdy, F; Winder, S J

2008-11-01

Dystroglycan is an important structural and signalling protein that is expressed in most human cells. alpha-Dystroglycan has been investigated and found to be reduced in human cancers, but there is only one published study on the expression of beta-dystroglycan in human cancer and that was only on small numbers of breast and prostatic cancers. The aim was to conduct a comprehensive immunohistochemical survey of the expression of beta-dystroglycan in normal human tissues and common cancers. Triplicate tissue microarrays of 681 samples of normal human tissues and common cancers were stained using an antibody directed against the cytoplasmic component of beta-dystroglycan. beta-Dystroglycan was strongly expressed at the intercellular junctions and basement membranes of all normal human epithelia. Expression of beta-dystroglycan was absent or markedly reduced in 100% of oesophageal adenocarcinomas, 97% of colonic cancers, 100% of transitional cell carcinomas of the urothelium and 94% of breast cancers. In the breast cancers, the only tumours that showed any retention of beta-dystroglycan expression were small low-grade oestrogen receptor-positive tumours. The only cancers that showed retention of beta-dystroglycan expression were cutaneous basal cell carcinomas. There is loss or marked reduction of beta-dystroglycan expression (by immunohistochemistry) in the vast majority of human cancers surveyed. Since beta-dystroglycan is postulated to have a tumour suppressor effect, this loss may have important functional significance.

Annual Progress Report FY 93 (Walter Reed Army Medical Center, Department of Clinical Investigation). Clinical Investigation Program. Volume 2

DTIC Science & Technology

1993-01-01

Effect of Methotrexate on Expression of Interceilular Adhesion Molecule I in Interleukin- 1 Stimulated Cultured Human Cells KEYWORDS: methotrexate...on interleukin- 1 ( IL -I) induced ICAM-l expression in cultured human fibroblasts and adenocarcinoma cells. TECHNICAL APPROACH Expression of ICAM- 1 will...incubated with IL -l to stimulate ICAM-l expression. In general, the cells responded well. ICAM- 1 was readily detectable with monoclonal antibodies and flow

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.

Comparison of two eukaryotic systems for the expression of VP6 protein of rotavirus specie A: transient gene expression in HEK293-T cells and insect cell-baculovirus system.

PubMed

da Silva Junior, Haroldo Cid; da Silva E Mouta Junior, Sérgio; de Mendonça, Marcos César Lima; de Souza Pereira, Mirian Claudia; da Rocha Nogueira, Alanderson; de Azevedo, Maria Luiza Borges; Leite, José Paulo Gagliardi; de Moraes, Márcia Terezinha Baroni

2012-09-01

The VP6 protein of rotavirus A (RVA) is a target antigen used for diagnostic assays and also for the development of new RVA vaccines. We have compared the expression of VP6 protein in human embryonic kidney (HEK293-T) cells with results obtained using a well-established insect cell-baculovirus system. The recombinant VP6 (rVP6) expressed in HEK293-T cells did not present degradation and also retained the ability to form trimers. In the insect cell-baculovirus system, rVP6 was expressed at higher levels and with protein degradation as well as partial loss of ability to form trimers was observed. Therefore, HEK293-T cells represent a less laborious alternative system than insect cells for expression of rVP6 from human RVA.

MERP1: a mammalian ependymin-related protein gene differentially expressed in hematopoietic cells.

PubMed

Gregorio-King, Claudia C; McLeod, Janet L; Collier, Fiona McL; Collier, Gregory R; Bolton, Karyn A; Van Der Meer, Gavin J; Apostolopoulos, Jim; Kirkland, Mark A

2002-03-20

We have utilized differential display polymerase chain reaction to investigate the gene expression of hematopoietic progenitor cells from adult bone marrow and umbilical cord blood. A differentially expressed gene was identified in CD34+ hematopoietic progenitor cells, with low expression in CD34- cells. We have obtained the full coding sequence of this gene which we designated human mammalian ependymin-related protein 1 (MERP1). Expression of MERP1 was found in a variety of normal human tissues, and is 4- and 10-fold higher in adult bone marrow and umbilical cord blood CD34+ cells, respectively, compared to CD34- cells. Additionally, MERP1 expression in a hematopoietic stem cell enriched population was down-regulated with proliferation and differentiation. Conceptual translation of the MERP1 open reading frame reveals significant homology to two families of glycoprotein calcium-dependant cell adhesion molecules: ependymins and protocadherins.

Primary human cervical carcinoma cells require human papillomavirus E6 and E7 expression for ongoing proliferation

PubMed Central

Magaldi, Thomas G.; Almstead, Laura L.; Bellone, Stefania; Prevatt, Edward G.; Santin, Alessandro D.; DiMaio, Daniel

2011-01-01

Repression of human papillomavirus (HPV) E6 and E7 oncogenes in established cervical carcinoma cell lines causes senescence due to reactivation of cellular tumor suppressor pathways. Here, we determined whether ongoing expression of HPV16 or HPV18 oncogenes is required for the proliferation of primary human cervical carcinoma cells in serum-free conditions at low passage number after isolation from patients. We used an SV40 viral vector expressing the bovine papillomavirus E2 protein to repress E6 and E7 in these cells. To enable efficient SV40 infection and E2 gene delivery, we first incubated the primary cervical cancer cells with the ganglioside GM1, a cell-surface receptor for SV40 limiting in these cells. Repression of HPV in primary cervical carcinoma cells caused them to undergo senescence, but the E2 protein had little effect on HPV-negative primary cells. These data suggest that E6 and E7 dependence is an inherent property of human cervical cancer cells. PMID:22056390

Functional properties of cells obtained from human cord blood CD34+ stem cells and mouse cardiac myocytes in coculture.

PubMed

Orlandi, Alessia; Pagani, Francesca; Avitabile, Daniele; Bonanno, Giuseppina; Scambia, Giovanni; Vigna, Elisa; Grassi, Francesca; Eusebi, Fabrizio; Fucile, Sergio; Pesce, Maurizio; Capogrossi, Maurizio C

2008-04-01

Prior in vitro studies suggested that different types of hematopoietic stem cells may differentiate into cardiomyocytes. The present work examined whether human CD34(+) cells from the human umbilical cord blood (hUCB), cocultured with neonatal mouse cardiomyocytes, acquire the functional properties of myocardial cells and express human cardiac genes. hUCB CD34(+) cells were cocultured onto cardiomyocytes following an infection with a lentivirus-encoding enhanced green fluorescent protein (EGFP). After 7 days, mononucleated EGFP(+) cells were tested for their electrophysiological features by patch clamp and for cytosolic [Ca(2+)] ([Ca(2+)](i)) homeostasis by [Ca(2+)](i) imaging of X-rhod1-loaded cells. Human Nkx2.5 and GATA-4 expression was examined in cocultured cell populations by real-time RT-PCR. EGFP(+) cells were connected to surrounding cells by gap junctions, acquired electrophysiological properties similar to those of cardiomyocytes, and showed action potential-associated [Ca(2+)](i) transients. These cells also exhibited spontaneous sarcoplasmic reticulum [Ca(2+)](i) oscillations and the associated membrane potential depolarization. However, RT-PCR of both cell populations showed no upregulation of human-specific cardiac genes. In conclusion, under our experimental conditions, hUCB CD34(+) cells cocultured with murine cardiomyocytes formed cells that exhibited excitation-contraction coupling features similar to those of cardiomyocytes. However, the expression of human-specific cardiac genes was undetectable by RT-PCR.

Transcriptomic Analysis and Meta-Analysis of Human Granulosa and Cumulus Cells

PubMed Central

Burnik Papler, Tanja; Vrtacnik Bokal, Eda; Maver, Ales; Kopitar, Andreja Natasa; Lovrečić, Luca

2015-01-01

Specific gene expression in oocytes and its surrounding cumulus (CC) and granulosa (GC) cells is needed for successful folliculogenesis and oocyte maturation. The aim of the present study was to compare genome-wide gene expression and biological functions of human GC and CC. Individual GC and CC were derived from 37 women undergoing IVF procedures. Gene expression analysis was performed using microarrays, followed by a meta-analysis. Results were validated using quantitative real-time PCR. There were 6029 differentially expressed genes (q < 10−4); of which 650 genes had a log2 FC ≥ 2. After the meta-analysis there were 3156 genes differentially expressed. Among these there were genes that have previously not been reported in human somatic follicular cells, like prokineticin 2 (PROK2), higher expressed in GC, and pregnancy up-regulated nonubiquitous CaM kinase (PNCK), higher expressed in CC. Pathways like inflammatory response and angiogenesis were enriched in GC, whereas in CC, cell differentiation and multicellular organismal development were among enriched pathways. In conclusion, transcriptomes of GC and CC as well as biological functions, are distinctive for each cell subpopulation. By describing novel genes like PROK2 and PNCK, expressed in GC and CC, we upgraded the existing data on human follicular biology. PMID:26313571

Rapid alterations of cell cycle control proteins in human T lymphocytes in microgravity

PubMed Central

2012-01-01

In our study we aimed to identify rapidly reacting gravity-responsive mechanisms in mammalian cells in order to understand if and how altered gravity is translated into a cellular response. In a combination of experiments using "functional weightlessness" provided by 2D-clinostats and real microgravity provided by several parabolic flight campaigns and compared to in-flight-1g-controls, we identified rapid gravity-responsive reactions inside the cell cycle regulatory machinery of human T lymphocytes. In response to 2D clinorotation, we detected an enhanced expression of p21 Waf1/Cip1 protein within minutes, less cdc25C protein expression and enhanced Ser147-phosphorylation of cyclinB1 after CD3/CD28 stimulation. Additionally, during 2D clinorotation, Tyr-15-phosphorylation occurred later and was shorter than in the 1 g controls. In CD3/CD28-stimulated primary human T cells, mRNA expression of the cell cycle arrest protein p21 increased 4.1-fold after 20s real microgravity in primary CD4+ T cells and 2.9-fold in Jurkat T cells, compared to 1 g in-flight controls after CD3/CD28 stimulation. The histone acetyltransferase (HAT) inhibitor curcumin was able to abrogate microgravity-induced p21 mRNA expression, whereas expression was enhanced by a histone deacetylase (HDAC) inhibitor. Therefore, we suppose that cell cycle progression in human T lymphocytes requires Earth gravity and that the disturbed expression of cell cycle regulatory proteins could contribute to the breakdown of the human immune system in space. PMID:22273506

Human T-cell leukemia virus type 1 Tax oncoprotein represses the expression of the BCL11B tumor suppressor in T-cells

PubMed Central

Takachi, Takayuki; Takahashi, Masahiko; Takahashi-Yoshita, Manami; Higuchi, Masaya; Obata, Miki; Mishima, Yukio; Okuda, Shujiro; Tanaka, Yuetsu; Matsuoka, Masao; Saitoh, Akihiko; Green, Patrick L; Fujii, Masahiro

2015-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia (ATL), which is an aggressive form of T-cell malignancy. HTLV-1 oncoproteins, Tax and HBZ, play crucial roles in the immortalization of T-cells and/or leukemogenesis by dysregulating the cellular functions in the host. Recent studies show that HTLV-1-infected T-cells have reduced expression of the BCL11B tumor suppressor protein. In the present study, we explored whether Tax and/or HBZ play a role in downregulating BCL11B in HTLV-1-infected T-cells. Lentiviral transduction of Tax in a human T-cell line repressed the expression of BCL11B at both the protein and mRNA levels, whereas the transduction of HBZ had little effect on the expression. Tax mutants with a decreased activity for the NF-κB, CREB or PDZ protein pathways still showed a reduced expression of the BCL11B protein, thereby implicating a different function of Tax in BCL11B downregulation. In addition, the HTLV-2 Tax2 protein reduced the BCL11B protein expression in T-cells. Seven HTLV-1-infected T-cell lines, including three ATL-derived cell lines, showed reduced BCL11B mRNA and protein expression relative to an uninfected T-cell line, and the greatest reductions were in the cells expressing Tax. Collectively, these results indicate that Tax is responsible for suppressing BCL11B protein expression in HTLV-1-infected T-cells; Tax-mediated repression of BCL11B is another mechanism that Tax uses to promote oncogenesis of HTLV-1-infected T-cells. PMID:25613934

The SGBS cell strain as a model for the in vitro study of obesity and cancer.

PubMed

Allott, Emma H; Oliver, Elizabeth; Lysaght, Joanne; Gray, Steven G; Reynolds, John V; Roche, Helen M; Pidgeon, Graham P

2012-10-01

The murine adipocyte cell line 3T3-L1 is well characterised and used widely, while the human pre-adipocyte cell strain, Simpson-Golabi-Behmel Syndrome (SGBS), requires validation for use in human studies. Obesity is currently estimated to account for up to 41 % of the worldwide cancer burden. A human in vitro model system is required to elucidate the molecular mechanisms for this poorly understood association. This work investigates the relevance of the SGBS cell strain for obesity and cancer research in humans. Pre-adipocyte 3T3-L1 and SGBS were differentiated according to standard protocols. Morphology was assessed by Oil Red O staining. Adipocyte-specific gene expression was measured by qPCR and biochemical function was assessed by glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity. Differential gene expression in oesophageal adenocarcinoma cell line OE33 following co-culture with SGBS or primary omental human adipocytes was investigated using Human Cancer Profiler qPCR arrays. During the process of differentiation, SGBS expressed higher levels of adipocyte-specific transcripts and fully differentiated SGBS expressed more similar morphology, transcript levels and biochemical function to primary omental adipocytes, relative to 3T3-L1. Co-culture with SGBS or primary omental adipocytes induced differential expression of genes involved in adhesion (ITGB3), angiogenesis (IGF1, TEK, TNF, VEGFA), apoptosis (GZMA, TERT) and invasion and metastasis (MMP9, TIMP3) in OE33 tumour cells. Comparable adipocyte-specific gene expression, biochemical function and a shared induced gene signature in co-cultured OE33 cells indicate that SGBS is a relevant in vitro model for obesity and cancer research in humans.

Glutathione S-transferase expression and isoenzyme composition during cell differentiation of Caco-2 cells.

PubMed

Scharmach, E; Hessel, S; Niemann, B; Lampen, A

2009-11-30

The human colon adenocarcinoma cell line Caco-2 is frequently used to study human intestinal metabolism and transport of xenobiotica. Previous studies have shown that both Caco-2 cells and human colon cells constitutively express the multigene family of detoxifying enzymes glutathione S-transferases (GSTs), particularly GST alpha and GST pi. GSTs may play a fundamental role in the molecular interplay between phase I, II enzymes and ABC-transporters. The gut fermentation product, butyrate, can modulate the potential for detoxification. The aim of this study was to investigate the basal expression of further cytosolic GSTs in Caco-2 cells during cell differentiation. In addition, a comparison was made with expression levels in MCF-7 and HepG2, two other cell types with barrier functions. Finally, the butyrate-mediated modulation of gene and protein expression was determined by real time PCR and western blot analysis. In Caco-2, gene and protein expression levels of GST alpha increased during cell differentiation. High levels of GSTO1 and GSTP1 were constantly expressed. No expression of GSTM5 and GSTT1 was detected. HepG2 expressed GSTO1 and MCF-7 GSTZ1 most intensively. No expression of GSTA5, GSTM5, or GSTP1 was detected in either cell. Incubation of Caco-2 cells with butyrate (5 mM) significantly induced GSTA1 and GSTM2 in proliferating Caco-2 cells. In differentiated cells, butyrate tended to increase GSTO1 and GSTP1. The results of this study show that a differentiation-dependent expression of GSTs in Caco-2 cells may reflect the in vivo situation and indicate the potential of butyrate to modify intestinal metabolism. GSTA1-A4 have been identified as good markers for cell differentiation. The Caco-2 cell line is a useful model for assessing the potential of food-related substances to modulate the GST expression pattern.

Cafestol overcomes ABT-737 resistance in Mcl-1-overexpressed renal carcinoma Caki cells through downregulation of Mcl-1 expression and upregulation of Bim expression.

PubMed

Woo, S M; Min, K-J; Seo, B R; Nam, J-O; Choi, K S; Yoo, Y H; Kwon, T K

2014-11-06

Although ABT-737, a small-molecule Bcl-2/Bcl-xL inhibitor, has recently emerged as a novel cancer therapeutic agent, ABT-737-induced apoptosis is often blocked in several types of cancer cells with elevated expression of Mcl-1. Cafestol, one of the major compounds in coffee beans, has been reported to have anti-carcinogenic activity and tumor cell growth-inhibitory activity, and we examined whether cafestol could overcome resistance against ABT-737 in Mcl-1-overexpressed human renal carcinoma Caki cells. ABT-737 alone had no effect on apoptosis, but cafestol markedly enhanced ABT-737-mediated apoptosis in Mcl-1-overexpressed Caki cells, human glioma U251MG cells, and human breast carcinoma MDA-MB231 cells. By contrast, co-treatment with ABT-737 and cafestol did not induce apoptosis in normal human skin fibroblast. Furthermore, combined treatment with cafestol and ABT-737 markedly reduced tumor growth compared with either drug alone in xenograft models. We found that cafestol inhibited Mcl-1 protein expression, which is important for ABT-737 resistance, through promotion of protein degradation. Moreover, cafestol increased Bim expression, and siRNA-mediated suppression of Bim expression reduced the apoptosis induced by cafestol plus ABT-737. Taken together, cafestol may be effectively used to enhance ABT-737 sensitivity in cancer therapy via downregulation of Mcl-1 expression and upregulation of Bim expression.

Unique and Common Features of Innate-Like Human Vδ2+ γδT Cells and Mucosal-Associated Invariant T Cells.

PubMed

Provine, Nicholas M; Binder, Benedikt; FitzPatrick, Michael E B; Schuch, Anita; Garner, Lucy C; Williamson, Kate D; van Wilgenburg, Bonnie; Thimme, Robert; Klenerman, Paul; Hofmann, Maike

2018-01-01

Mucosal-associated invariant T (MAIT) cells are innate-like T cells abundant in humans that can be activated in a TCR-independent manner by inflammatory and antiviral cytokines. In humans, the capacity for TCR-independent activation is functionally linked to a transcriptional program that can be identified by the expression of the C-type lectin receptor, CD161. In addition to MAIT cells, it has been demonstrated that a subset of γδT cells expresses CD161 and can be activated by TCR-independent cytokine stimulation. In this study, we sought to clarify the nature of cytokine-responsive human γδT cells. We could link CD161 expression on Vδ2 + versus Vδ1 + γδT cells to the observation that Vδ2 + γδT cells, but not Vδ1 + γδT cells, robustly produced IFN-γ upon stimulation with a variety of cytokine combinations. Interestingly, both CD161 + and CD161 - Vδ2 + γδT cells responded to these stimuli, with increased functionality within the CD161 + subset. This innate-like responsiveness corresponded to high expression of PLZF and IL-18Rα, analogous to MAIT cells. Vδ2 + γδT cells in human duodenum and liver maintained a CD161 + IL-18Rα + phenotype and produced IFN-γ in response to IL-12 and IL-18 stimulation. In contrast to MAIT cells, we could not detect IL-17A production but observed higher steady-state expression of Granzyme B by Vδ2 + γδT cells. Finally, we investigated the frequency and functionality of γδT cells in the context of chronic hepatitis C virus infection, as MAIT cells are reduced in frequency in this disease. By contrast, Vδ2 + γδT cells were maintained in frequency and displayed unimpaired IFN-γ production in response to cytokine stimulation. In sum, human Vδ2 + γδT cells are a functionally distinct population of cytokine-responsive innate-like T cells that is abundant in blood and tissues with similarities to human MAIT cells.

Tumor-derived exosomes regulate expression of immune function-related genes in human T cell subsets.

PubMed

Muller, Laurent; Mitsuhashi, Masato; Simms, Patricia; Gooding, William E; Whiteside, Theresa L

2016-02-04

Tumor cell-derived exosomes (TEX) suppress functions of immune cells. Here, changes in the gene profiles of primary human T lymphocytes exposed in vitro to exosomes were evaluated. CD4(+) Tconv, CD8(+) T or CD4(+) CD39(+) Treg were isolated from normal donors' peripheral blood and co-incubated with TEX or exosomes isolated from supernatants of cultured dendritic cells (DEX). Expression levels of 24-27 immune response-related genes in these T cells were quantified by qRT-PCR. In activated T cells, TEX and DEX up-regulated mRNA expression levels of multiple genes. Multifactorial data analysis of ΔCt values identified T cell activation and the immune cell type, but not exosome source, as factors regulating gene expression by exosomes. Treg were more sensitive to TEX-mediated effects than other T cell subsets. In Treg, TEX-mediated down-regulation of genes regulating the adenosine pathway translated into high expression of CD39 and increased adenosine production. TEX also induced up-regulation of inhibitory genes in CD4(+) Tconv, which translated into a loss of CD69 on their surface and a functional decline. Exosomes are not internalized by T cells, but signals they carry and deliver to cell surface receptors modulate gene expression and functions of human T lymphocytes.

Human embryonic stem cells express a unique set of microRNAs.

PubMed

Suh, Mi-Ra; Lee, Yoontae; Kim, Jung Yeon; Kim, Soo-Kyoung; Moon, Sung-Hwan; Lee, Ji Yeon; Cha, Kwang-Yul; Chung, Hyung Min; Yoon, Hyun Soo; Moon, Shin Yong; Kim, V Narry; Kim, Kye-Seong

2004-06-15

Human embryonic stem (hES) cells are pluripotent cell lines established from the explanted inner cell mass of human blastocysts. Despite their importance for human embryology and regenerative medicine, studies on hES cells, unlike those on mouse ES (mES) cells, have been hampered by difficulties in culture and by scant knowledge concerning the regulatory mechanism. Recent evidence from plants and animals indicates small RNAs of approximately 22 nucleotides (nt), collectively named microRNAs, play important roles in developmental regulation. Here we describe 36 miRNAs (from 32 stem-loops) identified by cDNA cloning in hES cells. Importantly, most of the newly cloned miRNAs are specifically expressed in hES cells and downregulated during development into embryoid bodies (EBs), while miRNAs previously reported from other human cell types are poorly expressed in hES cells. We further show that some of the ES-specific miRNA genes are highly related to each other, organized as clusters, and transcribed as polycistronic primary transcripts. These miRNA gene families have murine homologues that have similar genomic organizations and expression patterns, suggesting that they may operate key regulatory networks conserved in mammalian pluripotent stem cells. The newly identified hES-specific miRNAs may also serve as molecular markers for the early embryonic stage and for undifferentiated hES cells.

Leptin and Pro-Inflammatory Stimuli Synergistically Upregulate MMP-1 and MMP-3 Secretion in Human Gingival Fibroblasts.

PubMed

Williams, Rachel C; Skelton, Andrew J; Todryk, Stephen M; Rowan, Andrew D; Preshaw, Philip M; Taylor, John J

2016-01-01

Gingival fibroblast-mediated extracellular matrix remodelling is implicated in the pathogenesis of periodontitis, yet the stimuli that regulate this response are not fully understood. The immunoregulatory adipokine leptin is detectable in the gingiva, human gingival fibroblasts express functional leptin receptor mRNA and leptin is known to regulate extracellular matrix remodelling responses in cardiac fibroblasts. We therefore hypothesised that leptin would enhance matrix metalloproteinase secretion in human gingival fibroblasts. We used in vitro cell culture to investigate leptin signalling and the effect of leptin on mRNA and protein expression in human gingival fibroblasts. We confirmed human gingival fibroblasts expressed cell surface leptin receptor, found leptin increased matrix metalloproteinase-1, -3, -8 and -14 expression in human gingival fibroblasts compared to unstimulated cells, and observed that leptin stimulation activated MAPK, STAT1/3 and Akt signalling in human gingival fibroblasts. Furthermore, leptin synergised with IL-1 or the TLR2 agonist pam2CSK4 to markedly enhance matrix metalloproteinase-1 and -3 production by human gingival fibroblasts. Signalling pathway inhibition demonstrated ERK was required for leptin-stimulated matrix metalloproteinase-1 expression in human gingival fibroblasts; whilst ERK, JNK, p38 and STAT3 were required for leptin+IL-1- and leptin+pam2CSK4-induced matrix metalloproteinase-1 expression. A genome-wide expression array and gene ontology analysis confirmed genes differentially expressed in leptin+IL-1-stimulated human gingival fibroblasts (compared to unstimulated cells) were enriched for extracellular matrix organisation and disassembly, and revealed that matrix metalloproteinase-8 and -12 were also synergistically upregulated by leptin+IL-1 in human gingival fibroblasts. We conclude that leptin selectively enhances the expression and secretion of certain matrix metalloproteinases in human gingival fibroblasts, and suggest that gingival fibroblasts may have an ECM-degrading phenotype during conditions of hyperleptinaemia (e.g., obesity, type 2 diabetes mellitus, exogenous leptin therapy).

Leptin and Pro-Inflammatory Stimuli Synergistically Upregulate MMP-1 and MMP-3 Secretion in Human Gingival Fibroblasts

PubMed Central

Williams, Rachel C.; Skelton, Andrew J.; Todryk, Stephen M.; Rowan, Andrew D.; Preshaw, Philip M.; Taylor, John J.

2016-01-01

Introduction Gingival fibroblast-mediated extracellular matrix remodelling is implicated in the pathogenesis of periodontitis, yet the stimuli that regulate this response are not fully understood. The immunoregulatory adipokine leptin is detectable in the gingiva, human gingival fibroblasts express functional leptin receptor mRNA and leptin is known to regulate extracellular matrix remodelling responses in cardiac fibroblasts. We therefore hypothesised that leptin would enhance matrix metalloproteinase secretion in human gingival fibroblasts. Methods and Results We used in vitro cell culture to investigate leptin signalling and the effect of leptin on mRNA and protein expression in human gingival fibroblasts. We confirmed human gingival fibroblasts expressed cell surface leptin receptor, found leptin increased matrix metalloproteinase-1, -3, -8 and -14 expression in human gingival fibroblasts compared to unstimulated cells, and observed that leptin stimulation activated MAPK, STAT1/3 and Akt signalling in human gingival fibroblasts. Furthermore, leptin synergised with IL-1 or the TLR2 agonist pam2CSK4 to markedly enhance matrix metalloproteinase-1 and -3 production by human gingival fibroblasts. Signalling pathway inhibition demonstrated ERK was required for leptin-stimulated matrix metalloproteinase-1 expression in human gingival fibroblasts; whilst ERK, JNK, p38 and STAT3 were required for leptin+IL-1- and leptin+pam2CSK4-induced matrix metalloproteinase-1 expression. A genome-wide expression array and gene ontology analysis confirmed genes differentially expressed in leptin+IL-1-stimulated human gingival fibroblasts (compared to unstimulated cells) were enriched for extracellular matrix organisation and disassembly, and revealed that matrix metalloproteinase-8 and -12 were also synergistically upregulated by leptin+IL-1 in human gingival fibroblasts. Conclusions We conclude that leptin selectively enhances the expression and secretion of certain matrix metalloproteinases in human gingival fibroblasts, and suggest that gingival fibroblasts may have an ECM-degrading phenotype during conditions of hyperleptinaemia (e.g., obesity, type 2 diabetes mellitus, exogenous leptin therapy). PMID:26829555

Lactobacillus rhamnosus GG and its SpaC pilus adhesin modulate inflammatory responsiveness and TLR-related gene expression in the fetal human gut

PubMed Central

Ganguli, Kriston; Collado, Maria Carmen; Rautava, Jaana; Lu, Lei; Satokari, Reetta; von Ossowski, Ingemar; Reunanen, Justus; de Vos, Willem M.; Palva, Airi; Isolauri, Erika; Salminen, Seppo; Walker, W. Allan; Rautava, Samuli

2015-01-01

Background Bacterial contact in utero modulates fetal and neonatal immune responses. Maternal probiotic supplementation reduces the risk of immune-mediated disease in the infant. We investigated the immunomodulatory properties of live Lactobacillus rhamnosus GG and its SpaC pilus adhesin in human fetal intestinal models. Methods TNF-α mRNA expression was measured by qPCR in a human fetal intestinal organ culture model exposed to live L. rhamnosus GG and proinflammatory stimuli. Binding of recombinant SpaC pilus protein to intestinal epithelial cells was assessed in human fetal intestinal organ culture and the human fetal intestinal epithelial cell line H4 by immunohistochemistry and immunofluorescence, respectively. TLR-related gene expression in fetal ileal organ culture after exposure to recombinant SpaC was assessed by qPCR. Results Live L. rhamnosus GG significantly attenuates pathogen-induced TNF-α mRNA expression in the human fetal gut. Recombinant SpaC protein was found to adhere to the fetal gut and to modulate varying levels of TLR-related gene expression. Conclusion The human fetal gut is responsive to luminal microbes. L. rhamnosus GG significantly attenuates fetal intestinal inflammatory responses to pathogenic bacteria. The L. rhamnosus GG pilus adhesin SpaC binds to immature human intestinal epithelial cells and directly modulates intestinal epithelial cell innate immune gene expression. PMID:25580735

The Human Airway Epithelial Basal Cell Transcriptome

PubMed Central

Wang, Rui; Zwick, Rachel K.; Ferris, Barbara; Witover, Bradley; Salit, Jacqueline; Crystal, Ronald G.

2011-01-01

Background The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem/progenitor cells for the other airway cell types. The objective of this study is to better understand human airway epithelial basal cell biology by defining the gene expression signature of this cell population. Methodology/Principal Findings Bronchial brushing was used to obtain airway epithelium from healthy nonsmokers. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium in comparison to the transcriptome of the differentiated airway epithelium. This analysis identified the “human airway basal cell signature” as 1,161 unique genes with >5-fold higher expression level in basal cells compared to differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The basal cell signature displayed overlap with genes expressed in basal-like cells from other human tissues and with that of murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the human airway basal cell signature was characterized by genes encoding extracellular matrix components, growth factors and growth factor receptors, including genes related to the EGF and VEGF pathways. Interestingly, while the basal cell signature overlaps that of basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels. Conclusion/Significance The human airway epithelial basal cell signature identified in the present study provides novel insights into the molecular phenotype and biology of the stem/progenitor cells of the human airway epithelium. PMID:21572528

Enhanced expression of FNDC5 in human embryonic stem cell-derived neural cells along with relevant embryonic neural tissues.

PubMed

Ghahrizjani, Fatemeh Ahmadi; Ghaedi, Kamran; Salamian, Ahmad; Tanhaei, Somayeh; Nejati, Alireza Shoaraye; Salehi, Hossein; Nabiuni, Mohammad; Baharvand, Hossein; Nasr-Esfahani, Mohammad Hossein

2015-02-25

Availability of human embryonic stem cells (hESCs) has enhanced the capability of basic and clinical research in the context of human neural differentiation. Derivation of neural progenitor (NP) cells from hESCs facilitates the process of human embryonic development through the generation of neuronal subtypes. We have recently indicated that fibronectin type III domain containing 5 protein (FNDC5) expression is required for appropriate neural differentiation of mouse embryonic stem cells (mESCs). Bioinformatics analyses have shown the presence of three isoforms for human FNDC5 mRNA. To differentiate which isoform of FNDC5 is involved in the process of human neural differentiation, we have used hESCs as an in vitro model for neural differentiation by retinoic acid (RA) induction. The hESC line, Royan H5, was differentiated into a neural lineage in defined adherent culture treated by RA and basic fibroblast growth factor (bFGF). We collected all cell types that included hESCs, rosette structures, and neural cells in an attempt to assess the expression of FNDC5 isoforms. There was a contiguous increase in all three FNDC5 isoforms during the neural differentiation process. Furthermore, the highest level of expression of the isoforms was significantly observed in neural cells compared to hESCs and the rosette structures known as neural precursor cells (NPCs). High expression levels of FNDC5 in human fetal brain and spinal cord tissues have suggested the involvement of this gene in neural tube development. Additional research is necessary to determine the major function of FDNC5 in this process. Copyright © 2014 Elsevier B.V. All rights reserved.

Expression and structural features of endoglin (CD105), a transforming growth factor beta1 and beta3 binding protein, in human melanoma.

PubMed Central

Altomonte, M.; Montagner, R.; Fonsatti, E.; Colizzi, F.; Cattarossi, I.; Brasoveanu, L. I.; Nicotra, M. R.; Cattelan, A.; Natali, P. G.; Maio, M.

1996-01-01

Human endoglin (CD105) is a member of the transforming growth factor beta (TGF-beta) receptor family that binds TGF-beta1 and -beta3, but not TGF-beta2, on human endothelial cells. Immunohistochemical analyses demonstrated that CD105 is expressed on normal and neoplastic cells of the melanocytic lineage. The anti-CD105 MAb, MAEND3, stained 50, 25 and 34% of intradermal naevi, primary and metastatic melanomas investigated, respectively, and nine out of 12 melanoma cell lines. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that CD105 expressed by melanoma cells consists of a homodimeric protein with an apparent molecular weight of 180 and 95 kDa under non-reducing and reducing conditions. Cross-linking of 125I-labelled TGF-beta1 to melanoma cells, Mel 97, by disuccinimidyl suberate (DSS) demonstrated that CD105 expressed on pigmented cells binds TGF-beta1; the pattern of binding of TGF-beta1 to melanoma cells was found to be similar to that of human umbilical vein endothelial cells. The addition of exogenous, bioactive TGF-beta1 significantly (P<0.05) inhibited the growth of CD105-positive melanoma cells, Mel 97, but did not affect that of CD105-negative melanoma cells, F0-1. These data, altogether, demonstrate that CD105 is expressed on pigmented cells and might play a functionally relevant role in the biology of human melanoma cells by regulating their sensitivity to TGF-betas. Images Figure 1 Figure 3 Figure 4 PMID:8932339

The expression of the class 1 glucose transporter isoforms in human embryonic stem cells, and the potential use of GLUT2 as a marker for pancreatic progenitor enrichment.

PubMed

Segev, Hana; Fishman, Betina; Schulman, Rita; Itskovitz-Eldor, Joseph

2012-07-01

Even before the first appearance of the developing pancreas, glucose is the major substrate in the growing embryo. The transport of glucose across cell membranes is facilitated by a family of membranal glucose transporters (GLUT). We analyzed changes in expression of class 1 glucose transporters (GLUT1-4) during human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC) differentiation, from undifferentiated cells to 28-day-old embryoid bodies (EBs). We also examined the potential use of GLUT2 as a marker for differentiating pancreatic progenitor cells. Using quantitative real time polymerase chain reaction (qPCR), western blot, and immunofluorescence, we observed enhanced expression of GLUT1 and GLUT2 during differentiation, but only minor change in GLUT3 expression. GLUT4 expression was found to be very low both at the RNA and in the protein levels. Expression of the early pancreatic transcription factor, pancreatic duodenal homeobox gene 1 (PDX1), correlated with GLUT2 expression, suggesting the potential use of GLUT2 as a surface marker for tracking pancreatic precursor cells. After sorting EBs according to their membranal GLUT2 expression, GLUT2 and PDX1 expression were found elevated, as was expression of other endodermal markers such as PAX4, NGN3, CXCR4, and SOX17. This simple method may be used to differentiate embryonic stem cells and to isolate from them, using GLUT2 as a surface marker, an enriched pancreatic progenitor cell population in order to achieve insulin-producing cells. The sorted GLUT2 cells may potentially be used in the future as insulin-producing cells for beta cell therapies.

Design of a muscle cell-specific expression vector utilising human vascular smooth muscle alpha-actin regulatory elements.

PubMed

Keogh, M C; Chen, D; Schmitt, J F; Dennehy, U; Kakkar, V V; Lemoine, N R

1999-04-01

The facility to direct tissue-specific expression of therapeutic gene constructs is desirable for many gene therapy applications. We describe the creation of a muscle-selective expression vector which supports transcription in vascular smooth muscle, cardiac muscle and skeletal muscle, while it is essentially silent in other cell types such as endothelial cells, hepatocytes and fibroblasts. Specific transcriptional regulatory elements have been identified in the human vascular smooth muscle cell (VSMC) alpha-actin gene, and used to create an expression vector which directs the expression of genes in cis to muscle cells. The vector contains an enhancer element we have identified in the 5' flanking region of the human VSMC alpha-actin gene involved in mediating VSMC expression. Heterologous pairing experiments have shown that the enhancer does not interact with the basal transcription complex recruited at the minimal SV40 early promoter. Such a vector has direct application in the modulation of VSMC proliferation associated with intimal hyperplasia/restenosis.

Comparison of protein expression between human livers and the hepatic cell lines HepG2, Hep3B, and Huh7 using SWATH and MRM-HR proteomics: Focusing on drug-metabolizing enzymes.

PubMed

Shi, Jian; Wang, Xinwen; Lyu, Lingyun; Jiang, Hui; Zhu, Hao-Jie

2018-04-01

Human hepatic cell lines are widely used as an in vitro model for the study of drug metabolism and liver toxicity. However, the validity of this model is still a subject of debate because the expressions of various proteins in the cell lines, including drug-metabolizing enzymes (DMEs), can differ significantly from those in human livers. In the present study, we first conducted an untargeted proteomics analysis of the microsomes of the cell lines HepG2, Hep3B, and Huh7, and compared them to human livers using a sequential window acquisition of all theoretical mass spectra (SWATH) method. Furthermore, high-resolution multiple reaction monitoring (MRM-HR), a targeted proteomic approach, was utilized to compare the expressions of pre-selected DMEs between human livers and the cell lines. In general, the SWATH quantifications were in good agreement with the MRM-HR analysis. Over 3000 protein groups were quantified in the cells and human livers, and the proteome profiles of human livers significantly differed from the cell lines. Among the 101 DMEs quantified with MRM-HR, most were expressed at substantially lower levels in the cell lines. Thus, appropriate caution must be exercised when using these cell lines for the study of hepatic drug metabolism and toxicity. Copyright © 2018 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma.

PubMed

Kayamori, Kou; Katsube, Ken-Ichi; Sakamoto, Kei; Ohyama, Yoshio; Hirai, Hideaki; Yukimori, Akane; Ohata, Yae; Akashi, Takumi; Saitoh, Masao; Harada, Kiyoshi; Harada, Hiroyuki; Yamaguchi, Akira

2016-01-01

Recent studies have shown that Notch signaling is involved in many types of cancers, including oral squamous cell carcinomas (OSCCs). However, the role of Notch signaling in the tumor microenvironment is not yet fully understood. In this study, we investigated the roles of NOTCH3 signaling in cancer associated fibroblasts (CAFs) in OSCCs. Immunohistochemical study of 93 human tongue OSCC cases indicated that about one third of OSCCs showed NOTCH3 expression in CAFs, and that this expression significantly correlated with tumor-size. In vitro study showed that OSCC cell lines, especially HO1-N-1 cells stimulated NOTCH3 expression in normal human dermal fibroblasts (NHDFs) through direct cell-to-cell contact. Immunohistochemical and morphometric analysis using human OSCC samples demonstrated that NOTCH3 expression in CAFs significantly correlated with micro-vessel density in cancer stroma. In vitro angiogenesis assays involving co-culture of NHDFs with HO1-N-1 and human umbilical endothelial cells (HUVECs), and NOTCH3 knockdown in NHDFs using siRNA, demonstrated that HO1-N-1 cells significantly promoted tube formation dependent on NOTCH3-expression in NHDFs. Moreover, NOTCH3 expression in CAFs was related to poor prognosis of the OSCC patients. This work provides a new insight into the role of Notch signaling in CAFs associated with tumor angiogenesis and the possibility of NOTCH3-targeted molecular therapy in OSCCs.

NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

PubMed Central

Kayamori, Kou; Katsube, Ken-ichi; Sakamoto, Kei; Ohyama, Yoshio; Hirai, Hideaki; Yukimori, Akane; Ohata, Yae; Akashi, Takumi; Saitoh, Masao; Harada, Kiyoshi; Harada, Hiroyuki; Yamaguchi, Akira

2016-01-01

Recent studies have shown that Notch signaling is involved in many types of cancers, including oral squamous cell carcinomas (OSCCs). However, the role of Notch signaling in the tumor microenvironment is not yet fully understood. In this study, we investigated the roles of NOTCH3 signaling in cancer associated fibroblasts (CAFs) in OSCCs. Immunohistochemical study of 93 human tongue OSCC cases indicated that about one third of OSCCs showed NOTCH3 expression in CAFs, and that this expression significantly correlated with tumor-size. In vitro study showed that OSCC cell lines, especially HO1-N-1 cells stimulated NOTCH3 expression in normal human dermal fibroblasts (NHDFs) through direct cell-to-cell contact. Immunohistochemical and morphometric analysis using human OSCC samples demonstrated that NOTCH3 expression in CAFs significantly correlated with micro-vessel density in cancer stroma. In vitro angiogenesis assays involving co-culture of NHDFs with HO1-N-1 and human umbilical endothelial cells (HUVECs), and NOTCH3 knockdown in NHDFs using siRNA, demonstrated that HO1-N-1 cells significantly promoted tube formation dependent on NOTCH3-expression in NHDFs. Moreover, NOTCH3 expression in CAFs was related to poor prognosis of the OSCC patients. This work provides a new insight into the role of Notch signaling in CAFs associated with tumor angiogenesis and the possibility of NOTCH3-targeted molecular therapy in OSCCs. PMID:27124156

NLRP3 in Somatic Non-Immune Cells of Rodent and Primate Testes.

PubMed

Walenta, Lena; Schmid, Nina; Schwarzer, J U; Köhn, Frank-Michael; Urbanski, Henryk; Behr, Ruediger; Strauss, Leena; Poutanen, Matti; Mayerhofer, Artur

2018-06-15

NLRP3 is part of the NLRP3 inflammasome and a global sensor of cellular damage. It was recently discovered in rodent Sertoli cells. We investigated NLRP3 in mouse, human and non-human primate (marmoset and rhesus macaque) testes, employing immunohistochemistry. Sertoli cells of all species expressed NLRP3, and the expression preceded puberty. In addition, peritubular cells of the adult human testes expressed NLRP3. NLRP3 and associated genes (ASC, CASP1, IL1B) were also found in isolated human testicular peritubular cells (HTPCs) and the mouse Sertoli cell line TM4. Male infertility due to impairments of spermatogenesis may be related to sterile inflammatory events. We observed that the expression of NLRP3 was altered in the testes of patients suffering from mixed atrophy syndrome, in which tubules with impairments of spermatogenesis showed prominent NLRP3 staining. In order to explore a possible role of NLRP3 in male infertility, associated with sterile testicular inflammation, we studied a mouse model of male infertility. These human aromatase expressing transgenic mice (AROM+) develop testicular inflammation and impaired spermatogenesis during aging, and the present data show that this is associated with strikingly elevated Nlrp3 expression in the testes compared to wild type controls. Interference by aromatase inhibitor treatment significantly reduced increased Nlrp3 levels. Thus, throughout species NLRP3 is expressed by somatic cells of the testis, which are involved in testicular immune surveillance. We conclude that NLRP3 may be a novel player in testicular immune regulation. .

Picfeltarraenin IA inhibits lipopolysaccharide-induced inflammatory cytokine production by the nuclear factor-κB pathway in human pulmonary epithelial A549 cells.

PubMed

Shi, Rong; Wang, Qing; Ouyang, Yang; Wang, Qian; Xiong, Xudong

2016-02-01

The present study aimed to investigate the effect of picfeltarraenin IA (IA) on respiratory inflammation by analyzing its effect on interleukin (IL)-8 and prostaglandin E2 (PGE2) production. The expression of cyclooxygenase 2 (COX2) in human pulmonary adenocarcinoma epithelial A549 cells in culture was also examined. Human pulmonary epithelial A549 cells and the human monocytic leukemia THP-1 cell line were used in the current study. Cell viability was measured using a methylthiazol tetrazolium assay. The production of IL-8 and PGE2 was investigated using an enzyme-linked immunosorbent assay. The expression of COX2 and nuclear factor-κB (NF-κB)-p65 was examined using western blot analysis. Treatment with lipopolysaccharide (LPS; 10 µg/ml) resulted in the increased production of IL-8 and PGE2, and the increased expression of COX2 in the A549 cells. Furthermore, IA (0.1-10 µmol/l) significantly inhibited PGE2 production and COX2 expression in cells with LPS-induced IL-8, in a concentration-dependent manner. The results suggested that IA downregulates LPS-induced COX2 expression, and inhibits IL-8 and PGE2 production in pulmonary epithelial cells. Additionally, IA was observed to suppress the expression of COX2 in THP-1 cells, and also to regulate the expression of COX2 via the NF-κB pathway in the A549 cells, but not in the THP-1 cells. These results indicate that IA regulates LPS-induced cytokine release in A549 cells via the NF-κB pathway.

P16/p53 expression and telomerase activity in immortalized human dental pulp cells

PubMed Central

Egbuniwe, Obi; Idowu, Bernadine D; Funes, Juan M; Grant, Andrew D; Renton, Tara

2011-01-01

Introduction Residing within human dental pulp are cells of an ectomesenchymal origin that have the potential to differentiate into odontoblast-like cells. These cells have a limited growth potential owing to the effects of cell senescence. This study examines the effects of immortalizing odontoblast-like cells on cell proliferation and mineralization by comparing transformed dental pulp stem cells (tDPSCs) and non-transformed dental pulp stem cells (nDPSCs). Results With the exogenous expression of hTERT, tDPSCs maintained a continued expression of odontogenic markers for cell proliferation and mineralization (ALP, COL-1, DMP-1, DSPP, OCN and OPN), as did nDPSCs. Oncoprotein expression was seen in both groups except for a noted absence of p16 in the tDPSCs. nDPSCs also showed lower levels of total ALP and DNA activity in comparison to tDPSCs when assayed, as well as low telomerase activity readings. Methods Using a retroviral vector, exogenous human telomerase reverse transcriptase (hTERT) was expressed in tDPSCs. Both cell groups were cultured, and their telomerase activities were determined using a telomerase quantification assay. Also examined, were the expression of genes involved in proliferation and mineralization, such as human alkaline phosphatase (ALP), β-actin, collagen I (col-1), core binding factor (cbfa)-1, dentin matrix protein (DMP-1), dentin sialophosphoprotein (DSPP), GAPDH, hTERT, osteocalcin (OCN), osteopontin (OPN) as well as oncoproteins involved in senescence (p16, p21 and p53) using RT-PCR. DNA and alkaline phosphate activity was also assayed in both cell groups. Conclusion These results indicate maintenance of odontoblast-like differentiation characteristics after retroviral transformation with hTERT and suggest a possible link with a reduced p16 expression. PMID:22067611

3D-fibroblast tissues constructed by a cell-coat technology enhance tight-junction formation of human colon epithelial cells.

PubMed

Matsusaki, Michiya; Hikimoto, Daichi; Nishiguchi, Akihiro; Kadowaki, Koji; Ohura, Kayoko; Imai, Teruko; Akashi, Mitsuru

2015-02-13

Caco-2, human colon carcinoma cell line, has been widely used as a model system for intestinal epithelial permeability because Caco-2 cells express tight-junctions, microvilli, and a number of enzymes and transporters characteristic of enterocytes. However, the functional differentiation and polarization of Caco-2 cells to express sufficient tight-junctions (a barrier) usually takes over 21 days in culture. This may be due to the cell culture environment, for example inflammation induced by plastic petri dishes. Three-dimensional (3D) sufficient cell microenvironments similar to in vivo natural conditions (proteins and cells), will promote rapid differentiation and higher functional expression of tight junctions. Herein we report for the first time an enhancement in tight-junction formation by 3D-cultures of Caco-2 cells on monolayered (1L) and eight layered (8L) normal human dermal fibroblasts (NHDF). Trans epithelial electric resistance (TEER) of Caco-2 cells was enhanced in the 3D-cultures, especially 8L-NHDF tissues, depending on culture times and only 10 days was enough to reach the same TEER value of Caco-2 monolayers after a 21 day incubation. Relative mRNA expression of tight-junction proteins of Caco-2 cells on 3D-cultures showed higher values than those in monolayer structures. Transporter gene expression patterns of Caco-2 cells on 3D-constructs were almost the same as those of Caco-2 monolayers, suggesting that there was no effect of 3D-cultures on transporter protein expression. The expression correlation between carboxylesterase 1 and 2 in 3D-cultures represented similar trends with human small intestines. The results of this study clearly represent a valuable application of 3D-Caco-2 tissues for pharmaceutical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

Therapeutic effects of naringin on degenerative human nucleus pulposus cells for discogenic low back pain.

PubMed

Li, Nianhu; Whitaker, Camden; Xu, Zhanwang; Heggeness, Michael; Yang, Shang-You

2016-10-01

Over half the population of the world will suffer from moderate or severe low back pain (LBP) during their life span. Studies have shown that naringin, a major flavonoid in grapefruit and an active compound extracted from a Chinese herbal medicine (Rhizoma Drynariae) possesses many pharmacological effects. The aim of this study was to evaluate the influence of naringin on the growth of degenerative human nucleus pulposus (NP) cells, and its repair effects on protein and gene expressions of the cells. This was an in vitro investigation of the human NP cells isolated from degenerated intervertebral discs that were interacted with various concentrated of naringin. This study was exempted by the institutional Human Subjects Committee-2, University of Kansas School of Medicine-Wichita. Degenerative human NP cells were isolated from intervertebral discs of patients with discogenic LBP and cultured at 37°C with 5% CO 2 . The proliferation of NP cells was determined following treatment with various concentrations of naringin. The protein expressions of tumor necrosis factor-α (TNF-α) and Bone morphogenetic protein 2 (BMP-2) were tested using enzyme-linked immunosorbent assay. Aggrecan and type II collagen levels were measured by immunohistological staining. Further examination of the gene expression of aggrecan, Sox6, and MMP3 was performed after intervention with naringin for 3 days. The human NP cells were successfully propagated in culture and stained positive with toluidine blue staining. Naringin effectively enhanced the cell proliferation at an optimal concentration of 20 µg/mL. Naringin treatment resulted in significant inhibition of TNF-α, but elevated protein expressions of BMP-2, collagen II, and aggrecan. Naringin also increased disc matrix gene activity including aggrecan and Sox6, and decreased the gene expression of MMP3. Naringin effectively promotes the proliferation of degenerative human NP cells and improves the recuperation of the cells from degeneration by increasing expression of aggrecan, BMP-2, and Sox6 while inhibiting the expression of TNF-α and MMP3. This study suggests that naringin may represent an alternative therapeutic agent for disc degeneration. Copyright © 2016 Elsevier Inc. All rights reserved.

CCK processing by pituitary GH3 cells, human teratocarcinoma cells NT2 and hNT differentiated human neuronal cells evidence for a differentiation-induced change in enzyme expression and pro CCK processing.

PubMed

Beinfeld, Margery C; Wang, Wenge

2002-02-01

Human teratocarcinoma Ntera2/c 1.D1 (NT2) cells express very low levels of the prohormone convertase enzyme PC1, moderate levels of PC2 and significant levels of PC5. When infected with an adenovirus which expresses rat CCK mRNA, several glycine-extended forms were secreted that co-eluted with CCK 33, 22 and 12. Amidated CCK is not produced because these cells appear to lack the amidating enzyme. Pituitary GH3 cells express high levels of PC2 and PC5. CCK adenovirus-infected GH3 cells secrete amidated versions of the same peptides as NT2 cells. Differentiation of NT2 cells into hNT cells with retinoic acid and mitotic inhibitors increased expression of PC5 and decreased expression of PCI and PC2. CCK adenovirus-infected differentiated hNT cells also secrete glycine extended CCK products and the major molecular form produced co-eluted with CCK 8 Gly. These experiments demonstrate that the state of differentiation of this neuronal cell line influences its expression of PC 1,2, and 5 and its cleavage of pro CCK and suggests that these cells may make an interesting model to study how differentiation alters prohormone processing. These results also support the hypothesis that PC5 in differentiated neuronal cells is capable of processing pro CCK to glycine-extended CCK 8.

Tim-3-expressing macrophages are functionally suppressed and expanded in oral squamous cell carcinoma due to virus-induced Gal-9 expression.

PubMed

Dong, Jianfeng; Cheng, Lijun; Zhao, Minchao; Pan, Xiangfeng; Feng, Zhiqiang; Wang, Dawei

2017-05-01

Oropharyngeal head and neck squamous cell carcinoma is a common malignant tumor in the oral cavity. High-risk human papillomavirus 16 infection is a major cause of oropharyngeal head and neck squamous cell carcinoma development. Strong antitumor immune responses, especially CD8 + T cell responses, are thought to be essential to effective cancer treatment and are associated with better prognosis in oropharyngeal head and neck squamous cell carcinoma. In this study, we examined the role of the Tim-3/Gal-9 pathway in oropharyngeal head and neck squamous cell carcinoma patients. We found that Gal-9 expression by CD4 + T cells was increased in human papillomavirus-positive oropharyngeal head and neck squamous cell carcinoma patients, but not in human papillomavirus-negative oropharyngeal head and neck squamous cell carcinoma patients. Increased Gal-9 secretion by CD4 + T cells presented multiple immunosuppressive effects. Coculturing monocytes with high Gal-9-expressing CD4 + T cells resulted in the expansion of Tim-3 + monocytes, which suppressed interferon gamma production by activated CD8 + T cells. Subsequently, total monocytes incubated with exogenous Gal-9, or high Gal-9-expressing CD4 + T cells, suppressed the expression of interferon gamma by CD8 + T cells. Exogenous Gal-9 and high Gal-9-expressing CD4 + T cells also suppressed the secretion of both interleukin 10 and interleukin 12 by monocytes. These effects are Tim-3/Gal-9-dependent because blocking Tim-3 and/or Gal-9 could enhance the support of CD8 + T cell interferon gamma production and the interleukin 10 and interleukin 12 secretion by monocytes. Together, these data suggest that the high Tim-3 expression in monocytes could be utilized by tumor-promoting Gal-9 expression on CD4 + T cells. Immunotherapy in human papillomavirus-positive oropharyngeal head and neck squamous cell carcinoma patients therefore faces an additional challenge posed by Tim-3 and Gal-9 and likely requires the blockade of these molecules.

Downregulation of proapoptotic Bim augments IL-2-independent T-cell transformation by human T-cell leukemia virus type-1 Tax.

PubMed

Higuchi, Masaya; Takahashi, Masahiko; Tanaka, Yuetsu; Fujii, Masahiro

2014-12-01

Human T-cell leukemia virus type 1 (HTLV-1), an etiological agent of adult T-cell leukemia, immortalizes and transforms primary human T cells in vitro in both an interleukin (IL)-2-dependent and IL-2-independent manner. Expression of the HTLV-1 oncoprotein Tax transforms the growth of the mouse T-cell line CTLL-2 from being IL-2-dependent to IL-2-independent. Withdrawal of IL-2 from normal activated T cells induces apoptosis, which is mediated through the inducible expression of several proapoptotic proteins, including Bim. In this study, we found that Tax protects IL-2-depleted T cells against Bim-induced apoptosis. Withdrawal of IL-2 from CTLL-2 cells induced a prominent increase in the level of Bim protein in CTLL-2 cells, but not in Tax-transformed CTLL-2 cells. This inhibition of Bim in Tax-transformed CTLL-2 cells was mediated by two mechanisms: downregulation of Bim mRNA and posttranscriptional reduction of Bim protein. Transient expression of Tax in CTLL-2 cells also inhibited IL-2 depletion-induced expression of Bim, however, this decrease in Bim protein expression was not due to downregulation of Bim mRNA, thus indicating that Bim mRNA downregulation in Tax-transformed CTLL-2 occurs only after long-term expression of Tax. Transient expression of Tax in CTLL-2 cells also induced Erk activation, however, this was not involved in the reduction of Bim protein. Knockdown of Bim expression in CTLL-2 cells augmented Tax-induced IL-2-independent transformation. HTLV-1 infection of human T cells also reduced their levels of Bim protein, and restoring Bim expression in HTLV-1-infected cells reduced their proliferation by inducing apoptosis. Taken together, these results indicate that Tax-induced downregulation of Bim in HTLV-1-infected T cells promotes their IL-2-independent growth, thereby supporting the persistence of HTLV-1 infection in vivo. © 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Alternative functional in vitro models of human intestinal epithelia

PubMed Central

Kauffman, Amanda L.; Gyurdieva, Alexandra V.; Mabus, John R.; Ferguson, Chrissa; Yan, Zhengyin; Hornby, Pamela J.

2013-01-01

Physiologically relevant sources of absorptive intestinal epithelial cells are crucial for human drug transport studies. Human adenocarcinoma-derived intestinal cell lines, such as Caco-2, offer conveniences of easy culture maintenance and scalability, but do not fully recapitulate in vivo intestinal phenotypes. Additional sources of renewable physiologically relevant human intestinal cells would provide a much needed tool for drug discovery and intestinal physiology. We compared two alternative sources of human intestinal cells, commercially available primary human intestinal epithelial cells (hInEpCs) and induced pluripotent stem cell (iPSC)-derived intestinal cells to Caco-2, for use in in vitro transwell monolayer intestinal transport assays. To achieve this for iPSC-derived cells, intestinal organogenesis was adapted to transwell differentiation. Intestinal cells were assessed by marker expression through immunocytochemical and mRNA expression analyses, monolayer integrity through Transepithelial Electrical Resistance (TEER) measurements and molecule permeability, and functionality by taking advantage the well-characterized intestinal transport mechanisms. In most cases, marker expression for primary hInEpCs and iPSC-derived cells appeared to be as good as or better than Caco-2. Furthermore, transwell monolayers exhibited high TEER with low permeability. Primary hInEpCs showed molecule efflux indicative of P-glycoprotein (Pgp) transport. Primary hInEpCs and iPSC-derived cells also showed neonatal Fc receptor-dependent binding of immunoglobulin G variants. Primary hInEpCs and iPSC-derived intestinal cells exhibit expected marker expression and demonstrate basic functional monolayer formation, similar to or better than Caco-2. These cells could offer an alternative source of human intestinal cells for understanding normal intestinal epithelial physiology and drug transport. PMID:23847534

Alternative functional in vitro models of human intestinal epithelia.

PubMed

Kauffman, Amanda L; Gyurdieva, Alexandra V; Mabus, John R; Ferguson, Chrissa; Yan, Zhengyin; Hornby, Pamela J

2013-01-01

Physiologically relevant sources of absorptive intestinal epithelial cells are crucial for human drug transport studies. Human adenocarcinoma-derived intestinal cell lines, such as Caco-2, offer conveniences of easy culture maintenance and scalability, but do not fully recapitulate in vivo intestinal phenotypes. Additional sources of renewable physiologically relevant human intestinal cells would provide a much needed tool for drug discovery and intestinal physiology. We compared two alternative sources of human intestinal cells, commercially available primary human intestinal epithelial cells (hInEpCs) and induced pluripotent stem cell (iPSC)-derived intestinal cells to Caco-2, for use in in vitro transwell monolayer intestinal transport assays. To achieve this for iPSC-derived cells, intestinal organogenesis was adapted to transwell differentiation. Intestinal cells were assessed by marker expression through immunocytochemical and mRNA expression analyses, monolayer integrity through Transepithelial Electrical Resistance (TEER) measurements and molecule permeability, and functionality by taking advantage the well-characterized intestinal transport mechanisms. In most cases, marker expression for primary hInEpCs and iPSC-derived cells appeared to be as good as or better than Caco-2. Furthermore, transwell monolayers exhibited high TEER with low permeability. Primary hInEpCs showed molecule efflux indicative of P-glycoprotein (Pgp) transport. Primary hInEpCs and iPSC-derived cells also showed neonatal Fc receptor-dependent binding of immunoglobulin G variants. Primary hInEpCs and iPSC-derived intestinal cells exhibit expected marker expression and demonstrate basic functional monolayer formation, similar to or better than Caco-2. These cells could offer an alternative source of human intestinal cells for understanding normal intestinal epithelial physiology and drug transport.

Specific knockdown of Oct4 and beta2-microglobulin expression by RNA interference in human embryonic stem cells and embryonic carcinoma cells.

PubMed

Matin, Maryam M; Walsh, James R; Gokhale, Paul J; Draper, Jonathan S; Bahrami, Ahmad R; Morton, Ian; Moore, Harry D; Andrews, Peter W

2004-01-01

We have used RNA interference (RNAi) to downregulate beta2-microglobulin and Oct4 in human embryonal carcinoma (hEC) cells and embryonic stem (hES) cells, demonstrating that RNAi is an effective tool for regulating specific gene activity in these human stem cells. The knockdown of Oct4 but not beta2-microglobulin expression in both EC and ES cells resulted in their differentiation, as indicated by a marked change in morphology, growth rate, and surface antigen phenotype, with respect to SSEA1, SSEA3, and TRA-1-60 expression. Expression of hCG and Gcm1 was also induced following knockdown of Oct4 expression, in both 2102Ep hEC cells and in H7 and H14 hES cells, consistent with the conclusion that, as in the mouse, Oct4 is required to maintain the undifferentiated stem cell state, and that differentiation to trophectoderm occurs in its absence. NTERA2 hEC cells also differentiated, but not to trophectoderm, suggesting their equivalence to a later stage of embryogenesis than other hEC and hES cells.

Evolution and cell-type specificity of human-specific genes preferentially expressed in progenitors of fetal neocortex.

PubMed

Florio, Marta; Heide, Michael; Pinson, Anneline; Brandl, Holger; Albert, Mareike; Winkler, Sylke; Wimberger, Pauline; Huttner, Wieland B; Hiller, Michael

2018-03-21

Understanding the molecular basis that underlies the expansion of the neocortex during primate, and notably human, evolution requires the identification of genes that are particularly active in the neural stem and progenitor cells of the developing neocortex. Here, we have used existing transcriptome datasets to carry out a comprehensive screen for protein-coding genes preferentially expressed in progenitors of fetal human neocortex. We show that 15 human-specific genes exhibit such expression, and many of them evolved distinct neural progenitor cell-type expression profiles and levels compared to their ancestral paralogs. Functional studies on one such gene, NOTCH2NL , demonstrate its ability to promote basal progenitor proliferation in mice. An additional 35 human genes with progenitor-enriched expression are shown to have orthologs only in primates. Our study provides a resource of genes that are promising candidates to exert specific, and novel, roles in neocortical development during primate, and notably human, evolution. © 2018, Florio et al.

Evolution and cell-type specificity of human-specific genes preferentially expressed in progenitors of fetal neocortex

PubMed Central

Pinson, Anneline; Brandl, Holger; Albert, Mareike; Winkler, Sylke; Wimberger, Pauline

2018-01-01

Understanding the molecular basis that underlies the expansion of the neocortex during primate, and notably human, evolution requires the identification of genes that are particularly active in the neural stem and progenitor cells of the developing neocortex. Here, we have used existing transcriptome datasets to carry out a comprehensive screen for protein-coding genes preferentially expressed in progenitors of fetal human neocortex. We show that 15 human-specific genes exhibit such expression, and many of them evolved distinct neural progenitor cell-type expression profiles and levels compared to their ancestral paralogs. Functional studies on one such gene, NOTCH2NL, demonstrate its ability to promote basal progenitor proliferation in mice. An additional 35 human genes with progenitor-enriched expression are shown to have orthologs only in primates. Our study provides a resource of genes that are promising candidates to exert specific, and novel, roles in neocortical development during primate, and notably human, evolution. PMID:29561261

STEAP: A prostate-specific cell-surface antigen highly expressed in human prostate tumors

PubMed Central

Hubert, Rene S.; Vivanco, Igor; Chen, Emily; Rastegar, Shiva; Leong, Kahan; Mitchell, Steve C.; Madraswala, Rashida; Zhou, Yanhong; Kuo, James; Raitano, Arthur B.; Jakobovits, Aya; Saffran, Douglas C.; Afar, Daniel E. H.

1999-01-01

In search of novel genes expressed in metastatic prostate cancer, we subtracted cDNA isolated from benign prostatic hypertrophic tissue from cDNA isolated from a prostate cancer xenograft model that mimics advanced disease. One novel gene that is highly expressed in advanced prostate cancer encodes a 339-amino acid protein with six potential membrane-spanning regions flanked by hydrophilic amino- and carboxyl-terminal domains. This structure suggests a potential function as a channel or transporter protein. This gene, named STEAP for six-transmembrane epithelial antigen of the prostate, is expressed predominantly in human prostate tissue and is up-regulated in multiple cancer cell lines, including prostate, bladder, colon, ovarian, and Ewing sarcoma. Immunohistochemical analysis of clinical specimens demonstrates significant STEAP expression at the cell–cell junctions of the secretory epithelium of prostate and prostate cancer cells. Little to no staining was detected at the plasma membranes of normal, nonprostate human tissues, except for bladder tissue, which expressed low levels of STEAP at the cell membrane. Protein analysis located STEAP at the cell surface of prostate-cancer cell lines. Our results support STEAP as a cell-surface tumor-antigen target for prostate cancer therapy and diagnostic imaging. PMID:10588738

Microarray analysis of laser capture microdissected-anulus cells from the human intervertebral disc.

PubMed

Gruber, Helen E; Mougeot, Jean-Luc; Hoelscher, Gretchen; Ingram, Jane A; Hanley, Edward N

2007-05-15

Five Thompson Grade I/II discs (Group 1), 7 Grade III discs (Group 2), and 3 Grade IV discs (Group IV) were studied here in a project approved by the authors' Human Subjects Institutional Review Board. Our objective was to use laser capture microdissection (LCM) to harvest cells from the human anulus and to derive gene expression profiles using microarray analysis. Appropriate gene expression is essential in the intervertebral disc for maintenance of extracellular matrix (ECM), ECM remodeling, and maintenance of a viable disc cell population. During disc degeneration, cell numbers drop, making gene expression studies challenging. LCM was used to harvest cells from paraffin-embedded sections of human anulus tissue. Gene profiling used Affymetrix GeneChip Human X3P arrays. ANOVA and SAM permutation analysis were applied to dCHIP normalized, filtered, and log-transformed gene expression data ( approximately 33,500 probes), and data analyzed to identify genes that were significantly differentially expressed between the 3 groups. We identified 47 genes that were significantly differentially expressed between the 3 groups (P < 0.001 and lowest q values). Compared with the healthiest discs (Grade I/II), 13 genes were up-regulated and 19 down-regulated in both the Grade III and the Grade IV discs. Genes with biologic significance regulated during degeneration involved cell senescence, low cell division rates, hypoxia-related genes, heat-shock protein 70 interacting protein, neuropilin 2, and interleukin-23p19 (interleukin-12 family). Results expand our understanding of disc aging and degeneration and show that LCM is a valuable technique that can be used to collect mRNA amounts adequate for microarray analysis from the sparse cell population of the human anulus.

Overexpression of E2F3 promotes proliferation of functional human β cells without induction of apoptosis

PubMed Central

Rady, Brian; Chen, Yanmei; Vaca, Pilar; Wang, Qian; Wang, Yong; Salmon, Patrick; Oberholzer, José

2013-01-01

The mechanisms that control proliferation, or lack thereof, in adult human β cells are poorly understood. Controlled induction of proliferation could dramatically expand the clinical application of islet cell transplantation and represents an important component of regenerative approaches to a functional cure of diabetes. Adult human β cells are particularly resistant to common proliferative targets and often dedifferentiate during proliferation. Here we show that expression of the transcription factor E2F3 has a role in regulating β-cell quiescence and proliferation. We found human islets have virtually no expression of the pro-proliferative G1/S transcription factors E2F1–3, but an abundance of inhibitory E2Fs 4–6. In proliferative human insulinomas, inhibitory E2Fs were absent, while E2F3 is expressed. Using this pattern as a “roadmap” for proliferation, we demonstrated that ectopic expression of nuclear E2F3 induced significant expansion of insulin-positive cells in both rat and human islets. These cells did not undergo apoptosis and retained their glucose-responsive insulin secretion, showing the ability to reverse diabetes in mice. Our results suggest that E2F4–6 may help maintain quiescence in human β cells and identify E2F3 as a novel target to induce proliferation of functional β cells. Refinement of this approach may increase the islets available for cell-based therapies and research and could provide important cues for understanding in vivo proliferation of β cells. PMID:23907129

Tissue non-specific alkaline phosphatase production by human dental pulp stromal cells is enhanced by high density cell culture.

PubMed

Tomlinson, Matthew J; Dennis, Caitriona; Yang, Xuebin B; Kirkham, Jennifer

2015-08-01

The cell surface hydrolase tissue non-specific alkaline phosphatase (TNAP) (also known as MSCA-1) is used to identify a sub-population of bone marrow stromal cells (BMSCs) with high mineralising potential and is found on subsets of cells within the dental pulp. We aim to determine whether TNAP is co-expressed by human dental pulp stromal cells (hDPSCs) alongside a range of BMSC markers, whether this is an active form of the enzyme and the effects of culture duration and cell density on its expression. Cells from primary dental pulp and culture expanded hDPSCs expressed TNAP. Subsequent analyses revealed persistent TNAP expression and co-expression with BMSC markers such as CD73 and CD90. Flow cytometry and biochemical assays showed that increased culture durations and cell densities enhanced TNAP expression by hDPSCs. Arresting the hDPSC cell cycle also increased TNAP expression. These data confirm that TNAP is co-expressed by hDPSCs together with other BMSC markers and show that cell density affects TNAP expression levels. We conclude that TNAP is a potentially useful marker for hDPSC selection especially for uses in mineralised tissue regenerative therapies.

Ocular surface epithelium induces expression of human mucosal lymphocyte antigen (HML-1) on peripheral blood lymphocytes

PubMed Central

Gomes, J A P; Dua, H S; Rizzo, L V; Nishi, M; Joseph, A; Donoso, L A

2004-01-01

Background/aims: Peripheral blood CD8+ lymphocytes that home to mucosal surfaces express the human mucosal lymphocyte antigen (HML-1). At mucosal surfaces, including the ocular surface, only intraepithelial CD8+ lymphocytes express HML-1. These lymphocytes are retained in the intraepithelial compartment by virtue of the interaction between HML-1 and its natural ligand, E-cadherin, which is expressed on epithelial cells. The purpose of this study was to determine whether ocular surface epithelial cells (ocular mucosa) could induce the expression of human mucosal lymphocyte antigen on peripheral blood lymphocytes. Methods: Human corneal and conjunctival epithelial cells were co-cultured with peripheral blood lymphocytes. Both non-activated and activated lymphocytes were used in the experiments. After 7 days of incubation, lymphocytes were recovered and analysed for the antigens CD8/HML-1, CD4/HML-1, CD3/CD8, CD3/CD4, CD3/CD25, CD8/CD25, and CD4/CD25 by flowcytometry. Results: Significant statistical differences were observed in the CD8/HML-1 expression when conjunctival epithelial cells were co-cultured with non-activated and activated lymphocytes (p = 0.04 for each) and when corneal epithelial cells were co-cultured with non-activated lymphocytes (p = 0.03). Significant statistical difference in CD4/HML-1 expression was observed only when conjunctival epithelial cells were co-cultured with activated lymphocytes (p = 0.02). Conclusion: Ocular surface epithelial cells can induce the expression of human mucosal lymphocyte antigen on CD8+ (and to some extent on CD4+) lymphocytes. This may allow the retention of CD8+ and CD4+ lymphocytes within the epithelial compartment of the conjunctiva and play a part in mucosal homing of lymphocytes. PMID:14736792

Infection of human intestinal epithelial cells with invasive bacteria upregulates apical intercellular adhesion molecule-1 (ICAM)-1) expression and neutrophil adhesion.

PubMed Central

Huang, G T; Eckmann, L; Savidge, T C; Kagnoff, M F

1996-01-01

The acute host response to gastrointestinal infection with invasive bacteria is characterized by an accumulation of neutrophils in the lamina propria, and neutrophil transmigration to the luminal side of the crypts. Intestinal epithelial cells play an important role in the recruitment of inflammatory cells to the site of infection through the secretion of chemokines. However, little is known regarding the expression, by epithelial cells, of molecules that are involved in interactions between the epithelium and neutrophils following bacterial invasion. We report herein that expression of ICAM-1 on human colon epithelial cell lines, and on human enterocytes in an in vivo model system, is upregulated following infection with invasive bacteria. Increased ICAM-1 expression in the early period (4-9 h) after infection appeared to result mainly from a direct interaction between invaded bacteria and host epithelial cells since it co-localized to cells invaded by bacteria, and the release of soluble factors by epithelial cells played only a minor role in mediating increased ICAM-1 expression. Furthermore, ICAM-1 was expressed on the apical side of polarized intestinal epithelial cells, and increased expression was accompanied by increased neutrophil adhesion to these cells. ICAM-1 expression by intestinal epithelial cells following infection with invasive bacteria may function to maintain neutrophils that have transmigrated through the epithelium in close contact with the intestinal epithelium, thereby reducing further invasion of the mucosa by invading pathogens. PMID:8755670

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

PubMed Central

2009-01-01

Background Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. Methods A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Results Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene. Conclusion These studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency. PMID:20042112

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

Yang, Yi -Lin; Ni, Jian; Hsu, Ping -Chih

Malignant pleural mesothelioma (mesothelioma) is a highly aggressive cancer without an effective treatment. Cul4A, a scaffold protein that recruits substrates for degradation, is amplified in several human cancers, including mesothelioma. We have recently shown that Cul4A plays an oncogenic role in vitro and in a mouse model. In this study, we analysed clinical mesothelioma tumours and found moderate to strong expression of Cul4A in 70.9% (51/72) of these tumours, as shown by immunohistochemistry. In 72.2% mesothelioma tumours with increased Cul4A copy number identified by fluorescence in situ hybridization analysis, Cul4A protein expression was moderate to strong. Similarly, Cul4A was overexpressedmore » and Cul4A copy number was increased in human mesothelioma cell lines. Because Gli1 is highly expressed in human mesothelioma cells, we compared Cul4A and Gli1 expression in mesothelioma tumours and found their expression associated (P < 0.05, chi-square). In mesothelioma cell lines, inhibiting Cul4A by siRNA decreased Gli1 expression, suggesting that Gli1 expression is, at least in part, regulated by Cul4A in mesothelioma cells. Our results suggest a linkage between Cul4A and Gli1 expression in human mesothelioma.« less

[MACF1 knockdown in glioblastoma multiforme cells increases temozolomide-induced cytotoxicity].

PubMed

Xie, Si-di; Chen, Zi-Yang; Wang, Hai; He, Min-Yi; Lu, Yun-Tao; Lei, Bing-Xi; Li, He-Zhen; Liu, Ya-Wei; Qi, Song-Tao

2017-09-20

To investigate the role of microtubule-actin crosslinking factor 1 (MACF1) in the response of glioma cells to temozolomide (TMZ). TMZ was applied to a human gliomablastoma cell line (U87) and changes in the protein expression and cellular localization were determined with Western blot, RT-PCR, and immunofluorescence. The responses of the cells with MACF1 expression knockdown by RNA interference to TMZ were assessed. TMZ-induced effects on MACF1 expression were also assessed by immunohistochemistry in a nude mouse model bearing human glioblastoma xenografts. TMZ resulted in significantly increased MACF1 expression (by about 2 folds) and changes in its localization in the gliomablastoma cells both in vitro and in vivo (P<0.01). Knockdown of MACF1 reduced the proliferation (by 45%) of human glioma cell lines treated with TMZ (P<0.01). TMZ-induced changes in MACF1 expression was accompanied by cytoskeletal rearrangement. MACF1 may be a potential therapeutic target for glioblastoma.

Protein profile of basal prostate epithelial progenitor cells--stage-specific embryonal antigen 4 expressing cells have enhanced regenerative potential in vivo.

PubMed

Höfner, Thomas; Klein, Corinna; Eisen, Christian; Rigo-Watermeier, Teresa; Haferkamp, Axel; Sprick, Martin R

2016-04-01

The long-term propagation of basal prostate progenitor cells ex vivo has been very difficult in the past. The development of novel methods to expand prostate progenitor cells in vitro allows determining their cell surface phenotype in greater detail. Mouse (Lin(-)Sca-1(+) CD49f(+) Trop2(high)-phenotype) and human (Lin(-) CD49f(+) TROP2(high)) basal prostate progenitor cells were expanded in vitro. Human and mouse cells were screened using 242 anti-human or 176 antimouse monoclonal antibodies recognizing the cell surface protein profile. Quantitative expression was evaluated at the single-cell level using flow cytometry. Differentially expressed cell surface proteins were evaluated in conjunction with the known CD49f(+)/TROP2(high) phenotype of basal prostate progenitor cells and characterized by in vivo sandwich-transplantation experiments using nude mice. The phenotype of basal prostate progenitor cells was determined as CD9(+)/CD24(+)/CD29(+)/CD44(+)/CD47(+)/CD49f(+)/CD104(+)/CD147(+)/CD326(+)/Trop2(high) of mouse as well as human origin. Our analysis revealed several proteins, such as CD13, Syndecan-1 and stage-specific embryonal antigens (SSEAs), as being differentially expressed on murine and human CD49f(+) TROP2(+) basal prostate progenitor cells. Transplantation experiments suggest that CD49f(+) TROP2(high) SSEA-4(high) human prostate basal progenitor cells to be more potent to regenerate prostate tubules in vivo as compared with CD49f(+) TROP2(high) or CD49f(+) TROP2(high) SSEA-4(low) cells. Determination of the cell surface protein profile of functionally defined murine and human basal prostate progenitor cells reveals differentially expressed proteins that may change the potency and regenerative function of epithelial progenitor cells within the prostate. SSEA-4 is a candidate cell surface marker that putatively enables a more accurate identification of the basal PESC lineage. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Conserved expression of transposon-derived non-coding transcripts in primate stem cells.

PubMed

Ramsay, LeeAnn; Marchetto, Maria C; Caron, Maxime; Chen, Shu-Huang; Busche, Stephan; Kwan, Tony; Pastinen, Tomi; Gage, Fred H; Bourque, Guillaume

2017-02-28

A significant portion of expressed non-coding RNAs in human cells is derived from transposable elements (TEs). Moreover, it has been shown that various long non-coding RNAs (lncRNAs), which come from the human endogenous retrovirus subfamily H (HERVH), are not only expressed but required for pluripotency in human embryonic stem cells (hESCs). To identify additional TE-derived functional non-coding transcripts, we generated RNA-seq data from induced pluripotent stem cells (iPSCs) of four primate species (human, chimpanzee, gorilla, and rhesus) and searched for transcripts whose expression was conserved. We observed that about 30% of TE instances expressed in human iPSCs had orthologous TE instances that were also expressed in chimpanzee and gorilla. Notably, our analysis revealed a number of repeat families with highly conserved expression profiles including HERVH but also MER53, which is known to be the source of a placental-specific family of microRNAs (miRNAs). We also identified a number of repeat families from all classes of TEs, including MLT1-type and Tigger families, that contributed a significant amount of sequence to primate lncRNAs whose expression was conserved. Together, these results describe TE families and TE-derived lncRNAs whose conserved expression patterns can be used to identify what are likely functional TE-derived non-coding transcripts in primate iPSCs.

Transplantation of neurons derived from human iPS cells cultured on collagen matrix into guinea-pig cochleae.

PubMed

Ishikawa, Masaaki; Ohnishi, Hiroe; Skerleva, Desislava; Sakamoto, Tatsunori; Yamamoto, Norio; Hotta, Akitsu; Ito, Juichi; Nakagawa, Takayuki

2017-06-01

The present study examined the efficacy of a neural induction method for human induced pluripotent stem (iPS) cells to eliminate undifferentiated cells and to determine the feasibility of transplanting neurally induced cells into guinea-pig cochleae for replacement of spiral ganglion neurons (SGNs). A stepwise method for differentiation of human iPS cells into neurons was used. First, a neural induction method was established on Matrigel-coated plates; characteristics of cell populations at each differentiation step were assessed. Second, neural stem cells were differentiated into neurons on a three-dimensional (3D) collagen matrix, using the same protocol of culture on Matrigel-coated plates; neuron subtypes in differentiated cells on a 3D collagen matrix were examined. Then, human iPS cell-derived neurons cultured on a 3D collagen matrix were transplanted into intact guinea-pig cochleae, followed by histological analysis. In vitro analyses revealed successful induction of neural stem cells from human iPS cells, with no retention of undifferentiated cells expressing OCT3/4. After the neural differentiation of neural stem cells, approximately 70% of cells expressed a neuronal marker, 90% of which were positive for vesicular glutamate transporter 1 (VGLUT1). The expression pattern of neuron subtypes in differentiated cells on a 3D collagen matrix was identical to that of the differentiated cells on Matrigel-coated plates. In addition, the survival of transplant-derived neurons was achieved when inflammatory responses were appropriately controlled. Our preparation method for human iPS cell-derived neurons efficiently eliminated undifferentiated cells and contributed to the settlement of transplant-derived neurons expressing VGLUT1 in guinea-pig cochleae. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Dual-Color Fluorescence Imaging to Monitor CYP3A4 and CYP3A7 Expression in Human Hepatic Carcinoma HepG2 and HepaRG Cells

PubMed Central

Kubiura, Musashi; Hayashi, Ayaka; Ohbayashi, Tetsuya; Kazuki, Yasuhiro; Chesné, Christophe; Oshimura, Mitsuo; Tada, Masako

2014-01-01

Human adult hepatocytes expressing CYP3A4, a major cytochrome P450 enzyme, are required for cell-based assays to evaluate the potential risk of drug-drug interactions caused by transcriptional induction of P450 enzymes in early-phase drug discovery and development. However, CYP3A7 is preferentially expressed in premature hepatoblasts and major hepatic carcinoma cell lines. The human hepatocellular carcinoma cell line HepaRG possesses a high self-renewal capacity and can differentiate into hepatic cells similar to human adult hepatocytes in vitro. Transgenic HepaRG cells, in which the expression of fluorescent reporters is regulated by 35 kb regulatory elements of CYP3A4, have a distinct advantage over human hepatocytes isolated by collagenase perfusion, which are unstable in culture. Thus, we created transgenic HepaRG and HepG2 cells by replacing the protein-coding regions of human CYP3A4 and CYP3A7 with enhanced green fluorescent protein (EGFP) and DsRed reporters, respectively, in a bacterial artificial chromosome vector that included whole regulatory elements. The intensity of DsRed fluorescence was initially high during the proliferation of transgenic HepaRG cells. However, most EGFP-positive cells were derived from those in which DsRed fluorescence was extinguished. Comparative analyses in these transgenic clones showed that changes in the total fluorescence intensity of EGFP reflected fold changes in the mRNA level of endogenous CYP3A4. Moreover, CYP3A4 induction was monitored by the increase in EGFP fluorescence. Thus, this assay provides a real-time evaluation system for quality assurance of hepatic differentiation into CYP3A4-expressing cells, unfavourable CYP3A4 induction, and fluorescence-activated cell sorting-mediated enrichment of CYP3A4-expressing hepatocytes based on the total fluorescence intensities of fluorescent reporters, without the need for many time-consuming steps. PMID:25101946

Dual-color fluorescence imaging to monitor CYP3A4 and CYP3A7 expression in human hepatic carcinoma HepG2 and HepaRG cells.

PubMed

Tsuji, Saori; Kawamura, Fumihiko; Kubiura, Musashi; Hayashi, Ayaka; Ohbayashi, Tetsuya; Kazuki, Yasuhiro; Chesné, Christophe; Oshimura, Mitsuo; Tada, Masako

2014-01-01

Human adult hepatocytes expressing CYP3A4, a major cytochrome P450 enzyme, are required for cell-based assays to evaluate the potential risk of drug-drug interactions caused by transcriptional induction of P450 enzymes in early-phase drug discovery and development. However, CYP3A7 is preferentially expressed in premature hepatoblasts and major hepatic carcinoma cell lines. The human hepatocellular carcinoma cell line HepaRG possesses a high self-renewal capacity and can differentiate into hepatic cells similar to human adult hepatocytes in vitro. Transgenic HepaRG cells, in which the expression of fluorescent reporters is regulated by 35 kb regulatory elements of CYP3A4, have a distinct advantage over human hepatocytes isolated by collagenase perfusion, which are unstable in culture. Thus, we created transgenic HepaRG and HepG2 cells by replacing the protein-coding regions of human CYP3A4 and CYP3A7 with enhanced green fluorescent protein (EGFP) and DsRed reporters, respectively, in a bacterial artificial chromosome vector that included whole regulatory elements. The intensity of DsRed fluorescence was initially high during the proliferation of transgenic HepaRG cells. However, most EGFP-positive cells were derived from those in which DsRed fluorescence was extinguished. Comparative analyses in these transgenic clones showed that changes in the total fluorescence intensity of EGFP reflected fold changes in the mRNA level of endogenous CYP3A4. Moreover, CYP3A4 induction was monitored by the increase in EGFP fluorescence. Thus, this assay provides a real-time evaluation system for quality assurance of hepatic differentiation into CYP3A4-expressing cells, unfavourable CYP3A4 induction, and fluorescence-activated cell sorting-mediated enrichment of CYP3A4-expressing hepatocytes based on the total fluorescence intensities of fluorescent reporters, without the need for many time-consuming steps.

Derivation of mouse embryonic stem cell lines from tyrosine hydroxylase reporter mice crossed with a human SNCA transgenic mouse model of Parkinson's disease.

PubMed

Chumarina, Margarita; Azevedo, Carla; Bigarreau, Julie; Vignon, Clémentine; Kim, Kwang-Soo; Li, Jia-Yi; Roybon, Laurent

2017-03-01

Mouse embryonic stem cell (mESC) lines were derived by crossing heterozygous transgenic (tg) mice expressing green fluorescent protein (GFP) under the control of the rat tyrosine hydroxylase (TH) promoter, with homozygous alpha-synuclein (aSYN) mice expressing human mutant SNCA A53T under the control of the mouse Prion promoter (MoPrP), or wildtype (WT) mice. The expression of GFP and human aSYN was validated by immunocytochemistry in midbrain neuron cultures upon differentiation of mESC lines using stromal cell-derived inducing activity. These mESC lines can help to study the impact of human aSYN expression in neurons and oligodendrocytes, and also trace GFP-expressing midbrain neurons. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Circular RNA HIPK3 promotes gallbladder cancer cell growth by sponging microRNA-124.

PubMed

Kai, Ding; Yannian, Liao; Yitian, Chen; Dinghao, Gong; Xin, Zhao; Wu, Ji

2018-06-18

Recent studies have implied that circHIPK3, an abundant circular RNA (circRNA), participates in tumorigenesis and cancer progression. Its expression and potential functions in human gallbladder cancer were examined in this study. We show that circHIPK3 is upregulated in human gallbladder cancer cells. But its level is low in gallbladder epithelial cells. circHIPK3 silencing by targeted siRNA potently inhibited survival and proliferation of established and primary human gallbladder cancer cells, while inducing cell apoptosis. Conversely, ectopic over-expression of circHIPK3 can further promote cancer cell proliferation. In gallbladder cancer cells, circHIPK3 sponged the tumor-suppressive microRNA-124 (miR-124) to sequester and inhibit its activity, thereby leading to increased expression of miR-124 targets, including ROCK1 (rho-associated protein kinase 1) and CDK6 (rho-associated protein kinase). Ectopic over-expression of miR-124 b y a lentiviral vector mimicked and abolished actions by circHIPK3 siRNA in gallbladder cancer cells. At last, we show that circHIPK3 is upregulated in human gallbladder cancer tissues, which is correlated with miR-124 downregulation and ROCK1-CDK6 upregulation. Together, we conclude that circHIPK3 promotes gallbladder cancer cell growth possibly by sponging miR-124. The over-expressed circHIPK3 could be a novel therapeutic target and diagnosis marker of human gallbladder cancer. Copyright © 2018. Published by Elsevier Inc.

Aloe-emodin inhibits HER-2 expression through the downregulation of Y-box binding protein-1 in HER-2-overexpressing human breast cancer cells.

PubMed

Ma, Jui-Wen; Hung, Chao-Ming; Lin, Ying-Chao; Ho, Chi-Tang; Kao, Jung-Yie; Way, Tzong-Der

2016-09-13

Human epidermal growth factor receptor-2 (HER-2)-positive breast cancer tends to be aggressive, highly metastatic, and drug resistant and spreads rapidly. Studies have indicated that emodin inhibits HER-2 expression. This study compared the HER-2-inhibitory effects of two compounds extracted from rhubarb roots: aloe-emodin (AE) and rhein. Our results indicated that AE exerted the most potent inhibitory effect on HER-2 expression. Treatment of HER-2-overexpressing breast cancer cells with AE reduced tumor initiation, cell migration, and cell invasion. AE was able to suppress YB-1 expression, further suppressing downstream HER-2 expression. AE suppressed YB-1 expression through the inhibition of Twist in HER-2-overexpressing breast cancer cells. Our data also found that AE inhibited cancer metastasis and cancer stem cells through the inhibition of EMT. Interestingly, AE suppressed YB-1 expression through the downregulation of the intracellular integrin-linked kinase (ILK)/protein kinase B (Akt)/mTOR signaling pathway in HER-2-overexpressing breast cancer cells. In vivo study showed the positive result of antitumor activity of AE in nude mice injected with human HER-2-overexpressing breast cancer cells. These findings suggest the possible application of AE in the treatment of HER-2-positive breast cancer.

Effects of irradiation on human leukocyte antigen class I expression in human papillomavirus positive and negative base of tongue and mobile tongue squamous cell carcinoma cell lines.

PubMed

Haeggblom, Linnea; Nordfors, Cecilia; Tertipis, Nikolaos; Bersani, Cinzia; Ramqvist, Torbjörn; Näsman, Anders; Dalianis, Tina

2017-03-16

Human papillomavirus (HPV) infection is a risk factor for oropharyngeal cancer, besides smoking and alcohol. Patients with HPV-positive tumors have a better prognosis than those with HPV-negative tumors. Furthermore, patients with HPV-positive tumors, with high CD8+ tumor infiltrating lymphocyte counts or absent/low human leukocyte antigen (HLA) class I expression have the best outcome. The latter is paradoxical, since HLA class I expression is important for tumor recognition. Below, the hypothesis that radiation therapy increases HLA class I expression was tested. HPV16 positive head and neck cancer cell lines UPCI-SCC-154, UPCI-SCC-090 and UM-SCC-47, and the HPV-negative cancer cell line UT-SCC-14, were treated with 2-10 Gray (Gy) and tested for HLA class I expression, cell cycle changes and apoptosis by flow cytometry. HPV16 E5, E7 and HLA-A mRNA expression was tested by quantitative PCR. A dose of 10 Gy resulted in a tendency of increased HLA class I cell surface expression for all cell lines and reached statistical significance for UPCI-SCC-154 and UPCI-SCC-090. There were, however, no significant changes in HLA-A mRNA expression in any of the cell lines, or HPV16 E5, or E7 mRNA expression for UPCI-SCC-47 and UPCI-SCC-154, while for UPCI-SCC-090 HPV16 E5 mRNA decreased. In all cell lines there was a shift towards G2/M phase and increased apoptosis after irradiation with 10 Gy. To conclude, irradiation with 10 Gy increased HLA class I expression in the HPV-positive cell lines UPCI-SCC-154 and UPCI-SCC-090. A similar tendency was observed for HPV-positive UM-SCC-47 and HPV-negative UT-SCC-14.

[Expression of human-mouse chimeric antibody directed against Chikungunya virus with site-specific integration system].

PubMed

Li, Jian-min; Chen, Wei; Jia, Xiu-jie; An, Xiao-ping; Li, Bing; Fan, Ying-ru; Tong, Yi-gang

2005-05-01

To obtain CHO/dhfr(-) cells line with integrated FRT sequence in the chromosome transcription active site and to express human-mouse chimeric antibody directed against Chikungunya Virus by using the cell line. The fusion gene of FRT and HBsAg was constructed by PCR and cloned into the MCS of pCI-neo to construct pCI-FRT-HBsAg. The pCI-FRT-HBsAg was transfected into CHO/dhfr(-) cells and cell clones with high expression of HBsAg were screened by detecting the amount of HBsAg with ELISA. A CHO cell clone with the highest expression was chosen and named as CHO/dhfr(-) FRT(+). pAFRT HFLF, a expression plasmid of chimeric antibody with RFT sequence was transfected into CHO/dhfr(-) FRT(+) cells and cell clones with high expression of the chimeric antibody were screened by increasing concentration of MTX. A CHO cell clone with high expression of the chimeric antibody was cultured in large scale and supernatant was collected from which the chimeric antibody was purified. The purified chimeric antibody was analyzed by SDS-PAGE, Western blot and IFA. A CHO/dhfr(-) cells line with integrated FRT sequence in the chromosome transcription active site was obtained successfully. A cell clone with yield of 5 mg/L of chimeric antibody was obtained, as compared with routine CHO cell expression system with a yield of 2 mg/L. A cell line with integrated FRT sequence in the chromosome transcription active site was obtained and with it human-mouse chimeric antibody directed against Chikungunya virus was expressed. This system lays a solid foundation which can be used for expressing antibodies and other proteins.

The epithelial-mesenchymal transition generates cells with properties of stem cells.

PubMed

Mani, Sendurai A; Guo, Wenjun; Liao, Mai-Jing; Eaton, Elinor Ng; Ayyanan, Ayyakkannu; Zhou, Alicia Y; Brooks, Mary; Reinhard, Ferenc; Zhang, Cheng Cheng; Shipitsin, Michail; Campbell, Lauren L; Polyak, Kornelia; Brisken, Cathrin; Yang, Jing; Weinberg, Robert A

2008-05-16

The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. We here report that the induction of an EMT in immortalized human mammary epithelial cells (HMLEs) results in the acquisition of mesenchymal traits and in the expression of stem-cell markers. Furthermore, we show that those cells have an increased ability to form mammospheres, a property associated with mammary epithelial stem cells. Independent of this, stem cell-like cells isolated from HMLE cultures form mammospheres and express markers similar to those of HMLEs that have undergone an EMT. Moreover, stem-like cells isolated either from mouse or human mammary glands or mammary carcinomas express EMT markers. Finally, transformed human mammary epithelial cells that have undergone an EMT form mammospheres, soft agar colonies, and tumors more efficiently. These findings illustrate a direct link between the EMT and the gain of epithelial stem cell properties.

Osthole Suppresses the Migratory Ability of Human Glioblastoma Multiforme Cells via Inhibition of Focal Adhesion Kinase-Mediated Matrix Metalloproteinase-13 Expression

PubMed Central

Tsai, Cheng-Fang; Yeh, Wei-Lan; Chen, Jia-Hong; Lin, Chingju; Huang, Shiang-Suo; Lu, Dah-Yuu

2014-01-01

Glioblastoma multiforme (GBM) is the most common type of primary and malignant tumor occurring in the adult central nervous system. GBM often invades surrounding regions of the brain during its early stages, making successful treatment difficult. Osthole, an active constituent isolated from the dried C. monnieri fruit, has been shown to suppress tumor migration and invasion. However, the effects of osthole in human GBM are largely unknown. Focal adhesion kinase (FAK) is important for the metastasis of cancer cells. Results from this study show that osthole can not only induce cell death but also inhibit phosphorylation of FAK in human GBM cells. Results from this study show that incubating GBM cells with osthole reduces matrix metalloproteinase (MMP)-13 expression and cell motility, as assessed by cell transwell and wound healing assays. This study also provides evidence supporting the potential of osthole in reducing FAK activation, MMP-13 expression, and cell motility in human GBM cells. PMID:24599080

Increase in apoptosis by combination of metformin with silibinin in human colorectal cancer cells

PubMed Central

Tsai, Cheng-Chia; Chuang, Tang-Wei; Chen, Li-Jen; Niu, Ho-Shan; Chung, Kun-Ming; Cheng, Juei-Tang; Lin, Kao-Chang

2015-01-01

AIM: To investigate the effect of metformin on silibinin-induced apoptosis in human colorectal cancer (COLO 205) cells. METHODS: MTT assays were performed to quantify cell viability. Western blot assays were applied to identify the expression of signaling proteins. RESULTS: The combined treatment of COLO 205 cells with metformin and silibinin decreased cell survival at a dose insufficient to influence the non-malignant cells [Human colonic epithelial cells (HCoEpiC)]. Silibinin and metformin increased phosphatase and tensin homolog and 5’-adenosine monophosphate-activated protein kinase expression in COLO 205 cells and inhibited the phosphorylation of mammol/Lalian target of rapamycin. This combined treatment resulted in an increase in the expression of activated caspase 3 and apoptosis inducing factor, indicating apoptosis. CONCLUSION: The combined treatment of human colorectal cancer cells with silibinin and metformin may induce apoptosis at a dose that does not affect HCoEpiC. This finding reveals a potential therapeutic strategy for the treatment of colorectal cancer. PMID:25892866

Ganoderma lucidum inhibits proliferation of human ovarian cancer cells by suppressing VEGF expression and up-regulating the expression of connexin 43.

PubMed

Dai, Shuyan; Liu, Jingjing; Sun, Xiaofei; Wang, Ning

2014-11-05

Ganoderma lucidum (G. lucidum, Reishimax) is an herbal mushroom known to have inhibitory effect on tumor cell growth. However, the molecular mechanisms responsible for its anti-proliferative effects on the ovarian cancer have not been fully elucidated. Human ovarian cancer cells HO 8910 (HOCC) and human primary ovarian cells (HPOC) were treated with G. lucidum. Effects of G. lucidum treatment on cell proliferation were studied by MTT assay. The expression of vascular endothelial growth factor (VEGF) and connexin 43 (Cx43) were measured by immunohistochemistry and real time polymerase chain reaction. To study the molecular mechanism of CX43 mediated anti-tumor activity, small interference RNA (siRNA) was used to knockdown Cx43 expression in HOCC. G. lucidum treatment resulted in reduced proliferation of HOCC. Inhibition of proliferation was accompanied by a decrease in VEGF expression and increase in Cx43 expression in the cancer cells. The extent of immune-reactivity of Cx43 or VEGF in cancer cells were correlated with the concentrations of G. lucidum used for treatment. Furthermore, knockdown of Cx43 expression in HOCC abrogated the effect of G. lucidum on cell proliferation without alteration of G. lucidum-induced attenuation of VEGF expression. G. lucidum inhibits ovarian cancer by down-regulating the expression of VEGF and up-regulating the downstream Cx43 expression. G. lucidum may be a promising therapeutic agent for the treatment of ovarian cancer.

Studies of the expression of human poly(ADP-ribose) polymerase-1 in Saccharomyces cerevisiae and identification of PARP-1 substrates by yeast proteome microarray screening.

PubMed

Tao, Zhihua; Gao, Peng; Liu, Hung-Wen

2009-12-15

Poly(ADP-ribosyl)ation of various nuclear proteins catalyzed by a family of NAD(+)-dependent enzymes, poly(ADP-ribose) polymerases (PARPs), is an important posttranslational modification reaction. PARP activity has been demonstrated in all types of eukaryotic cells with the exception of yeast, in which the expression of human PARP-1 was shown to lead to retarded cell growth. We investigated the yeast growth inhibition caused by human PARP-1 expression in Saccharomyces cerevisiae. Flow cytometry analysis reveals that PARP-1-expressing yeast cells accumulate in the G(2)/M stage of the cell cycle. Confocal microscopy analysis shows that human PARP-1 is distributed throughout the nucleus of yeast cells but is enriched in the nucleolus. Utilizing yeast proteome microarray screening, we identified 33 putative PARP-1 substrates, six of which are known to be involved in ribosome biogenesis. The poly(ADP-ribosyl)ation of three of these yeast proteins, together with two human homologues, was confirmed by an in vitro PARP-1 assay. Finally, a polysome profile analysis using sucrose gradient ultracentrifugation demonstrated that the ribosome levels in yeast cells expressing PARP-1 are lower than those in control yeast cells. Overall, our data suggest that human PARP-1 may affect ribosome biogenesis by modifying certain nucleolar proteins in yeast. The artificial PARP-1 pathway in yeast may be used as a simple platform to identify substrates and verify function of this important enzyme.

Single-cell gene expression profiling reveals functional heterogeneity of undifferentiated human epidermal cells

PubMed Central

Tan, David W. M.; Jensen, Kim B.; Trotter, Matthew W. B.; Connelly, John T.; Broad, Simon; Watt, Fiona M.

2013-01-01

Human epidermal stem cells express high levels of β1 integrins, delta-like 1 (DLL1) and the EGFR antagonist LRIG1. However, there is cell-to-cell variation in the relative abundance of DLL1 and LRIG1 mRNA transcripts. Single-cell global gene expression profiling showed that undifferentiated cells fell into two clusters delineated by expression of DLL1 and its binding partner syntenin. The DLL1+ cluster had elevated expression of genes associated with endocytosis, integrin-mediated adhesion and receptor tyrosine kinase signalling. Differentially expressed genes were not independently regulated, as overexpression of DLL1 alone or together with LRIG1 led to the upregulation of other genes in the DLL1+ cluster. Overexpression of DLL1 and LRIG1 resulted in enhanced extracellular matrix adhesion and increased caveolin-dependent EGFR endocytosis. Further characterisation of CD46, one of the genes upregulated in the DLL1+ cluster, revealed it to be a novel cell surface marker of human epidermal stem cells. Cells with high endogenous levels of CD46 expressed high levels of β1 integrin and DLL1 and were highly adhesive and clonogenic. Knockdown of CD46 decreased proliferative potential and β1 integrin-mediated adhesion. Thus, the previously unknown heterogeneity revealed by our studies results in differences in the interaction of undifferentiated basal keratinocytes with their environment. PMID:23482486

Pokemon reduces Bcl-2 expression through NF-κ Bp65: A possible mechanism of hepatocellular carcinoma.

PubMed

Zhao, Xinkai; Ning, Qiaoming; Sun, Xiaoning; Tian, De'an

2011-06-01

To investigate the relationship among Pokemon, NF-κ B p65 and Bcl-2 in hepatoma cells. HCC cell HepG2, SMMC7721 and human fetal liver cell line LO2 cells were used, and expression of Pokemon, NF-κ B p65 and Bcl-2 in three cells were detected by real-time PCR and western blot. Then siRNA of Pokemon was applied to inhibit the expression of Pokemon and NF-κ B p65 and apoptotic rate was determined by flow cytometric analysis. Expressions of Pokemon, NF-κ B p65 and Bcl-2 in human hepatoma cell HepG2, SMMC7721 expression were significantly higher than those in human embryonic stem cells LO2. siRNA of Pokemon inhibited the expression of Pokemon, NF-κ B p65 and Bcl-2 in liver cancer cells, and significantly increased apoptosis of liver cells. While siRNA of NF-κ B p65 inhibited the expression of NF-κ B p65 and Bcl-2, but Pokemon expression in hepatoma cells had no significant change. The proto-oncogene Pokemon can inhibit P14ARF by specific transcription regulation of cell cycle and can induce tumors. In addition, Pokemon can regulate NF-κ B p65 through the expression of apoptosis repressor, and promote the development of liver cancer. It suggests signal network in the liver include the regulation of new non-classical NF-κ B regulatory pathway. Copyright © 2011 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

CD52 is expressed on human mast cells and is a potential therapeutic target in Waldenstrom's Macroglobulinemia and mast cell disorders.

PubMed

Santos, Daniel Ditzel; Hatjiharissi, Evdoxia; Tournilhac, Olivier; Chemaly, Mariana Z A; Leleu, Xavier; Xu, Lian; Patterson, Christopher; Branagan, Andrew R; Manning, Robert J; Ho, Allen W; Hunter, Zachary R; Dimmock, Elizabeth A; Kutok, Jeffery L; Churchill, Winthrop H; Castells, Mariana C; Tai, Yu-Tzu; Anderson, Kenneth C; Treon, Steven P

2006-05-01

Alemtuzumab is a monoclonal antibody used in the treatment of CD52-expressing B-cell malignancies, including Waldenstrom's macroglobulinemia (WM). Recent studies demonstrate high levels of alemtuzumab activity in relapsed/refractory disease. One potential target of alemtuzumab is bone marrow mast cells (BMMCs), which provide growth and survival signaling for WM lymphoplasmacytic cells. We therefore examined BMMCs (FceRI+, CD117+) from WM and other mast cell (MC) disorders for expression of CD52. We identified cell surface antigen expression by multicolor flow cytometric analysis and found CD52 expressed on human mast-derived cell line-1 (HMC-1) and LAD2 MC lines, on BMMC from 13 of 15 patients with WM, and on BMMCs from 4 of 4 patients with systemic mastocytosis (SM). None of 4 healthy donors expressed CD52. Reverse-transcriptase polymerase chain reaction analysis confirmed CD52 expression in the HMC-1 and LAD2 MC lines, in BMMCs from 14 of 15 patients with WM, and 3 of 3 patients with SM. CD52 transcripts were also detected in BMMCs from 6 of 6 healthy donors, despite the absence of CD52 cell surface expression. Importantly, we observed high levels of alemtuzumab-mediated, antibody-dependent, cell-mediated cytotoxicity against LAD2 MCs and BMMCs from patients with WM and SM. These studies demonstrate that CD52 is widely expressed on human MCs and WM bone marrow lymphoplasmacytic cells and provide the preclinical rationale for the use of alemtuzumab in the treatment of WM and possibly other MC-related disorders.

The expression of ADAM12 (meltrin alpha) in human giant cell tumours of bone.

PubMed

Tian, B L; Wen, J M; Zhang, M; Xie, D; Xu, R B; Luo, C J

2002-12-01

To examine the expression of ADAM12 (meltrin alpha), a member of the disintegrin and metalloprotease (ADAM) family, in human giant cell tumours of the bone, skeletal muscle tissue from human embryos, and human adult skeletal muscle tissue. ADAM12 mRNA was detected by reverse transcription polymerase chain reaction and in situ hybridisation. ADAM12 mRNA was detected in 14 of the 20 giant cell tumours of bone and in three of the six tumour cell cultures. The expression of ADAM12 in cells cultured from the tumour was linked to the presence of multinucleated giant cells. ADAM12 mRNA could not be detected in the five adult skeletal muscle tissue samples, although it was found in the two embryonic skeletal muscle tissue samples. ADAM12 mRNA was localised to the cytoplasm of multinucleated giant cells and some mononuclear stromal cells. These results indicate that multinucleated giant cells are formed by the cell fusion of mononuclear stromal cells in giant cell tumours of bone and that ADAM12 is involved in the cell fusion process.

Effects of 1,25-dihydroxyvitamin D3 and vitamin D3 on the expression of the vitamin D receptor in human skeletal muscle cells

USDA-ARS?s Scientific Manuscript database

Vitamin D receptor (VDR) expression and action in non-human skeletal muscle have recently been reported in several studies, yet data on the activity and expression of VDR in human muscle cells are scarce. We conducted a series of studies to examine the (1) effect of 1,25-dihydroxyvitamin D3 (1,25(OH...

Simultaneous Overexpression of Functional Human HO-1, E5NT and ENTPD1 Protects Murine Fibroblasts against TNF-α-Induced Injury In Vitro

PubMed Central

Cinti, Alessandro; De Giorgi, Marco; Chisci, Elisa; Arena, Claudia; Galimberti, Gloria; Farina, Laura; Bugarin, Cristina; Rivolta, Ilaria; Gaipa, Giuseppe; Smolenski, Ryszard Tom; Cerrito, Maria Grazia; Lavitrano, Marialuisa; Giovannoni, Roberto

2015-01-01

Several biomedical applications, such as xenotransplantation, require multiple genes simultaneously expressed in eukaryotic cells. Advances in genetic engineering technologies have led to the development of efficient polycistronic vectors based on the use of the 2A self-processing oligopeptide. The aim of this work was to evaluate the protective effects of the simultaneous expression of a novel combination of anti-inflammatory human genes, ENTPD1, E5NT and HO-1, in eukaryotic cells. We produced an F2A system-based multicistronic construct to express three human proteins in NIH3T3 cells exposed to an inflammatory stimulus represented by tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine which plays an important role during inflammation, cell proliferation, differentiation and apoptosis and in the inflammatory response during ischemia/reperfusion injury in several organ transplantation settings. The protective effects against TNF-α-induced cytotoxicity and cell death, mediated by HO-1, ENTPD1 and E5NT genes were better observed in cells expressing the combination of genes as compared to cells expressing each single gene and the effect was further improved by administrating enzymatic substrates of the human genes to the cells. Moreover, a gene expression analyses demonstrated that the expression of the three genes has a role in modulating key regulators of TNF-α signalling pathway, namely Nemo and Tnfaip3, that promoted pro-survival phenotype in TNF-α injured cells. These results could provide new insights in the research of protective mechanisms in transplantation settings. PMID:26513260

Altered serotonin physiology in human breast cancers favors paradoxical growth and cell survival

PubMed Central

2009-01-01

Introduction The breast microenvironment can either retard or accelerate the events associated with progression of latent cancers. However, the actions of local physiological mediators in the context of breast cancers are poorly understood. Serotonin (5-HT) is a critical local regulator of epithelial homeostasis in the breast and other organs. Herein, we report complex alterations in the intrinsic mammary gland serotonin system of human breast cancers. Methods Serotonin biosynthetic capacity was analyzed in human breast tumor tissue microarrays using immunohistochemistry for tryptophan hydroxylase 1 (TPH1). Serotonin receptors (5-HT1-7) were analyzed in human breast tumors using the Oncomine database. Serotonin receptor expression, signal transduction, and 5-HT effects on breast cancer cell phenotype were compared in non-transformed and transformed human breast cells. Results In the context of the normal mammary gland, 5-HT acts as a physiological regulator of lactation and involution, in part by favoring growth arrest and cell death. This tightly regulated 5-HT system is subverted in multiple ways in human breast cancers. Specifically, TPH1 expression undergoes a non-linear change during progression, with increased expression during malignant progression. Correspondingly, the tightly regulated pattern of 5-HT receptors becomes dysregulated in human breast cancer cells, resulting in both ectopic expression of some isoforms and suppression of others. The receptor expression change is accompanied by altered downstream signaling of 5-HT receptors in human breast cancer cells, resulting in resistance to 5-HT-induced apoptosis, and stimulated proliferation. Conclusions Our data constitutes the first report of direct involvement of 5-HT in human breast cancer. Increased 5-HT biosynthetic capacity accompanied by multiple changes in 5-HT receptor expression and signaling favor malignant progression of human breast cancer cells (for example, stimulated proliferation, inappropriate cell survival). This occurs through uncoupling of serotonin from the homeostatic regulatory mechanisms of the normal mammary epithelium. The findings open a new avenue for identification of diagnostic and prognostic markers, and valuable new therapeutic targets for managing breast cancer. PMID:19903352

p21Waf1/Cip1/Sdi1 Prevents Apoptosis as Well as Stimulates Growth in Cells Transformed or Immortalized by Human T-Cell Leukemia Virus Type 1-Encoded Tax

PubMed Central

Kawata, Sanae; Ariumi, Yasuo; Shimotohno, Kunitada

2003-01-01

Human T-cell leukemia virus type 1 (HTLV-1) Tax regulates the expression of virally encoded genes, as well as various endogenous host genes in trans. Tax-mediated regulation of gene expression is important for the immortalization of normal human T lymphocytes and the transformation of fibroblast cells, such as Rat-1 cells. Tax has the ability to transactivate p21Waf1/Cip1/Sdi1, resulting in high expression levels in HTLV-1-immortalized cells. Since p21 expression is suppressed due to methylation of the promoter region in Rat-l cell line, p21 may not be critical for the transformation of this cell line by Tax. To further understand the role of p21 for the proliferation of Tax-transformed Rat-1 cells, we examined the effect of ectopic expression of p21 in these cells. Here, we observed that p21 expression enhanced the transformation of this cell line via at least two mechanisms: (i) the enhancement of NF-κB activation and/or CREB signaling and (ii) the excitation of antiapoptotic machinery. To analyze the role of p21 that is overexpressed in HTLV-1-immortalized lymphocytes, p21 expression was suppressed by using an antisense oligonucleotide specific for p21 mRNA; these cells then became sensitive to apoptotic induction. These results suggest that p21 plays an important role in the proliferation of Tax-expressing cells through the regulation of at least two independent mechanisms. PMID:12805427

Phenotypic and Functional Changes in Blood Monocytes Following Adherence to Endothelium

PubMed Central

Tso, Colin; Rye, Kerry-Anne; Barter, Philip

2012-01-01

Objective Blood monocytes are known to express endothelial-like genes during co-culture with endothelium. In this study, the time-dependent change in the phenotype pattern of primary blood monocytes after adhering to endothelium is reported using a novel HLA-A2 mistyped co-culture model. Methods and Results Freshly isolated human PBMCs were co-cultured with human umbilical vein endothelial cells or human coronary arterial endothelial cells of converse human leukocyte antigen A2 (HLA-A2) status. This allows the tracking of the PBMC-derived cells by HLA-A2 expression and assessment of their phenotype pattern over time. PBMCs that adhered to the endothelium at the start of the co-culture were predominantly CD11b+ blood monocytes. After 24 to 72 hours in co-culture, the endothelium-adherent monocytes acquired endothelial-like properties including the expression of endothelial nitric oxide synthase, CD105, CD144 and vascular endothelial growth factor receptor 2. The expression of monocyte/macrophage lineage antigens CD14, CD11b and CD36 were down regulated concomitantly. The adherent monocytes did not express CD115 after 1 day of co-culture. By day 6, the monocyte-derived cells expressed vascular cell adhesion molecule 1 in response to tumour necrosis factor alpha. Up to 10% of the PBMCs adhered to the endothelium. These monocyte-derived cells contributed up to 30% of the co-cultured cell layer and this was dose-dependent on the PBMC seeding density. Conclusions Human blood monocytes undergo rapid phenotype change to resemble endothelial cells after adhering to endothelium. PMID:22615904

Cell–specific Variation in E-selectin Ligand Expression Among Human Peripheral Blood Mononuclear Cells: Implications for Immunosurveillance and Pathobiology

PubMed Central

Silva, Mariana; Fung, Ronald Kam Fai; Donnelly, Conor Brian; Videira, Paula Alexandra; Sackstein, Robert

2017-01-01

Both host defense and immunopathology are shaped by the ordered recruitment of circulating leukocytes to affected sites, a process initiated by binding of blood-borne cells to E-selectin displayed at target endothelial beds. Accordingly, knowledge of the expression and function of leukocyte E-selectin ligands is key to understanding the tempo and specificity of immunoreactivity. Here, we performed E-selectin adherence assays under hemodynamic flow conditions coupled with flow cytometry and western blot analysis to elucidate the function and structural biology of glycoprotein E-selectin ligands expressed on human peripheral blood mononuclear cells (PBMCs). Circulating monocytes uniformly express high levels of the canonical E-selectin binding determinant sLeX and display markedly greater adhesive interactions with E-selectin than do circulating lymphocytes, which exhibit variable E-selectin binding among CD4+ and CD8+ T-cells but no binding by B-cells. Monocytes prominently present sLeX decorations on an array of protein scaffolds including PSGL-1, CD43, and CD44 (rendering the E-selectin ligands CLA, CD43E, and HCELL, respectively), and B-cells altogether lack E-selectin ligands. Quantitative PCR gene expression studies of glycosyltransferases that regulate display of sLeX reveal high transcript levels among circulating monocytes and low levels among circulating B-cells, and, commensurately, cell surface α(1,3)-fucosylation reveals that acceptor sialyllactosaminyl glycans convertible into sLeX are abundantly expressed on human monocytes yet are relatively deficient on B-cells. Collectively, these findings unveil distinct cell-specific patterns of E-selectin ligand expression among human PBMCs, indicating that circulating monocytes are specialized to engage E-selectin and providing key insights into the molecular effectors mediating recruitment of these cells at inflammatory sites. PMID:28330896

Antiviral Responses by Swine Primary Bronchoepithelial Cells Are Limited Compared to Human Bronchoepithelial Cells Following Influenza Virus Infection

PubMed Central

Hauser, Mary J.; Dlugolenski, Daniel; Culhane, Marie R.; Wentworth, David E.; Tompkins, S. Mark; Tripp, Ralph A.

2013-01-01

Swine generate reassortant influenza viruses because they can be simultaneously infected with avian and human influenza; however, the features that restrict influenza reassortment in swine and human hosts are not fully understood. Type I and III interferons (IFNs) act as the first line of defense against influenza virus infection of respiratory epithelium. To determine if human and swine have different capacities to mount an antiviral response the expression of IFN and IFN-stimulated genes (ISG) in normal human bronchial epithelial (NHBE) cells and normal swine bronchial epithelial (NSBE) cells was evaluated following infection with human (H3N2), swine (H1N1), and avian (H5N3, H5N2, H5N1) influenza A viruses. Expression of IFNλ and ISGs were substantially higher in NHBE cells compared to NSBE cells following H5 avian influenza virus infection compared to human or swine influenza virus infection. This effect was associated with reduced H5 avian influenza virus replication in human cells at late times post infection. Further, RIG-I expression was lower in NSBE cells compared to NHBE cells suggesting reduced virus sensing. Together, these studies identify key differences in the antiviral response between human and swine respiratory epithelium alluding to differences that may govern influenza reassortment. PMID:23875024

The experimental study of genetic engineering human neural stem cells mediated by lentivirus to express multigene.

PubMed

Cai, Pei-qiang; Tang, Xun; Lin, Yue-qiu; Martin, Oudega; Sun, Guang-yun; Xu, Lin; Yang, Yun-kang; Zhou, Tian-hua

2006-02-01

To explore the feasibility to construct genetic engineering human neural stem cells (hNSCs) mediated by lentivirus to express multigene in order to provide a graft source for further studies of spinal cord injury (SCI). Human neural stem cells from the brain cortex of human abortus were isolated and cultured, then gene was modified by lentivirus to express both green fluorescence protein (GFP) and rat neurotrophin-3 (NT-3); the transgenic expression was detected by the methods of fluorescence microscope, dorsal root ganglion of fetal rats and slot blot. Genetic engineering hNSCs were successfully constructed. All of the genetic engineering hNSCs which expressed bright green fluorescence were observed under the fluorescence microscope. The conditioned medium of transgenic hNSCs could induce neurite flourishing outgrowth from dorsal root ganglion (DRG). The genetic engineering hNSCs expressed high level NT-3 which could be detected by using slot blot. Genetic engineering hNSCs mediated by lentivirus can be constructed to express multigene successfully.

Laminin γ3 plays an important role in retinal lamination, photoreceptor organisation and ganglion cell differentiation.

PubMed

Dorgau, Birthe; Felemban, Majed; Sharpe, Alexander; Bauer, Roman; Hallam, Dean; Steel, David H; Lindsay, Susan; Mellough, Carla; Lako, Majlinda

2018-05-23

Laminins are heterotrimeric glycoproteins of the extracellular matrix. Eleven different laminin chains have been identified in vertebrates. They are ubiquitously expressed in the human body, with a distinct tissue distribution. Laminin expression in neural retina and their functional role during human retinogenesis is still unknown. This study investigated the laminin expression in human developing and adult retina, showing laminin α1, α5, β1, β2 and γ1 to be predominantly expressed in Bruch's membrane and the inner limiting membrane. Laminin-332 and laminin γ3 expression were mainly observed in the neural retina during retinal histogenesis. These expression patterns were largely conserved in pluripotent stem cell-derived retinal organoids. Blocking of laminin γ3 function in retinal organoids resulted in the disruption of laminar organisation and synapse formation, the loss of photoreceptor organisation and retinal ganglion cells. Our data demonstrate a unique temporal and spatial expression for laminins and reveal a novel role for laminin γ3 during human retinogenesis.

Mechanism of Hormonal Regulation of CAD Gene Expression in Human Breast Cancer Cells

DTIC Science & Technology

2006-05-01

showed that PGJ2, troglitazone, and ciglitazone inhibit E2-stimulated cell proliferation of a leiomyoma -derived cell line and human primary leiomyoma ...cultures and also inhibit ER-mediated gene expression and protein expression. These results suggested that in uterine leiomyomas PPARγ activation is...peroxisome proliferator-activated receptor gamma ligands in uterine leiomyoma . Cancer Res. 63, 1221-1227. Hulka, BS., Liu, ET., and Lininger, RA., 1994

Reprogramming human gallbladder cells into insulin-producing β-like cells

PubMed Central

Benedetti, Eric; Wang, Yuhan; Pelz, Carl; Schug, Jonathan; Kaestner, Klaus H.; Grompe, Markus

2017-01-01

The gallbladder and cystic duct (GBCs) are parts of the extrahepatic biliary tree and share a common developmental origin with the ventral pancreas. Here, we report on the very first genetic reprogramming of patient-derived human GBCs to β-like cells for potential autologous cell replacement therapy for type 1 diabetes. We developed a robust method for large-scale expansion of human GBCs ex vivo. GBCs were reprogrammed into insulin-producing pancreatic β-like cells by a combined adenoviral-mediated expression of hallmark pancreatic endocrine transcription factors PDX1, MAFA, NEUROG3, and PAX6 and differentiation culture in vitro. The reprogrammed GBCs (rGBCs) strongly induced the production of insulin and pancreatic endocrine genes and these responded to glucose stimulation in vitro. rGBCs also expressed an islet-specific surface marker, which was used to enrich for the most highly reprogrammed cells. More importantly, global mRNA and microRNA expression profiles and protein immunostaining indicated that rGBCs adopted an overall β-like state and these rGBCs engrafted in immunodeficient mice. Furthermore, comparative global expression analyses identified putative regulators of human biliary to β cell fate conversion. In summary, we have developed, for the first time, a reliable and robust genetic reprogramming and culture expansion of primary human GBCs—derived from multiple unrelated donors—into pancreatic β-like cells ex vivo, thus showing that human gallbladder is a potentially rich source of reprogrammable cells for autologous cell therapy in diabetes. PMID:28813430

Evaluation of Accessory Lacrimal Gland in Muller’s Muscle Conjunctival Resection Specimens for Precursor Cell Markers and Biological Markers of Dry Eye Disease

PubMed Central

Ali, Marwan; Shah, Dhara; Pasha, Zeeshan; Jassim, Sarmad H.; Jaboori, Assraa Jassim; Setabutr, Pete; Aakalu, Vinay K.

2017-01-01

Purpose The accessory lacrimal glands (ALG) are an understudied component of the tear functional unit, even though they are important in the development of dry eye syndrome (DES). To advance our understanding of aging changes, regenerative potential and histologic correlates to human characteristics, we investigated human ALG tissue from surgical samples to determine the presence or absence of progenitor cell markers and lacrimal epithelial markers and to correlate marker expression to relevant patient characteristics. Materials and Methods ALG tissues obtained from Muller’s Muscle Conjunctival Resection (MMCR) specimens were created using tissue microarrays (TMAs). Immunofluorescence staining of MMCR sections was performed using primary antibodies specific to cell protein markers. Cell marker localization in TMAs was then assessed by two blinded observers using a standardized scoring system. Patient characteristics including age, race, and status of ocular surface health were then compared against expression of stem cell markers. Results Human ALG expressed a number of epithelial markers, and in particular, histatin-1 was well correlated with the expression of epithelial markers and was present in most acini. In addition, we noted the presence of precursor cell markers nestin, ABCG2 and CD90 in ALG tissue. There was a decrease in precursor cell marker expression with increasing age. Finally, we noted that a negative association was present between histatin-1 expression and DES. Conclusions Thus, we report for the first time that human ALG tissues contain precursor marker positive cells and that this marker expression may decrease with increasing age. Moreover, histatin-1 expression may be decreased in DES. Future studies will be performed to use these cell markers to isolate and culture lacrimal epithelial cells from heterogeneous tissues, determine the relevance of histatin-1 expression to DES and isolate candidate precursor cells from ALG tissue. PMID:27612554

Cytokine stimulation of MUC4 expression in human female reproductive tissue carcinoma cell lines and endometrial cancer.

PubMed

Chapela, Patricia J; Broaddus, Russell R; Hawkins, Shannon M; Lessey, Bruce A; Carson, Daniel D

2015-11-01

MUC4, a transmembrane glycoprotein, interferes with cell adhesion, and promotes EGFR signaling in cancer. Studies in rat models have demonstrated steroid hormonal regulation of endometrial MUC4 expression. In this study, qRT-PCR screening of mouse tissues determined that Muc4 mRNA also was robustly expressed in mouse uteri. Previous studies from our labs have demonstrated MUC4 mRNA was expressed at levels <1% of MUC1 mRNA in human endometrium and endometriotic tissue. Multiple human endometrial adenocarcinoma cell lines were assayed for MUC4 mRNA expression revealing extremely low basal expression in the Ishikawa, RL-95-2, AN3CA, and KLE lines. Moderate to high expression was observed in HEC50 and HEC-1A cells. MUC4 mRNA expression was not affected by progesterone and/or estrogen treatment, but was greatly stimulated at both mRNA and protein levels by proinflammatory cytokines (IFN-γ and TNF-α), particularly when used in combination. In endometrial tissue, MUC4 mRNA levels did not change significantly between normal or cancerous samples; although, a subset of patients with grade 1 and 2 tumors displayed substantially higher expression. Likewise, immunostaining of human endometrial adenocarcinoma tissues revealed little to no staining in many patients (low MUC4), but strong staining in some patients (high MUC4) independent of cancer grade. In cases where staining was observed, it was heterogeneous with some cells displaying robust MUC4 expression and others displaying little or no staining. Collectively, these observations demonstrate that while MUC4 is highly expressed in the mouse uterus, it is not a major mucin in normal human endometrium. Rather, MUC4 is a potential marker of endometrial adenocarcinoma in a subset of patients. © 2015 Wiley Periodicals, Inc.

Sox10 Expressing Cells in the Lateral Wall of the Aged Mouse and Human Cochlea

PubMed Central

Hao, Xinping; Xing, Yazhi; Moore, Michael W.; Zhang, Jianning; Han, Demin; Schulte, Bradley A.; Dubno, Judy R.; Lang, Hainan

2014-01-01

Age-related hearing loss (presbycusis) is a common human disorder, affecting one in three Americans aged 60 and over. Previous studies have shown that presbyacusis is associated with a loss of non-sensory cells in the cochlear lateral wall. Sox10 is a transcription factor crucial to the development and maintenance of neural crest-derived cells including some non-sensory cell types in the cochlea. Mutations of the Sox10 gene are known to cause various combinations of hearing loss and pigmentation defects in humans. This study investigated the potential relationship between Sox10 gene expression and pathological changes in the cochlear lateral wall of aged CBA/CaJ mice and human temporal bones from older donors. Cochlear tissues prepared from young adult (1–3 month-old) and aged (2–2.5 year-old) mice, and human temporal bone donors were examined using quantitative immunohistochemical analysis and transmission electron microscopy. Cells expressing Sox10 were present in the stria vascularis, outer sulcus and spiral prominence in mouse and human cochleas. The Sox10+ cell types included marginal and intermediate cells and outer sulcus cells, including those that border the scala media and those extending into root processes (root cells) in the spiral ligament. Quantitative analysis of immunostaining revealed a significant decrease in the number of Sox10+ marginal cells and outer sulcus cells in aged mice. Electron microscopic evaluation revealed degenerative alterations in the surviving Sox10+ cells in aged mice. Strial marginal cells in human cochleas from donors aged 87 and older showed only weak immunostaining for Sox10. Decreases in Sox10 expression levels and a loss of Sox10+ cells in both mouse and human aged ears suggests an important role of Sox10 in the maintenance of structural and functional integrity of the lateral wall. A loss of Sox10+ cells may also be associated with a decline in the repair capabilities of non-sensory cells in the aged ear. PMID:24887110

MURC/cavin-4 Is Co-Expressed with Caveolin-3 in Rhabdomyosarcoma Tumors and Its Silencing Prevents Myogenic Differentiation in the Human Embryonal RD Cell Line

PubMed Central

Faggi, Fiorella; Codenotti, Silvia; Poliani, Pietro Luigi; Cominelli, Manuela; Chiarelli, Nicola; Colombi, Marina; Vezzoli, Marika; Monti, Eugenio; Bono, Federica; Tulipano, Giovanni; Fiorentini, Chiara; Zanola, Alessandra; Lo, Harriet P.; Parton, Robert G.; Keller, Charles; Fanzani, Alessandro

2015-01-01

The purpose of this study was to investigate whether MURC/cavin-4, a plasma membrane and Z-line associated protein exhibiting an overlapping distribution with Caveolin-3 (Cav-3) in heart and muscle tissues, may be expressed and play a role in rhabdomyosarcoma (RMS), an aggressive myogenic tumor affecting childhood. We found MURC/cavin-4 to be expressed, often concurrently with Cav-3, in mouse and human RMS, as demonstrated through in silico analysis of gene datasets and immunohistochemical analysis of tumor samples. In vitro expression studies carried out using human cell lines and primary mouse tumor cultures showed that expression levels of both MURC/cavin-4 and Cav-3, while being low or undetectable during cell proliferation, became robustly increased during myogenic differentiation, as detected via semi-quantitative RT-PCR and immunoblotting analysis. Furthermore, confocal microscopy analysis performed on human RD and RH30 cell lines confirmed that MURC/cavin-4 mostly marks differentiated cell elements, colocalizing at the cell surface with Cav-3 and labeling myosin heavy chain (MHC) expressing cells. Finally, MURC/cavin-4 silencing prevented the differentiation in the RD cell line, leading to morphological cell impairment characterized by depletion of myogenin, Cav-3 and MHC protein levels. Overall, our data suggest that MURC/cavin-4, especially in combination with Cav-3, may play a consistent role in the differentiation process of RMS. PMID:26086601

MURC/cavin-4 Is Co-Expressed with Caveolin-3 in Rhabdomyosarcoma Tumors and Its Silencing Prevents Myogenic Differentiation in the Human Embryonal RD Cell Line.

PubMed

Faggi, Fiorella; Codenotti, Silvia; Poliani, Pietro Luigi; Cominelli, Manuela; Chiarelli, Nicola; Colombi, Marina; Vezzoli, Marika; Monti, Eugenio; Bono, Federica; Tulipano, Giovanni; Fiorentini, Chiara; Zanola, Alessandra; Lo, Harriet P; Parton, Robert G; Keller, Charles; Fanzani, Alessandro

2015-01-01

The purpose of this study was to investigate whether MURC/cavin-4, a plasma membrane and Z-line associated protein exhibiting an overlapping distribution with Caveolin-3 (Cav-3) in heart and muscle tissues, may be expressed and play a role in rhabdomyosarcoma (RMS), an aggressive myogenic tumor affecting childhood. We found MURC/cavin-4 to be expressed, often concurrently with Cav-3, in mouse and human RMS, as demonstrated through in silico analysis of gene datasets and immunohistochemical analysis of tumor samples. In vitro expression studies carried out using human cell lines and primary mouse tumor cultures showed that expression levels of both MURC/cavin-4 and Cav-3, while being low or undetectable during cell proliferation, became robustly increased during myogenic differentiation, as detected via semi-quantitative RT-PCR and immunoblotting analysis. Furthermore, confocal microscopy analysis performed on human RD and RH30 cell lines confirmed that MURC/cavin-4 mostly marks differentiated cell elements, colocalizing at the cell surface with Cav-3 and labeling myosin heavy chain (MHC) expressing cells. Finally, MURC/cavin-4 silencing prevented the differentiation in the RD cell line, leading to morphological cell impairment characterized by depletion of myogenin, Cav-3 and MHC protein levels. Overall, our data suggest that MURC/cavin-4, especially in combination with Cav-3, may play a consistent role in the differentiation process of RMS.

Human telomerase reverse transcriptase regulates vascular endothelial growth factor expression via human papillomavirus oncogene E7 in HPV-18-positive cervical cancer cells.

PubMed

Li, Fang; Cui, Jinquan

2015-07-01

Human papillomavirus (HPV) infection induces chronic and precancerous lesions and results in invasive cervical cancer. Human telomerase as well as inflammatory and angiogenic factors such as telomerase reverse transcriptase (hTERT) or vascular endothelial growth factor (VEGF) could play a role in regulating HPV-induced cervical cancer. This study investigated underlying molecular events in HPV-induced HPV-positive cervical cancer through hTERT and VEGF in vitro. Expressions of hTERT, a rate-limiting subunit of telomerase, and VEGF mRNA and proteins were, respectively, assessed by qRT-PCR, ELISA, and TRAP-ELISA in HPV-positive tissue samples and cervical cancer cell lines. To assess hTERT and VEGF secretion, hTERT overexpression and knockdown were conducted in HPV-18-positive Hela cells by hTERT cDNA and shRNA transfection, respectively. Then, the effect of HPV E6 and E7 on VEGF expressions was assessed in HPV-negative cervical cancer cells. Data have shown that VEGF expression levels are associated with hTERT expressions and telomerase activity in HPV-positive cervical cancer tissues and cells. Knockdown of hTERT expression down-regulated VEGF expressions, whereas overexpression of hTERT up-regulated VEGF expressions in HPV-18-positive Hela cells. Furthermore, HPV E7 oncoprotein was necessary for hTERT to up-regulate VEGF expressions in HPV-negative cervical cancer cells. Data from this current study indicate that HPV oncoproteins up-regulated hTERT and telomerase activity and in turn promoted VEGF expressions, which could be a key mechanism for HPV-induced cervical cancer development and progression.

Paucity of PD-L1 expression in prostate cancer: innate and adaptive immune resistance.

PubMed

Martin, A M; Nirschl, T R; Nirschl, C J; Francica, B J; Kochel, C M; van Bokhoven, A; Meeker, A K; Lucia, M S; Anders, R A; DeMarzo, A M; Drake, C G

2015-12-01

Primary prostate cancers are infiltrated with programmed death-1 (PD-1) expressing CD8+ T-cells. However, in early clinical trials, men with metastatic castrate-resistant prostate cancer did not respond to PD-1 blockade as a monotherapy. One explanation for this unresponsiveness could be that prostate tumors generally do not express programmed death ligand-1 (PD-L1), the primary ligand for PD-1. However, lack of PD-L1 expression in prostate cancer would be surprising, given that phosphatase and tensin homolog (PTEN) loss is relatively common in prostate cancer and several studies have shown that PTEN loss correlates with PD-L1 upregulation--constituting a mechanism of innate immune resistance. This study tested whether prostate cancer cells were capable of expressing PD-L1, and whether the rare PD-L1 expression that occurs in human specimens correlates with PTEN loss. Human prostate cancer cell lines were evaluated for PD-L1 expression and loss of PTEN by flow cytometry and western blotting, respectively. Immunohistochemical (IHC) staining for PTEN was correlated with PD-L1 IHC using a series of resected human prostate cancer samples. In vitro, many prostate cancer cell lines upregulated PD-L1 expression in response to inflammatory cytokines, consistent with adaptive immune resistance. In these cell lines, no association between PTEN loss and PD-L1 expression was apparent. In primary prostate tumors, PD-L1 expression was rare, and was not associated with PTEN loss. These studies show that some prostate cancer cell lines are capable of expressing PD-L1. However, in human prostate cancer, PTEN loss is not associated with PD-L1 expression, arguing against innate immune resistance as a mechanism that mitigates antitumor immune responses in this disease.

Anti-sense suppression of epidermal growth factor receptor expression alters cellular proliferation, cell-adhesion and tumorigenicity in ovarian cancer cells.

PubMed

Alper, O; De Santis, M L; Stromberg, K; Hacker, N F; Cho-Chung, Y S; Salomon, D S

2000-11-15

Over-expression of epidermal growth factor receptor (EGFR) in ovarian cancer has been well documented. Human NIH:OVCAR-8 ovarian carcinoma cells were transfected with an expression vector containing the anti-sense orientation of truncated human EGFR cDNA. EGFR anti-sense over-expression resulted in decreased EGFR protein and mRNA expression, cell proliferation and tumor formation in nude mice. In accordance with the reduced levels of EGFR in EGFR anti-sense-expressing cells, tyrosine phosphorylation of EGFR was decreased compared to untransfected parental cells treated with EGF. In EGFR anti-sense-transfected cells, expression of erbB-3, but not erbB-2, was increased. In addition, basal and heregulin-beta 1-stimulated tyrosine phosphorylation of erbB-3 was higher in EGFR anti-sense vector-transfected cells. A morphological alteration in EGFR anti-sense gene-expressing cells was correlated with a decrease in the expression of E-cadherin, alpha-catenin and, to a lesser extent, beta-catenin. Changes in the expression of these proteins were associated with a reduction in complex formation among E-cadherin, beta-catenin and alpha-catenin and between beta-catenin and EGFR in EGFR anti-sense-expressing cells compared to sense-transfected control cells. These results demonstrate that EGFR expression in ovarian carcinoma cells regulates expression of cell adhesion proteins that may enhance cell growth and invasiveness. Copyright 2000 Wiley-Liss, Inc.

Neurogenin 3 Expressing Cells in the Human Exocrine Pancreas Have the Capacity for Endocrine Cell Fate

PubMed Central

Gomez, Danielle L.; O’Driscoll, Marci; Sheets, Timothy P.; Hruban, Ralph H.; Oberholzer, Jose; McGarrigle, James J.; Shamblott, Michael J.

2015-01-01

Neurogenin 3 (NGN3) is necessary and sufficient for endocrine differentiation during pancreatic development and is expressed by a population of progenitor cells that give rise exclusively to hormone-secreting cells within islets. NGN3 protein can be detected in the adult rodent pancreas only following certain types of injury, when it is transiently expressed by exocrine cells undergoing reprogramming to an endocrine cell fate. Here, NGN3 protein can be detected in 2% of acinar and duct cells in living biopsies of histologically normal adult human pancreata and 10% in cadaveric biopsies of organ donor pancreata. The percentage and total number of NGN3+ cells increase during culture without evidence of proliferation or selective cell death. Isolation of highly purified and viable NGN3+ cell populations can be achieved based on coexpression of the cell surface glycoprotein CD133. Transcriptome and targeted expression analyses of isolated CD133+ / NGN3+ cells indicate that they are distinct from surrounding exocrine tissue with respect to expression phenotype and Notch signaling activity, but retain high level mRNA expression of genes indicative of acinar and duct cell function. NGN3+ cells have an mRNA expression profile that resembles that of mouse early endocrine progenitor cells. During in vitro differentiation, NGN3+ cells express genes in a pattern characteristic of endocrine development and result in cells that resemble beta cells on the basis of coexpression of insulin C-peptide, chromogranin A and pancreatic and duodenal homeobox 1. NGN3 expression in the adult human exocrine pancreas marks a dedifferentiating cell population with the capacity to take on an endocrine cell fate. These cells represent a potential source for the treatment of diabetes either through ex vivo manipulation, or in vivo by targeting mechanisms controlling their population size and endocrine cell fate commitment. PMID:26288179

Substance P stimulates production of inflammatory cytokines in human disc cells.

PubMed

Kepler, Christopher K; Markova, Dessislava Z; Hilibrand, Alan S; Vaccaro, Alexander R; Risbud, Makarand V; Albert, Todd J; Anderson, D Greg

2013-10-01

Laboratory study. The aims of this study were as follows: (1) to confirm that Substance P (SP) is expressed by nucleus pulposus (NP) and annulus fibrosus (AF) cells; (2) to determine the effect of SP on expression of inflammatory mediators in human disc cells and the effect of inflammatory mediators on the expression of SP; and (3) to characterize the relative expression of SP receptor isoforms in disc tissue and describe whether exposure to SP changes receptor expression. SP, classically described as a neurotransmitter, acts as an inflammatory regulator in other tissue types, but its role within the intervertebral disc has not been characterized. Human AF and NP cells from 7 individuals were expanded in monolayer and maintained in alginate bead culture. Cells were treated with SP or interleukin (IL)-1β/tumor necrosis factor-α (TNF-α). After treatment, the cells were recovered and then RNA was isolated and transcribed into cDNA. Quantitative reverse-transcriptase polymerase chain reaction was performed to evaluate expression of inflammatory mediators and SP and its receptors. Disc cells treated with SP demonstrated significant upregulation of IL-1β, IL-6, and IL-8 in NP and AF cells whereas significant upregulation of RANTES and TNF occurred only in the AF cells. AF and NP cells expressed SP at low levels; expression did not change significantly with SP treatment but was significantly upregulated after treatment with IL-1β/TNF-α. Both SP receptor isoforms were expressed by NP and AF cells. SP upregulates inflammatory mediators in disc cells. SP and its receptors were expressed in both NP and AF cells, and expression did not change after treatment with SP but increased after treatment with IL-1β/TNF-α. SP likely acts in an autocrine or paracrine manner in intervertebral disc cells and may be involved in "crosstalk" between disc cells and neurons, providing a potential mechanism for transmission of painful discogenic stimuli.

Craniopharyngiomas express embryonic stem cell markers (SOX2, OCT4, KLF4, and SOX9) as pituitary stem cells but do not coexpress RET/GFRA3 receptors.

PubMed

Garcia-Lavandeira, Montserrat; Saez, Carmen; Diaz-Rodriguez, Esther; Perez-Romero, Sihara; Senra, Ana; Dieguez, Carlos; Japon, Miguel A; Alvarez, Clara V

2012-01-01

Adult stem cells maintain some markers expressed by embryonic stem cells and express other specific markers depending on the organ where they reside. Recently, stem/progenitor cells in the rodent and human pituitary have been characterized as expressing GFRA2/RET, PROP1, and stem cell markers such as SOX2 and OCT4 (GPS cells). Our objective was to detect other specific markers of the pituitary stem cells and to investigate whether craniopharyngiomas (CRF), a tumor potentially derived from Rathke's pouch remnants, express similar markers as normal pituitary stem cells. We conducted mRNA and Western blot studies in pituitary extracts, and immunohistochemistry and immunofluorescence on sections from normal rat and human pituitaries and 20 CRF (18 adamantinomatous and two papillary). Normal pituitary GPS stem cells localized in the marginal zone (MZ) express three key embryonic stem cell markers, SOX2, OCT4, and KLF4, in addition to SOX9 and PROP1 and β-catenin overexpression. They express the RET receptor and its GFRA2 coreceptor but also express the coreceptor GFRA3 that could be detected in the MZ of paraffin pituitary sections. CRF maintain the expression of SOX2, OCT4, KLF4, SOX9, and β-catenin. However, RET and GFRA3 expression was altered in CRF. In 25% (five of 20), both RET and GFRA3 were detected but not colocalized in the same cells. The other 75% (15 of 20) lose the expression of RET, GFRA3, or both proteins simultaneously. Human pituitary adult stem/progenitor cells (GPS) located in the MZ are characterized by expression of embryonic stem cell markers SOX2, OCT4, and KLF4 plus the specific pituitary embryonic factor PROP1 and the RET system. Redundancy in RET coreceptor expression (GFRA2 and GFRA3) suggest an important systematic function in their physiological behavior. CRF share the stem cell markers suggesting a common origin with GPS. However, the lack of expression of the RET/GFRA system could be related to the cell mislocation and deregulated growth of CRF.

HGF and c-Met Interaction Promotes Migration in Human Chondrosarcoma Cells

PubMed Central

Tsou, Hsi-Kai; Chen, Hsien-Te; Hung, Ya-Huey; Chang, Chia-Hao; Li, Te-Mao; Fong, Yi-Chin; Tang, Chih-Hsin

2013-01-01

Chondrosarcoma is a type of highly malignant tumor with a potent capacity for local invasion and causing distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Hepatocyte growth factor (HGF) has been demonstrated to stimulate cancer proliferation, migration, and metastasis. However, the effect of HGF on migration activity of human chondrosarcoma cells is not well known. Here, we found that human chondrosarcoma tissues demonstrated significant expression of HGF, which was higher than that in normal cartilage. We also found that HGF increased the migration and expression of matrix metalloproteinase (MMP)-2 in human chondrosarcoma cells. c-Met inhibitor and siRNA reduced HGF-increased cell migration and MMP-2 expression. HGF treatment resulted in activation of the phosphatidylinositol 3′-kinase (PI3K)/Akt/PKCδ/NF-κB pathway, and HGF-induced expression of MMP-2 and cell migration was inhibited by specific inhibitors or siRNA-knockdown of PI3K, Akt, PKCδ, and NF-κB cascades. Taken together, our results indicated that HGF enhances migration of chondrosarcoma cells by increasing MMP-2 expression through the c-Met receptor/PI3K/Akt/PKCδ/NF-κB signal transduction pathway. PMID:23320110

Quercetin induces the apoptosis of human ovarian carcinoma cells by upregulating the expression of microRNA-145.

PubMed

Zhou, Junbo; Gong, Jian; Ding, Chun; Chen, Guiqin

2015-08-01

Ovarian cancer is one of the most malignant types of cancer of the female human reproductive track, posing a severe threat to the health of the female population. Numerous previous studies have demonstrated that microRNA (miR)-145 is downregulated in ovarian cancer, and that quercetin can inhibit the growth of cancer cells via regulating the expression of miRs. Therefore, the present study investigated the effect of quercetin on the expression of miR-145 in SKOV-3 and A2780 human ovarian cancer cell lines. The results revealed that the expression levels of cleaved caspase-3 in the SKOV-3 and A2780 cells were significantly increased following treatment to induce overexpression of miR-145 compared with treatment with quercetin alone (P<0.01). However, the expression of cleaved caspase-3 in the anti-miR-145 (miR-145 inhibitor) group of cells was markedly decreased compared with that in the miR-145 overexpression group (P<0.01). Taken together, the results suggested that treatment with quercetin induced the apoptosis of human ovarian carcinoma cells through activation of the extrinsic death receptor mediated and intrinsic mitochondrial apoptotic pathways.

The therapeutic response of CDDO-Me in the esophageal squamous cell carcinoma (ESCC) cells is mediated by CaMKIIα.

PubMed

Wang, Yan-Yang; Zhou, Shun; Zhao, Ren; Hai, Ping; Zhe, Hong

2016-01-01

CDDO-Me has exhibited a potent anticancer effect in human esophageal squamous cell carcinoma (ESCC) cells in our previous study, but the molecular interactome remains elusive. We applied the approach of stable-isotope labeling by amino acids in cell culture (SILAC) to assess the proteomic responses of CDDO-Me treatment in human ESCC Ec109 cells. The data were subsequently validated using Western blot assay. The results of our study revealed that CDDO-Me increased the expression level of 543 protein molecules, but decreased the expression level of 709 protein molecules in Ec109 cells. Among these modulated protein molecules, calcium/calmodulin-dependent protein kinase type II subunit α (CaMKIIα) was highly expressed in all tested ESCC cell lines, whereas its expression levels were substantially lower in normal control cell line. Its silencing by small interfering RNA inhibited CDDO-Me induced apoptosis and autophagy in ESCC cells. Collectively, these data demonstrate that the therapeutic response of CDDO-Me in the human ESCC cells is mediated by CaMKIIα.

PEST-containing nuclear protein mediates the proliferation, migration, and invasion of human neuroblastoma cells through MAPK and PI3K/AKT/mTOR signaling pathways.

PubMed

Wu, Dong-Dong; Gao, Ying-Ran; Li, Tao; Wang, Da-Yong; Lu, Dan; Liu, Shi-Yu; Hong, Ya; Ning, Hui-Bin; Liu, Jun-Ping; Shang, Jia; Shi, Jun-Feng; Wei, Jian-She; Ji, Xin-Ying

2018-05-02

PEST-containing nuclear protein (PCNP), a novel nuclear protein, is involved in cell proliferation and tumorigenesis. However, the precise mechanism of action of PCNP in the process of tumor growth has not yet been fully elucidated. ShRNA knockdown and overexpression of PCNP were performed in human neuroblastoma cells. Tumorigenic and metastatic effects of PCNP were examined by tumor growth, migration, and invasion assays in vitro, as well as xenograft tumor assay in vivo. PCNP over-expression decreased the proliferation, migration, and invasion of human neuroblastoma cells and down-regulation of PCNP showed reverse effects. PCNP over-expression increased protein expressions of cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, and cleaved poly adenosine diphosphate-ribose polymerase, as well as ratios of B-cell lymphoma-2 (Bcl-2)-associated X protein/Bcl-2 and Bcl-2-associated death promoter/B-cell lymphoma-extra large in human neuroblastoma cells, however PCNP knockdown exhibited reverse trends. PCNP over-expression increased phosphorylations of extracellular signal-regulated protein kinase 1/2, p38, c-Jun N-terminal kinase, as well as decreased phosphorylations of phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR), nevertheless PCNP knockdown exhibited opposite effects. Furthermore, PCNP over-expression significantly reduced the growth of human neuroblastoma xenograft tumors by down-regulating angiogenesis, whereas PCNP knockdown markedly promoted the growth of human neuroblastoma xenograft tumors through up-regulation of angiogenesis. PCNP mediates the proliferation, migration, and invasion of human neuroblastoma cells through mitogen-activated protein kinase and PI3K/AKT/mTOR signaling pathways, implying that PCNP is a therapeutic target for patients with neuroblastoma.

Csa-19, a radiation-responsive human gene, identified by an unbiased two-gel cDNA library screening method in human cancer cells

NASA Technical Reports Server (NTRS)

Balcer-Kubiczek, E. K.; Meltzer, S. J.; Han, L. H.; Zhang, X. F.; Shi, Z. M.; Harrison, G. H.; Abraham, J. M.

1997-01-01

A novel polymerase chain reaction (PCR)-based method was used to identify candidate genes whose expression is altered in cancer cells by ionizing radiation. Transcriptional induction of randomly selected genes in control versus irradiated human HL60 cells was compared. Among several complementary DNA (cDNA) clones recovered by this approach, one cDNA clone (CL68-5) was downregulated in X-irradiated HL60 cells but unaffected by 12-O-tetradecanoyl phorbol-13-acetate, forskolin, or cyclosporin-A. DNA sequencing of the CL68-5 cDNA revealed 100% nucleotide sequence homology to the reported human Csa-19 gene. Northern blot analysis of RNA from control and irradiated cells revealed the expression of a single 0.7-kilobase (kb) messenger RNA (mRNA) transcript. This 0.7-kb Csa-19 mRNA transcript was also expressed in a variety of human adult and corresponding fetal normal tissues. Moreover, when the effect of X- or fission neutron-irradiation on Csa-19 mRNA was compared in cultured human cells differing in p53 gene status (p53-/- versus p53+/+), downregulation of Csa-19 by X-rays or fission neutrons was similar in p53-wild type and p53-null cell lines. Our results provide the first known example of a radiation-responsive gene in human cancer cells whose expression is not associated with p53, adenylate cyclase or protein kinase C.

Development of a Stable Cell Line, Overexpressing Human T-cell Immunoglobulin Mucin 1

PubMed Central

Ebrahimi, Mina; Kazemi, Tohid; Ganjalikhani-hakemi, Mazdak; Majidi, Jafar; khanahmad, Hossein; Rahimmanesh, Ilnaz; Homayouni, Vida; Kohpayeh, Shirin

2015-01-01

Background Recent researches have demonstrated that human T-cell immunoglobulin mucin 1 (TIM-1) glycoprotein plays important roles in regulation of autoimmune and allergic diseases, as well as in tumor immunity and response to viral infections. Therefore, targeting TIM-1 could be a potential therapeutic approach against such diseases. Objectives In this study, we aimed to express TIM-1 protein on Human Embryonic kidney (HEK) 293T cell line in order to have an available source of the TIM-1 antigen. Materials and Methods The cDNA was synthesized after RNA extraction from peripheral blood mononuclear cells (PBMC) and TIM-1 cDNA was amplified by PCR with specific primers. The PCR product was cloned in pcDNA™3.1/Hygro (+) and transformed in Escherichia coli TOP 10 F’. After cloning, authenticity of DNA sequence was checked and expressed in HEK 293T cells. Finally, expression of TIM-1 was analyzed by flow cytometry and real-time PCR. Results The result of DNA sequencing demonstrated correctness of TIM-1 DNA sequence. The flow cytometry results indicated that TIM-1 was expressed in about 90% of transfected HEK 293T cells. The real-time PCR analysis showed TIM-1 mRNA expression increased 195-fold in transfected cells compared with un-transfected cells. Conclusions Findings of present study demonstrated the successful cloning and expression of TIM-1 on HEK 293T cells. These cells could be used as an immunogenic source for production of specific monoclonal antibodies, nanobodies and aptamers against human TIM-1. PMID:28959306

Antagonism of the Protein Kinase R Pathway in Human Cells by Rhesus Cytomegalovirus.

PubMed

Child, Stephanie J; Hickson, Sarah E; Bayer, Avraham; Malouli, Daniel; Früh, Klaus; Geballe, Adam P

2018-03-15

While cytomegalovirus (CMV) infections are often limited in host range by lengthy coevolution with a single host species, a few CMVs are known to deviate from this rule. For example, rhesus macaque CMV (RhCMV), a model for human CMV (HCMV) pathogenesis and vaccine development, can replicate in human cells, as well as in rhesus cells. Both HCMV and RhCMV encode species-specific antagonists of the broadly acting host cell restriction factor protein kinase R (PKR). Although the RhCMV antagonist of PKR, rTRS1, has very limited activity against human PKR, here, we show it is essential for RhCMV replication in human cells because it prevents human PKR from phosphorylating the translation initiation factor eIF2α, thereby allowing continued translation and viral replication. Although rTRS1 is necessary for RhCMV replication, it is not sufficient to rescue replication of HCMV lacking its own PKR antagonists in human fibroblasts. However, overexpression of rTRS1 in human fibroblasts enabled HCMV expressing rTRS1 to replicate, indicating that elevated levels or early expression of a weak antagonist can counteract a resistant restriction factor like human PKR. Exploring potential mechanisms that might allow RhCMV to replicate in human cells revealed that RhCMV makes no less double-stranded RNA than HCMV. Rather, in human cells, RhCMV expresses rTRS1 at levels 2 to 3 times higher than those of the HCMV-encoded PKR antagonists during HCMV infection. These data suggest that even a modest increase in expression of this weak PKR antagonist is sufficient to enable RhCMV replication in human cells. IMPORTANCE Rhesus macaque cytomegalovirus (RhCMV) offers a valuable model for studying congenital human cytomegalovirus (HCMV) pathogenesis and vaccine development. Therefore, it is critical to understand variations in how each virus infects and affects its host species to be able to apply insights gained from the RhCMV model to HCMV. While HCMV is capable only of infecting cells from humans and very closely related species, RhCMV displays a wider host range, including human as well as rhesus cells. RhCMV expresses an antagonist of a broadly acting antiviral factor present in all mammalian cells, and its ability to counter both the rhesus and human versions of this host factor is a key component of RhCMV's ability to cross species barriers. Here, we examine the molecular mechanisms that allow this RhCMV antagonist to function against a human restriction factor. Copyright © 2018 American Society for Microbiology.

Human type II pneumocyte chemotactic responses to CXCR3 activation are mediated by splice variant A.

PubMed

Ji, Rong; Lee, Clement M; Gonzales, Linda W; Yang, Yi; Aksoy, Mark O; Wang, Ping; Brailoiu, Eugen; Dun, Nae; Hurford, Matthew T; Kelsen, Steven G

2008-06-01

Chemokine receptors control several fundamental cellular processes in both hematopoietic and structural cells, including directed cell movement, i.e., chemotaxis, cell differentiation, and proliferation. We have previously demonstrated that CXCR3, the chemokine receptor expressed by Th1/Tc1 inflammatory cells present in the lung, is also expressed by human airway epithelial cells. In airway epithelial cells, activation of CXCR3 induces airway epithelial cell movement and proliferation, processes that underlie lung repair. The present study examined the expression and function of CXCR3 in human alveolar type II pneumocytes, whose destruction causes emphysema. CXCR3 was present in human fetal and adult type II pneumocytes as assessed by immunocytochemistry, immunohistochemistry, and Western blotting. CXCR3-A and -B splice variant mRNA was present constitutively in cultured type II cells, but levels of CXCR3-B greatly exceeded CXCR3-A mRNA. In cultured type II cells, I-TAC, IP-10, and Mig induced chemotaxis. Overexpression of CXCR3-A in the A549 pneumocyte cell line produced robust chemotactic responses to I-TAC and IP-10. In contrast, I-TAC did not induce chemotactic responses in CXCR3-B and mock-transfected cells. Finally, I-TAC increased cytosolic Ca(2+) and activated the extracellular signal-regulated kinase, p38, and phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B kinases only in CXCR3-A-transfected cells. These data indicate that the CXCR3 receptor is expressed by human type II pneumocytes, and the CXCR3-A splice variant mediates chemotactic responses possibly through Ca(2+) activation of both mitogen-activated protein kinase and PI 3-kinase signaling pathways. Expression of CXCR3 in alveolar epithelial cells may be important in pneumocyte repair from injury.

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

Lee, Seung-Hwan; Kim, Dong-Young; Jing, Feifeng

Developmental endothelial locus-1 (Del-1) is an endogenous anti-inflammatory molecule that is highly expressed in the lung and the brain and limits leukocyte migration to these tissues. We previously reported that the expression of Del-1 is positively regulated by p53 in lung endothelial cells. Although several reports have implicated the altered expression of Del-1 gene in cancer patients, little is known about its role in tumor cells. We here investigated the effect of Del-1 on the features of human lung carcinoma cells. Del-1 mRNA was found to be significantly decreased in the human lung adenocarcinoma cell lines A549 (containing wild typemore » of p53), H1299 (null for p53) and EKVX (mutant p53), compared to in human normal lung epithelial BEAS-2B cells and MRC-5 fibroblasts. The decrease of Del-1 expression was dependent on the p53 activity in the cell lines, but not on the expression of p53. Neither treatment with recombinant human Del-1 protein nor the introduction of adenovirus expressing Del-1 altered the expression of the apoptosis regulators BAX, PUMA and Bcl-2. Unexpectedly, the adenovirus-mediated overexpression of Del-1 gene into the lung carcinoma cell lines promoted proliferation and invasion of the lung carcinoma cells, as revealed by BrdU incorporation and transwell invasion assays, respectively. In addition, overexpression of the Del-1 gene enhanced features of epithelial–mesenchymal transition (EMT), such as increasing vimentin while decreasing E-cadherin in A549 cells, and increases in the level of Slug, an EMT-associated transcription regulator. Our findings demonstrated for the first time that there are deleterious effects of high levels of Del-1 in lung carcinoma cells, and suggest that Del-1 may be used as a diagnostic or prognostic marker for cancer progression, and as a novel therapeutic target for lung carcinoma. - Highlights: • Developmental Endothelial Locus-1 (Del-1) expression is downregulated in human lung cancer cells. • Overexpression of the Del-1 gene potentiates proliferation and invasion of lung carcinoma cells. • Del-1 may be used as a diagnostic or prognostic marker for lung cancer progression.« less

PD-L1 Expression Promotes Epithelial to Mesenchymal Transition in Human Esophageal Cancer.

PubMed

Chen, Lujun; Xiong, Yuqi; Li, Jing; Zheng, Xiao; Zhou, Qi; Turner, Abbey; Wu, Changping; Lu, Binfeng; Jiang, Jingting

2017-01-01

PD-L1 (Programmed cell death 1 ligand 1, PD-L1), an essential immune checkpoint molecule in the tumor microenvironment, is an important target for cancer immunotherapy. We have previously reported that its expression in human gastric and esophageal cancer tissues is significantly associated with cancer progression and patients' postoperative prognoses. Its expression in cancer cells is well known to inhibit the T cell-mediated anti-tumor response, and this mechanism of action has been targeted for cancer immunotherapy. As of now, the autonomous effect of PD-L1 on cancer cells is not well understood, thus our present study aimed to examine the role of PD-L1 intervention in cellular biological functions, especially epithelial to mesenchymal transition (EMT), of the human esophageal cancer cell line, Eca-109 cells. Immunohistochemistry assay was used to investigate the correlation between expression of PD-L1 and EMT markers in human esophageal cancer tissues. Intervention of PD-L1 by using RNAi and over-expression methods were used to study the role of PD-L1 in regulation of biological behaviors and EMT in Eca-109 cells. Our clinical and pathological data demonstrated that tumor samples in the EMT positive subgroup had higher PD-L1 expression than those in the EMT negative subgroup. By manipulating PD-L1 expression in Eca-109 cells either through ablation or overexpression of wild type and the cytoplasmic domain-truncated mutant, we demonstrated that PD-L1 expression significantly promoted the cell viability, migration and EMT phenotype. Furthermore, our study also indicated that PD-1 fusion protein mediated stimulation of PD-L1 and the cytoplasmic domain of PD-L1 played a critical role in promoting EMT phenotype of Eca-109 cells, thereby suggesting that PD-1 receptor usually by triggering the reverse signaling can effect PD-L1 mediated regulation of esophageal cancer cell response. Our present study reveals a tumor cell-autonomous role of PD-L1 signaling in promoting EMT in human esophageal cancer. © 2017 The Author(s). Published by S. Karger AG, Basel.

The RUNX1 +24 enhancer and P1 promoter identify a unique subpopulation of hematopoietic progenitor cells derived from human pluripotent stem cells

PubMed Central

Ferrell, Patrick I; Xi, Jiafei; Ma, Chao; Adlakha, Mitali; Kaufman, Dan S.

2016-01-01

Derivation of hematopoietic stem cells from human pluripotent stem cells remains a key goal for the fields of developmental biology and regenerative medicine. Here, we use a novel genetic reporter system to prospectively identify and isolate early hematopoietic cells derived from human embryonic stem cells (hESCs) and human induced pluripotent cells (iPSCs). Cloning the human RUNX1c P1 promoter and +24 enhancer to drive expression of tdTomato (tdTom) in hESCs and iPSCs, we demonstrate that tdTom expression faithfully enriches for RUNX1c-expressing hematopoietic progenitor cells. Time-lapse microscopy demonstrated the tdTom+ hematopoietic cells to emerge from adherent cells. Furthermore, inhibition of primitive hematopoiesis by blocking Activin/Nodal signaling promoted the expansion and/or survival of tdTom+ population. Notably, RUNX1c/tdTom+ cells represent only a limited subpopuation of CD34+CD45+ and CD34+CD43+ cells with a unique genetic signature. Using gene array analysis, we find significantly lower expression of Let-7 and mir181a microRNAs in the RUNX1c/tdTom+ cell population. These phenotypic and genetic analyses comparing the RUNX1c/tdTom+ population to CD34+CD45+ umbilical cord blood and fetal liver demonstrate several key differences that likely impact the development of HSCs capable of long-term multilineage engraftment from hESCs and iPSCs. PMID:25546363

Generation of a TALEN-mediated, p63 knock-in in human induced pluripotent stem cells.

PubMed

Kobayashi, Yuki; Hayashi, Ryuhei; Quantock, Andrew J; Nishida, Kohji

2017-12-01

The expression of p63 in surface ectodermal cells during development of the cornea, skin, oral mucosa and olfactory placodes is integral to the process of cellular self-renewal and the maintenance of the epithelial stem cell status. Here, we used TALEN technology to generate a p63 knock-in (KI) human induced pluripotent stem (hiPS) cell line in which p63 expression can be visualized via enhanced green fluorescent protein (EGFP) expression. The KI-hiPS cells maintained pluripotency and expressed the stem cell marker gene, ΔNp63α. They were also able to successfully differentiate into functional corneal epithelial cells as assessed by p63 expression in reconstructed corneal epithelium. This approach enables the tracing of p63-expressing cell lineages throughout epithelial development, and represents a promising application in the field of stem cell research. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Homing of human B cells to lymphoid organs and B-cell lymphoma engraftment are controlled by cell adhesion molecule JAM-C.

PubMed

Doñate, Carmen; Ody, Christiane; McKee, Thomas; Ruault-Jungblut, Sylvie; Fischer, Nicolas; Ropraz, Patricia; Imhof, Beat A; Matthes, Thomas

2013-01-15

Junctional adhesion molecule C (JAM-C) is expressed by vascular endothelium and human but not mouse B lymphocytes. The level of JAM-C expression defines B-cell differentiation stages and allows the classification of marginal zone-derived (JAM-C-positive) and germinal center-derived (JAM-C-negative) B-cell lymphomas. In the present study, we investigated the role of JAM-C in homing of human B cells, using a xenogeneic nonobese diabetic/severe combined immunodeficient mouse model. Treatment with anti-JAM-C antibodies in short-term experiments reduced migration of normal and malignant JAM-C-expressing B cells to bone marrow, lymph nodes, and spleen. Blocking homing to the spleen is remarkable, as most other antiadhesion antibodies reduce homing of B cells only to bone marrow and lymph nodes. Long-term administration of anti-JAM-C antibodies prevented engraftment of JAM-Cpos lymphoma cells in bone marrow, spleen, and lymph nodes of mice. Plasmon resonance studies identified JAM-B as the major ligand for JAM-C, whereas homotypic JAM-C interactions remained at background levels. Accordingly, anti-JAM-C antibodies blocked adhesion of JAM-C-expressing B cells to their ligand JAM-B, and immunofluorescence analysis showed the expression of JAM-B on murine and human lymphatic endothelial cells. Targeting JAM-C could thus constitute a new therapeutic strategy to prevent lymphoma cells from reaching supportive microenvironments not only in the bone marrow and lymph nodes but also in the spleen.

Human amnion epithelial cells expressing HLA-G as novel cell-based treatment for liver disease.

PubMed

Strom, Stephen C; Gramignoli, Roberto

2016-09-01

Despite routine liver transplantation and supporting medical therapies, thousands of patients currently wait for an organ and there is an unmet need for more refined and widely available regenerative strategies to treat liver diseases. Cell transplants attempt to maximize the potential for repair and/or regeneration in liver and other organs. Over 40years of laboratory pre-clinical research and 25years of clinical procedures have shown that certain liver diseases can be treated by the infusion of isolated cells (hepatocyte transplant). However, like organ transplants, hepatocyte transplant suffers from a paucity of tissues useful for cell production. Alternative sources have been investigated, yet with limited success. The tumorigenic potential of pluripotent stem cells together with their primitive level of hepatic differentiation, have limited the use of stem cell populations. Stem cell sources from human placenta, and the amnion tissue in particular are receiving renewed interest in the field of regenerative medicine. Unlike pluripotent stem cells, human amnion epithelial (AE) cells are easily available without ethical or religious concerns; they do not express telomerase and are not immortal or tumorigenic when transplanted. In addition, AE cells have been reported to express genes normally expressed in mature liver, when transplanted into the liver. Moreover, because of the possibility of an immune-privileged status related to their expression of HLA-G, it might be possible to transplant human AE cells without immunosuppression of the recipient. Copyright © 2016. Published by Elsevier Inc.

[Effect of forced E-cadherin expression on adhesion and proliferation of human breast carcinoma cells].

PubMed

Yang, Li-Juan; Liu, Yu-Qin; Gu, Bei; Bian, Xiao-Cui; Feng, Hai-Liang; Yang, Zhen-Li; Liu, Yan-Yan

2010-12-01

To investigate the role that E-cadherin (E-cad) plays on cell adhesion and proliferation of human breast carcinoma. E-cad expression vector was transfected into an E-cad-negative human breast carcinoma MDA-MB-231 cells. G418 was used to screen positive clones. E-cad, β-catenin (β-cat) and cyclin D1 expressions of these clones were confirmed by Western blot. Their cell-cell and cell-matrix adhesion abilities were detected. E-cad/β-catenin interaction was confirmed by immunoprecipitation. Cell proliferation was evaluated by MTT. Cell apoptosis was analyzed by flow cytometry. Direct two-step immunocytochemistry was used to detect the localization of β-cat. E-cad(+) cell strains Ecad-231-7 and Ecad-231-9 were established. When cultured in ultra-low-binding dishes Ecad-231 cells grow in suspension while Ecad-231-7 and Ecad-231-9 cells grow in large clamps. When co-cultured with HCT116 cells, the average adhesion rates at 30 min are 39.0%, 60.0% and 59.5% for MDA-MB-231, Ecad-231-7 and Ecad-231-9 respectively. The average detachment rates by EDTA for 5 min are 37.4%, 4.2% and 7.4% respectively. So E-cad expression enhanced hemotypic and heterotypic cell-cell adhesion and cell-matrix adhesion. Forced exogenously expressed E-cad could combine with endogenous β-cat, whereas down stream cyclin D1 expression was significantly decreased, as evidenced by Western blot. The rates of cell apoptosis of MDA-MB-231, Ecad-231-7 and Ecad-231-9 were 1.8%, 2.0% and 2.1%. Expression of E-cad had no obvious effect on the apoptosis of tumor cells with regular culture. β-cat increased in the cytoplasma. Two monoclonal tumor cell strains (Ecad-231-7 and Ecad-231-9) stably expressing E-cad were successfully established. E-cad could enhance adhesion and inhibit proliferation of human breast carcinoma cells through a pathway involving β-cat and cyclin D1.

Cul4A overexpression associated with Gli1 expression in malignant pleural mesothelioma

DOE PAGES

Yang, Yi -Lin; Ni, Jian; Hsu, Ping -Chih; ...

2015-07-27

Malignant pleural mesothelioma (mesothelioma) is a highly aggressive cancer without an effective treatment. Cul4A, a scaffold protein that recruits substrates for degradation, is amplified in several human cancers, including mesothelioma. We have recently shown that Cul4A plays an oncogenic role in vitro and in a mouse model. In this study, we analysed clinical mesothelioma tumours and found moderate to strong expression of Cul4A in 70.9% (51/72) of these tumours, as shown by immunohistochemistry. In 72.2% mesothelioma tumours with increased Cul4A copy number identified by fluorescence in situ hybridization analysis, Cul4A protein expression was moderate to strong. Similarly, Cul4A was overexpressedmore » and Cul4A copy number was increased in human mesothelioma cell lines. Because Gli1 is highly expressed in human mesothelioma cells, we compared Cul4A and Gli1 expression in mesothelioma tumours and found their expression associated (P < 0.05, chi-square). In mesothelioma cell lines, inhibiting Cul4A by siRNA decreased Gli1 expression, suggesting that Gli1 expression is, at least in part, regulated by Cul4A in mesothelioma cells. Our results suggest a linkage between Cul4A and Gli1 expression in human mesothelioma.« less

Expression and function of glycogen synthase kinase-3 in human hair follicles.

PubMed

Yamauchi, Koichi; Kurosaka, Akira

2010-05-01

Beta-catenin is involved in the hair follicle morphogenesis and stem cell differentiation, and inhibition of glycogen synthase kinase-3 (GSK-3) increases beta-catenin concentration in the cytoplasm. To examine the effects of GSK-3 inhibition on the hair follicle epithelium, we first examined the expression of GSK-3 in plucked human hair follicles by RT-PCR and found GSK-3 expression in hair follicles. Western blotting with a GSK-3beta-specific antibody, Y174, also demonstrated GSK-3beta expression in the follicles. Moreover, GSK-3beta immunostaining with Y174 showed that GSK-3beta colocalized with hair follicle bulge markers. Contrary to GSK-3beta, GSK-3 alpha was widely expressed throughout the follicles when immunostained with a specific antibody, EP793Y. We then investigated the influence of GSK-3 inhibition. A GSK-3 inhibitor, BIO, promoted the growth of human outer root sheath cells, which could be cultured for up to four passages. The BIO-treated cells exhibited smaller and more undifferentiated morphology than control cells. Moreover, in organ culture of plucked human hair, outer root sheath cells in the middle of a hair follicle proliferated when cultured with BIO. These results indicate that GSK-3beta is expressed in hair bulge stem cells and BIO promotes the growth of ORS cells, possibly by regulating the GSK-3 signaling pathway.

Effects of bisphenol A on the expression of cytochrome P450 aromatase (CYP19) in human fetal osteoblastic and granulosa cell-like cell lines.

PubMed

Watanabe, Masatada; Ohno, Shuji; Nakajin, Shizuo

2012-04-05

The effects of bisphenol A (BPA), an endocrine disruptor, on aromatase (CYP19) expression in human osteoblastic (SV-HFO) and ovarian granulosa-like (KGN) cell lines were examined. CYP19 enzyme activity was suppressed in the presence of BPA in a dose-dependent fashion in both cell lines. CYP19 gene transcript expression, as well as activities of promoter I.4 in SV-HFO and promoter II in KGN, was down-regulated by BPA, suggesting that BPA affects CYP19 at the gene-expression level. These data and the previous finding that BPA induced the down-regulation of promoter I.1 activity within the human placental cell line suggest that there may be a conserved signaling pathway that down-regulates CYP19 expression in response to BPA in both cell lines. Additionally, differences between promoter I.4 and II suggest that there may be cell- and promoter-specific down-regulating mechanisms downstream from the actions of BPA. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

CUL4A overexpression enhances lung tumor growth and sensitizes lung cancer cells to Erlotinib via transcriptional regulation of EGFR

DOE PAGES

Wang, Yunshan; Zhang, Pengju; Liu, Ziming; ...

2014-11-21

CUL4A has been proposed as oncogene in several types of human cancer, but its clinical significance and functional role in human non-small cell lung cancer (NSCLC) remain unclear. Expression level of CUL4A was examined by RT-PCR and Western blot. Forced expression of CUL4A was mediated by retroviruses, and CUL4A silencing by shRNAs expressing lentiviruses. Growth capacity of lung cancer cells was measured by MTT in vitro and tumorigenesis in vivo, respectively. We found that CUL4A was highly expressed in human lung cancer tissues and lung cancer cell lines, and this elevated expression positively correlated with disease progression and prognosis. Overexpressionmore » of CUL4A in human lung cancer cell lines increased cell proliferation, inhibited apoptosis, and subsequently conferred resistance to chemotherapy. On other hand, silencing CUL4A expression in NSCLC cells reduced proliferation, promoted apoptosis and resulted in tumor growth inhibition in cancer xenograft model. Mechanistically, we revealed CUL4A regulated EGFR transcriptional expression and activation, and subsequently activated AKT. Targeted inhibition of EGFR activity blocked these CUL4A induced oncogenic activities. In conclusion, our results highlight the significance of CUL4A in NSCLC and suggest that CUL4A could be a promising therapy target and a potential biomarker for prognosis and EGFR target therapy in NSCLC patients.« less

Predominant antitumor effects by fully human anti-TRAIL-receptor2 (DR5) monoclonal antibodies in human glioma cells in vitro and in vivo

PubMed Central

Nagane, Motoo; Shimizu, Saki; Mori, Eiji; Kataoka, Shiro; Shiokawa, Yoshiaki

2010-01-01

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL/Apo2 L) preferentially induces apoptosis in human tumor cells through its cognate death receptors DR4 or DR5, thereby being investigated as a potential agent for cancer therapy. Here, we applied fully human anti-human TRAIL receptor monoclonal antibodies (mAbs) to specifically target one of death receptors for TRAIL in human glioma cells, which could also reduce potential TRAIL-induced toxicity in humans. Twelve human glioma cell lines treated with several fully human anti-human TRAIL receptor mAbs were sensitive to only anti-DR5 mAbs, whereas they were totally insensitive to anti-DR4 mAb. Treatment with anti-DR5 mAbs exerted rapid cytotoxicity and lead to apoptosis induction. The cellular sensitivity was closely associated with cell-surface expression of DR5. Expression of c-FLIPL, Akt, and Cyclin D1 significantly correlated with sensitivity to anti-DR5 mAbs. Primary cultures of glioma cells were also relatively resistant to anti-DR5 mAbs, exhibiting both lower DR5 and higher c-FLIPL expression. Downregulation of c-FLIPL expression resulted in the sensitization of human glioma cells to anti-DR5 mAbs, whereas overexpression of c-FLIPL conferred resistance to anti-DR5 mAb. Treatment of tumor-burden nude mice with the direct agonist anti-DR5 mAb KMTR2 significantly suppressed growth of subcutaneous glioma xenografts leading to complete regression. Similarly, treatment of nude mice bearing intracerebral glioma xenografts with KMTR2 significantly elongated lifespan without tumor recurrence. These results suggest that DR5 is the predominant TRAIL receptor mediating apoptotic signals in human glioma cells, and sensitivity to anti-DR5 mAbs was determined at least in part by the expression level of c-FLIPL and Akt. Specific targeting of death receptor pathway through DR5 using fully human mAbs might provide a novel therapeutic strategy for intractable malignant gliomas. PMID:20511188

FLIP switches Fas-mediated glucose signaling in human pancreatic cells from apoptosis to cell replication

NASA Astrophysics Data System (ADS)

Maedler, Kathrin; Fontana, Adriano; Ris, Frédéric; Sergeev, Pavel; Toso, Christian; Oberholzer, José; Lehmann, Roger; Bachmann, Felix; Tasinato, Andrea; Spinas, Giatgen A.; Halban, Philippe A.; Donath, Marc Y.

2002-06-01

Type 2 diabetes mellitus results from an inadequate adaptation of the functional pancreatic cell mass in the face of insulin resistance. Changes in the concentration of glucose play an essential role in the regulation of cell turnover. In human islets, elevated glucose concentrations impair cell proliferation and induce cell apoptosis via up-regulation of the Fas receptor. Recently, it has been shown that the caspase-8 inhibitor FLIP may divert Fas-mediated death signals into those for cell proliferation in lymphatic cells. We observed expression of FLIP in human pancreatic cells of nondiabetic individuals, which was decreased in tissue sections of type 2 diabetic patients. In vitro exposure of islets from nondiabetic organ donors to high glucose levels decreased FLIP expression and increased the percentage of apoptotic terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL)-positive cells; FLIP was no longer detectable in such TUNEL-positive cells. Up-regulation of FLIP, by incubation with transforming growth factor or by transfection with an expression vector coding for FLIP, protected cells from glucose-induced apoptosis, restored cell proliferation, and improved cell function. The beneficial effects of FLIP overexpression were blocked by an antagonistic anti-Fas antibody, indicating their dependence on Fas receptor activation. The present data provide evidence for expression of FLIP in the human cell and suggest a novel approach to prevent and treat diabetes by switching Fas signaling from apoptosis to proliferation.

Anaplastic lymphoma kinase is expressed in different subtypes of human breast cancer

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

Perez-Pinera, Pablo; Chang, Y.; Astudillo, A.

2007-06-29

Pleiotrophin (PTN, Ptn) is an 18 kDa cytokine expressed in human breast cancers. Since inappropriate expression of Ptn stimulates progression of breast cancer in transgenic mice and a dominant negative PTN reverses the transformed phenotype of human breast cancer cells that inappropriately express Ptn, it is suggested that constitutive PTN signaling in breast cancer cells that inappropriately express Ptn activates pathways that promote a more aggressive breast cancer phenotype. Pleiotrophin signals by inactivating its receptor, the receptor protein tyrosine phosphatase (RPTP){beta}/{zeta}, and, recently, PTN was found to activate anaplastic lymphoma kinase (ALK) through the PTN/RPTP{beta}/{zeta} signaling pathway in PTN-stimulated cells,more » not through a direct interaction of PTN with ALK and thus not through the PTN-enforced dimerization of ALK. Since full-length ALK is activated in different malignant cancers and activated ALK is a potent oncogenic protein, we examined human breast cancers to test the possibility that ALK may be expressed in breast cancers and potentially activated through the PTN/RPTP{beta}/{zeta} signaling pathway; we now demonstrate that ALK is strongly expressed in different histological subtypes of human breast cancer; furthermore, ALK is expressed in both nuclei and cytoplasm and, in the 'dotted' pattern characteristic of ALK fusion proteins in anaplastic large cell lymphoma. This study thus supports the possibility that activated ALK may be important in human breast cancers and potentially activated either through the PTN/RPTP{beta}/{zeta} signaling pathway, or, alternatively, as an activated fusion protein to stimulate progression of breast cancer in humans.« less

Hypoexpression and epigenetic regulation of candidate tumor suppressor gene CADM-2 in human prostate cancer.

PubMed

Chang, Guimin; Xu, Shuping; Dhir, Rajiv; Chandran, Uma; O'Keefe, Denise S; Greenberg, Norman M; Gingrich, Jeffrey R

2010-11-15

Cell adhesion molecules (CADM) comprise a newly identified protein family whose functions include cell polarity maintenance and tumor suppression. CADM-1, CADM-3, and CADM-4 have been shown to act as tumor suppressor genes in multiple cancers including prostate cancer. However, CADM-2 expression has not been determined in prostate cancer. The CADM-2 gene was cloned and characterized and its expression in human prostatic cell lines and cancer specimens was analyzed by reverse transcription-PCR and an immunohistochemical tissue array, respectively. The effects of adenovirus-mediated CADM-2 expression on prostate cancer cells were also investigated. CADM-2 promoter methylation was evaluated by bisulfite sequencing and methylation-specific PCR. We report the initial characterization of CADM-2 isoforms: CADM-2a and CADM-2b, each with separate promoters, in human chromosome 3p12.1. Prostate cancer cell lines, LNCaP and DU145, expressed negligible CADM-2a relative to primary prostate tissue and cell lines, RWPE-1 and PPC-1, whereas expression of CADM-2b was maintained. Using immunohistochemistry, tissue array results from clinical specimens showed statistically significant decreased expression in prostate carcinoma compared with normal donor prostate, benign prostatic hyperplasia, prostatic intraepithelial neoplasia, and normal tissue adjacent to tumor (P < 0.001). Adenovirus-mediated CADM-2a expression suppressed DU145 cell proliferation in vitro and colony formation in soft agar. The decrease in CADM-2a mRNA in cancer cell lines correlated with promoter region hypermethylation as determined by bisulfite sequencing and methylation-specific PCR. Accordingly, treatment of cells with the demethylating agent 5-aza-2'-deoxycytidine alone or in combination with the histone deacetylase inhibitor trichostatin A resulted in the reactivation of CADM-2a expression. CADM-2a protein expression is significantly reduced in prostate cancer. Its expression is regulated in part by promoter methylation and implicates CADM-2 as a previously unrecognized tumor suppressor gene in a proportion of human prostate cancers. ©2010 AACR.

Liver-specific gene expression in cultured human hematopoietic stem cells.

PubMed

Fiegel, Henning C; Lioznov, Michael V; Cortes-Dericks, Lourdes; Lange, Claudia; Kluth, Dietrich; Fehse, Boris; Zander, Axel R

2003-01-01

Hematopoietic and hepatic stem cells share characteristic markers such as CD34, c-kit, and Thy1. Based on the recent observations that hepatocytes may originate from bone marrow, we investigated the potential of CD34(+) bone marrow cells to differentiate into hepatocytic cells in vitro. CD34(+) and CD34(-) human bone marrow cells were separated by magnetic cell sorting. Cells were cultured on a collagen matrix in a defined medium containing hepatocyte growth factor. Cell count and size were measured by flow cytometry, and reverse transcription polymerase chain reaction was carried out for the liver-specific markers CK-19 and albumin. During cell culture, CD34(+) cells showed an increasing cell number and proliferative activity as assessed by Ki-67 staining. Under the specified culture conditions, CD34(+) cells expressed albumin RNA and CK-19 RNA after 28 days, whereas CD34(-) cells did not show liver-specific gene expression. The results indicate that CD34(+) adult human bone marrow stem cells can differentiate into hepatocytic cells in vitro.

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

NOX4-mediated ROS production induces apoptotic cell death via down-regulation of c-FLIP and Mcl-1 expression in combined treatment with thioridazine and curcumin.

PubMed

Seo, Seung Un; Kim, Tae Hwan; Kim, Dong Eun; Min, Kyoung-Jin; Kwon, Taeg Kyu

2017-10-01

Thioridazine is known to have anti-tumor effects by inhibiting PI3K/Akt signaling, which is an important signaling pathway in cell survival. However, thioridazine alone does not induce apoptosis in head and neck squamous cell carcinoma (AMC-HN4), human breast carcinoma (MDA-MB231), and human glioma (U87MG) cells. Therefore, we investigated whether combined treatment with thioridazine and curcumin induces apoptosis. Combined treatment with thioridazine and curcumin markedly induced apoptosis in cancer cells without inducing apoptosis in human normal mesangial cells and human normal umbilical vein cells (EA.hy926). We found that combined treatment with thioridazine and curcumin had synergistic effects in AMC-HN4 cells. Among apoptosis-related proteins, thioridazine plus curcumin induced down-regulation of c-FLIP and Mcl-1 expression at the post-translational levels in a proteasome-dependent manner. Augmentation of proteasome activity was related to the up-regulation of proteasome subunit alpha 5 (PSMA5) expression in curcumin plus thioridazine-treated cells. Combined treatment with curcumin and thioridazine produced intracellular ROS in a NOX4-dependent manner, and ROS-mediated activation of Nrf2/ARE signaling played a critical role in the up-regulation of PSMA5 expression. Furthermore, ectopic expression of c-FLIP and Mcl-1 inhibited apoptosis in thioridazine and curcumin-treated cells. Therefore, we demonstrated that thioridazine plus curcumin induces proteasome activity by up-regulating PSMA5 expression via NOX4-mediated ROS production and that down-regulation of c-FLIP and Mcl-1 expression post-translationally is involved in apoptosis. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Monomethylfumarate Induces γ-Globin Expression and Fetal Hemoglobin Production in Cultured Human Retinal Pigment Epithelial (RPE) and Erythroid Cells, and in Intact Retina

PubMed Central

Promsote, Wanwisa; Makala, Levi; Li, Biaoru; Smith, Sylvia B.; Singh, Nagendra; Ganapathy, Vadivel; Pace, Betty S.; Martin, Pamela M.

2014-01-01

Purpose. Sickle retinopathy (SR) is a major cause of vision loss in sickle cell disease (SCD). There are no strategies to prevent SR and treatments are extremely limited. The present study evaluated (1) the retinal pigment epithelial (RPE) cell as a hemoglobin producer and novel cellular target for fetal hemoglobin (HbF) induction, and (2) monomethylfumarate (MMF) as an HbF-inducing therapy and abrogator of oxidative stress and inflammation in SCD retina. Methods. Human globin gene expression was evaluated by RT–quantitative (q)PCR in the human RPE cell line ARPE-19 and in primary RPE cells isolated from Townes humanized SCD mice. γ-Globin promoter activity was monitored in KU812 stable dual luciferase reporter expressing cells treated with 0 to 1000 μM dimethylfumarate, MMF, or hydroxyurea (HU; positive control) by dual luciferase assay. Reverse transcriptase–qPCR, fluorescence-activated cell sorting (FACS), immunofluorescence, and Western blot techniques were used to evaluate γ-globin expression and HbF production in primary human erythroid progenitors, ARPE-19, and normal hemoglobin producing (HbAA) and homozygous βs mutation (HbSS) RPE that were treated similarly, and in MMF-injected (1000 μM) HbAA and HbSS retinas. Dihydroethidium labeling and nuclear factor (erythroid-derived 2)-like 2 (Nrf2), IL-1β, and VEGF expression were also analyzed. Results. Retinal pigment epithelial cells express globin genes and synthesize adult and fetal hemoglobin MMF stimulated γ-globin expression and HbF production in cultured RPE and erythroid cells, and in HbSS mouse retina where it also reduced oxidative stress and inflammation. Conclusions. The production of hemoglobin by RPE suggests the potential involvement of this cell type in the etiology of SR. Monomethylfumarate influences multiple parameters consistent with improved retinal health in SCD and may therefore be of therapeutic potential in SR treatment. PMID:24825111

Silencing NKD2 by Promoter Region Hypermethylation Promotes Esophageal Cancer Progression by Activating Wnt Signaling.

PubMed

Cao, Baoping; Yang, Weili; Jin, Yongshuai; Zhang, Meiying; He, Tao; Zhan, Qimin; Herman, James G; Zhong, Guanglin; Guo, Mingzhou

2016-11-01

Naked cuticle homolog 2 (NKD2) was found to be frequently methylated in human breast and gastric cancers. However, the epigenetic changes and mechanisms of NKD2 in human esophageal cancer remain unclear. Nine esophageal cancer cell lines and 154 cases of primary esophageal cancer samples were analyzed using methylation-specific polymerase chain reaction, immunohistochemical analysis, Western blot, and xenograft mouse models. Loss of NKD2 expression and complete methylation were found in KYSE150 and TE1 cells. Reduced NKD2 expression and partial methylation of the promoter region were observed in KYSE30, KYSE70, KYSE410, KYSE140, and COLO680 cells. High levels of NKD2 expression and unmethylation were detected in KYSE450 and TE8 cells. Reexpression of NKD2 was induced by 5-aza-2'-deoxycytidine in cells in which NKD2 was not expressed or cells in which NKD2 expression was reduced. NKD2 was methylated in 53.2% of human primary esophageal cancer samples (82 of 154), and promoter region hypermethylation was significantly associated with reduced expression of NKD2 (p < 0.01). NKD2 methylation was associated with tumor, node, and metastasis stage and lymph node metastasis (p < 0.01). Our results suggest that NKD2 is regulated by promoter region methylation and that methylation of NKD2 may serve as a prognostic marker in esophageal cancer. Our further studies demonstrate that NKD2 suppresses cell proliferation, colony formation, cell invasion, and migration and also induces G1/S checkpoint arrest in esophageal cancer cells. NKD2 suppressed xenograft tumor growth and inhibited Wnt signaling in human esophageal cancer cells. NKD2 is frequently methylated in human esophageal cancer, and the expression of NKD2 is regulated by promoter region methylation. NKD2 suppresses esophageal cancer progression by inhibiting Wnt signaling both in vitro and in vivo. Copyright © 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

DNA methyl transferases are differentially expressed in the human anterior eye segment.

PubMed

Bonnin, Nicolas; Belville, Corinne; Chiambaretta, Frédéric; Sapin, Vincent; Blanchon, Loïc

2014-08-01

DNA methylation is an epigenetic mark involved in the control of genes expression. Abnormal epigenetic events have been reported in human pathologies but weakly documented in eye diseases. The purpose of this study was to establish DNMT mRNA and protein expression levels in the anterior eye segment tissues and their related (primary or immortalized) cell cultures as a first step towards future in vivo and in vitro methylomic studies. Total mRNA was extracted from human cornea, conjunctiva, anterior lens capsule, trabeculum and related cell cultures (cornea epithelial, trabecular meshwork, keratocytes for primary cells; and HCE, Chang, B-3 for immortalized cells). cDNA was quantified by real-time PCR using specific primers for DNMT1, 2, 3A, 3B and 3L. Immunolocalization assays were carried out on human cornea using specific primary antibodies for DNMT1, 2 and 3A, 3B and 3L. All DNMT transcripts were detected in human cornea, conjunctiva, anterior lens capsule, trabeculum and related cells but showed statistically different expression patterns between tissues and cells. DNMT2 protein presented a specific and singular expression pattern in corneal endothelium. This study produced the first inventory of the expression patterns of DNMTs in human adult anterior eye segment. Our research highlights that DNA methylation cannot be ruled out as a way to bring new insights into well-known ocular diseases. In addition, future DNA methylation studies using various cells as experimental models need to be conducted with attention to approach the results analysis from a global tissue perspective. © 2014 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

Repression of hTERT transcription by the introduction of chromosome 3 into human oral squamous cell carcinoma

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

Nishio, Sachiyo; Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago, Tottori, 683-8503; Ohira, Takahito

Telomerase is a ribonucleoprotein enzyme that maintains telomere length. Telomerase activity is primarily attributed to the expression of telomerase reverse transcriptase (TERT). It has been reported that introduction of an intact human chromosome 3 into the human oral squamous cell carcinoma cell line HSC3 suppresses the tumorigenicity of these cells. However, the mechanisms that regulate tumorigenicity have not been elucidated. To determine whether this reduction in tumorigenicity was accompanied by a reduction in telomerase activity, we investigated the transcriptional activation of TERT in HSC3 microcell hybrid clones with an introduced human chromosome 3 (HSC3#3). HSC#3 cells showed inhibition of hTERT transcriptionmore » compared to that of the parental HSC3 cells. Furthermore, cell fusion experiments showed that hybrids of HSC3 cells and cells of the RCC23 renal carcinoma cell line, which also exhibits suppression of TERT transcription by the introduction of human chromosome 3, also displayed suppressed TERT transcription. These results suggested that human chromosome 3 may carry functionally distinct, additional TERT repressor genes. - Highlights: • hTERT mRNA expression level decreased in the chromosome 3 introduced HSC3 clones. • hTERT mRNA expression level was tend to suppressed in HSC3 and RCC23 hybrid cells. • We provide evidence that human chromosome 3 carries at least two distinct hTERT regulatory factors.« less

Enhanced proliferation of human hepatoma cells by PAR-2 agonists via the ERK/AP-1 pathway.

PubMed

Xie, Liqun; Zheng, Yanmin; Li, Xuan; Zhao, Junyan; Chen, Xiaoyi; Chen, Li; Zhou, Jing; Hai, Ou; Li, Fei

2012-11-01

To investigate the expression and role of PAR-2 in the proliferation of the human hepatoma cell line HepG2, PAR-2 protein and mRNA expression were evaluated by immuno-histochemistry, immunofluorescence and RT-PCR analysis. The signaling pathways downstream of PAR-2 activation that lead to hepatoma cell proliferation were analyzed. The results showed that PAR-2 is expressed in human hepatoma cells and PAR-2 mRNA expression was found to be upregulated in cells treated with trypsin or SLIGKV-NH2 (P<0.001). The proliferation rate of HepG2 cells treated with trypsin or SLIGKV-NH2 was significantly increased (P<0.001). The percentage of S phase, G2/M phase and the proliferation index (PI) of HepG2 cells treated with trypsin or SLIGKV-NH2 were significantly elevated (P<0.001). The proliferative responses of HepG2 to trypsin and SLIGKV-NH2 were associated with the upregulation of c-fos and PCNA, which were significantly blocked by PD98059 pretreatment. In conclusion, our results indicate that PAR-2 enhances proliferation of human hepatoma cells possibly via the ERK/AP-1 pathway.

BCL6 antagonizes NOTCH2 to maintain survival of human follicular lymphoma cells

PubMed Central

Valls, Ester; Lobry, Camille; Geng, Huimin; Wang, Ling; Cardenas, Mariano; Rivas, Martín; Cerchietti, Leandro; Oh, Philmo; Yang, Shao Ning; Oswald, Erin; Graham, Camille W.; Jiang, Yanwen; Hatzi, Katerina; Agirre, Xabier; Perkey, Eric; Li, Zhuoning; Tam, Wayne; Bhatt, Kamala; Leonard, John P.; Zweidler-McKay, Patrick A.; Maillard, Ivan; Elemento, Olivier; Ci, Weimin; Aifantis, Iannis; Melnick, Ari

2017-01-01

Summary Although the BCL6 transcriptional repressor is frequently expressed in human follicular lymphomas (FL), its biological role in this disease remains unknown. Herein we comprehensively identify the set of gene promoters directly targeted by BCL6 in primary human FLs. We noted that BCL6 binds and represses NOTCH2 and Notch pathway genes. Moreover, BCL6 and NOTCH2 pathway gene expression is inversely correlated in FL. Notably BCL6 up-regulation is associated with repression of Notch2 and its target genes in primary human and murine germinal center cells. Repression of Notch2 is an essential function of BCL6 in FL and GC B-cells since inducible expression of Notch2 abrogated GC formation in mice and kills FL cells. Indeed BCL6-targeting compounds or gene silencing leads to the induction of NOTCH2 activity and compromises survival of FL cells whereas NOTCH2 depletion or pathway antagonists rescue FL cells from such effects. Moreover, BCL6 inhibitors induced NOTCH2 expression and suppressed growth of human FL xenografts in vivo and primary human FL specimens ex vivo. These studies suggest that established FLs are thus dependent on BCL6 through its suppression of NOTCH2. PMID:28232365

Increased expression of human leucocyte antigen class I free heavy chains on monocytes of patients with spondyloarthritis and cells transfected with HLA-B27.

PubMed

Ding, Jin; Feng, Yuan; Zheng, Zhao Hui; Li, Xue Yi; Wu, Zhen Biao; Zhu, Ping

2015-02-01

Human leucocyte antigen (HLA)-B27 expression is correlated with spondyloarthritis (SpA), but its role in disease pathogenesis remains unclear. The aim of the study was to determine whether HLA-B27 free heavy chain (FHC) contributes to SpA pathogenesis. Flow cytometry was used to analyse the FHC expression on CD3+ and CD14+ cells in the peripheral blood (PB) and synovial fluid (SF) from SpA patients, healthy controls, and rheumatoid arthritis (RA) patients. Human monocytic U937 cell lines stably expressing enhanced green fluorescence protein (EGFP)/HLA-B27, EGFP/HLA-A2 or EGFP alone were created to further investigate the relation between HLA-B27 and FHC expression. The relative FHC level on CD14+ PB cells was significantly higher in SpA patients than in controls, but lower than on the SF cells of SpA patients. No significant correlation was found for relative FHC expression with HLA-B27 or β2-microglobulin expression. HLA-B27-transfected U937 cells expressed higher FHC levels than either EGFP/HLA-A2- or EGFP-transfected cells. HLA class I FHC expression was significantly increased on monocytes of SpA patients and HLA-B27-transfected cells, implying that FHC, perhaps mostly derived from HLA-B27, plays an important role in SpA pathogenesis. © 2014 John Wiley & Sons Ltd.

Comparative analysis of human protein-coding and noncoding RNAs between brain and 10 mixed cell lines by RNA-Seq.

PubMed

Chen, Geng; Yin, Kangping; Shi, Leming; Fang, Yuanzhang; Qi, Ya; Li, Peng; Luo, Jian; He, Bing; Liu, Mingyao; Shi, Tieliu

2011-01-01

In their expression process, different genes can generate diverse functional products, including various protein-coding or noncoding RNAs. Here, we investigated the protein-coding capacities and the expression levels of their isoforms for human known genes, the conservation and disease association of long noncoding RNAs (ncRNAs) with two transcriptome sequencing datasets from human brain tissues and 10 mixed cell lines. Comparative analysis revealed that about two-thirds of the genes expressed between brain and cell lines are the same, but less than one-third of their isoforms are identical. Besides those genes specially expressed in brain and cell lines, about 66% of genes expressed in common encoded different isoforms. Moreover, most genes dominantly expressed one isoform and some genes only generated protein-coding (or noncoding) RNAs in one sample but not in another. We found 282 human genes could encode both protein-coding and noncoding RNAs through alternative splicing in the two samples. We also identified more than 1,000 long ncRNAs, and most of those long ncRNAs contain conserved elements across either 46 vertebrates or 33 placental mammals or 10 primates. Further analysis showed that some long ncRNAs differentially expressed in human breast cancer or lung cancer, several of those differentially expressed long ncRNAs were validated by RT-PCR. In addition, those validated differentially expressed long ncRNAs were found significantly correlated with certain breast cancer or lung cancer related genes, indicating the important biological relevance between long ncRNAs and human cancers. Our findings reveal that the differences of gene expression profile between samples mainly result from the expressed gene isoforms, and highlight the importance of studying genes at the isoform level for completely illustrating the intricate transcriptome.

Induction of Human Blood Group A Antigen Expression on Mouse Cells, Using Lentiviral Gene Transduction

PubMed Central

Fan, Xiaohu; Lang, Haili; Zhou, Xianpei; Zhang, Li; Yin, Rong; Maciejko, Jessica; Giannitsos, Vasiliki; Motyka, Bruce; Medin, Jeffrey A.; Platt, Jeffrey L.

2010-01-01

Abstract The ABO histo-blood group system is the most important antigen system in transplantation medicine, yet no small animal model of the ABO system exists. To determine the feasibility of developing a murine model, we previously subcloned the human α-1,2-fucosyltransferase (H-transferase, EC 2.4.1.69) cDNA and the human α-1,3-N-acetylgalactosaminyltransferase (A-transferase, EC 2.4.1.40) cDNA into lentiviral vectors to study their ability to induce human histo-blood group A antigen expression on mouse cells. Herein we investigated the optimal conditions for human A and H antigen expression in murine cells. We determined that transduction of a bicistronic lentiviral vector (LvEF1-AH-trs) resulted in the expression of A antigen in a mouse endothelial cell line. We also studied the in vivo utility of this vector to induce human A antigen expression in mouse liver. After intrahepatic injection of LvEF1-AH-trs, A antigen expression was observed on hepatocytes as detected by immunohistochemistry and real-time RT-PCR. In human group A erythrocyte-sensitized mice, A antigen expression in the liver was associated with tissue damage, and deposition of antibody and complement. These results suggest that this gene transfer strategy can be used to simulate the human ABO blood group system in a murine model. This model will facilitate progress in the development of interventions for ABO-incompatible transplantation and transfusion scenarios, which are difficult to develop in clinical or large animal settings. PMID:20163247

Magnolol affects expression of IGF-1 and associated binding proteins in human prostate cancer cells in vitro.

PubMed

McKeown, Brendan T; Hurta, Robert A R

2014-11-01

This study investigated the effects of magnolol, a compound from Magnolia officinalis, on the behavior of LNCaP and PC3 human prostate cancer cells in vitro. In vitro cell culture approach with biochemical tests and Western blot analyses was used. Magnolol, (80 μM, 6 hour exposure) was found to affect the expression of insulin-like growth factor-1 (IGF-1) and associated proteins. In both cell lines, protein expression of IGF-1 and insulin-like growth factor binding protein-5 (IGFBP-5) were significantly decreased, while protein expression of IGFBP-3 was significantly increased. Additionally, protein expression of insulin-like growth factor-1 receptor (IGF-1R) was significantly increased and the phosphorylated form of IGF-1 (p-IGF-1R) was significantly decreased in PC3 cells, while IGFBP-4 protein expression was significantly increased in LNCaP cells. This study has demonstrated for the first time that magnolol can alter the expression of IGF-1 and associated proteins in human prostate cancer cells in vitro and suggests that magnolol may have a potential role as a novel anti-prostate cancer agent. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Identification of a regulatory T cell specific cell surface molecule that mediates suppressive signals and induces Foxp3 expression.

PubMed

Wang, Rui; Wan, Qi; Kozhaya, Lina; Fujii, Hodaka; Unutmaz, Derya

2008-07-16

Regulatory T (T(reg)) cells control immune activation and maintain tolerance. How T(regs) mediate their suppressive function is unclear. Here we identified a cell surface molecule, called GARP, (or LRRC32), which within T cells is specifically expressed in T(regs) activated through the T cell receptor (TCR). Ectopic expression of GARP in human naïve T (T(N)) cells inhibited their proliferation and cytokine secretion upon TCR activation. Remarkably, GARP over-expression in T(N) cells induced expression of T(reg) master transcription factor Foxp3 and endowed them with a partial suppressive function. The extracellular but not the cytoplasmic region of GARP, was necessary for these functions. Silencing Foxp3 in human T(reg) cells reduced expression of GARP and attenuated their suppressive function. However, GARP function was not affected when Foxp3 was downregulated in GARP-overexpressing cells, while silencing GARP in Foxp3-overexpressing cells reduced their suppressive activity. These findings reveal a novel cell surface molecule-mediated regulatory mechanism, with implications for modulating aberrant immune responses.

[Expressions of CXCL16/CXCR6 and CXCL12/CXCR4 in first-trimester human trophoblast cells].

PubMed

Huang, Yu; Li, Da-jin; Wang, Ming-yan; Cheng, Hai-dong

2006-06-01

To investigate the transcription and protein expressions of chemokines CXCL16, CXCL12 and their receptors CXCR6, CXCR4 in first-trimester human cytotrophoblast cells and human choriocarcinoma cell line JAR. Transcriptions of CXCR6, CXCL16, CXCR4, CXCL12 in purified first-trimester human trophoblast cells and JAR line were assessed by semi-quantitative RT-PCR, and protein expressions of CXCR6, CXCL16, CXCR4, CXCL12 were analyzed in primary cultured villous cytotrophoblasts (VCT), extravillous cytotrophoblasts (EVCT), JAR line and placentas by immunostaining. CXCR6 and CXCR4 were highly transcribed in primary cultured trophoblast cells with mRNA relative level of 1.12 +/- 0.25 and 1.08 +/- 0.11 respectively, and their ligands CXCL16 and CXCL12 were transcribed moderately with mRNA relative level of 0.89 +/- 0.11 and 0.78 +/- 0.10 respectively. It was demonstrated that CXCL16, CXCL12, CXCR6 and CXCR4 were expressed in primary cultured VCT, EVCT, JAR line and placentas by immunostaining. The co-expression of CXCL16/CXCR6 and CXCL12/CXCR4 in trophoblast cells may play a role in the proliferation and differentiation of first-trimester trophoblast cells in a manner of autocrine.

Hepatic leukemia factor promotes resistance to cell death: Implications for therapeutics and chronotherapy

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

Waters, Katrina M.; Sontag, Ryan L.; Weber, Thomas J., E-mail: Thomas.Weber@pnl.gov

Physiological variation related to circadian rhythms and aberrant gene expression patterns are believed to modulate therapeutic efficacy, but the precise molecular determinants remain unclear. Here we examine the regulation of cell death by hepatic leukemia factor (HLF), which is an output regulator of circadian rhythms and is aberrantly expressed in human cancers, using an ectopic expression strategy in JB6 mouse epidermal cells and human keratinocytes. Ectopic HLF expression inhibited cell death in both JB6 cells and human keratinocytes, as induced by serum-starvation, tumor necrosis factor alpha and ionizing radiation. Microarray analysis indicates that HLF regulates a complex multi-gene transcriptional programmore » encompassing upregulation of anti-apoptotic genes, downregulation of pro-apoptotic genes, and many additional changes that are consistent with an anti-death program. Collectively, our results demonstrate that ectopic expression of HLF, an established transcription factor that cycles with circadian rhythms, can recapitulate many features associated with circadian-dependent physiological variation. - Highlights: ► Circadian-dependent physiological variation impacts therapeutic efficacy. ► Hepatic leukemia factor inhibits cell death and is a candidate circadian factor. ► Hepatic leukemia factor anti-death program is conserved in murine and human cells. ► Transcriptomics indicates the anti-death program results from a systems response.« less

Expression of adhesion and extracellular matrix genes in human blastocysts upon attachment in a 2D co-culture system.

PubMed

Aberkane, A; Essahib, W; Spits, C; De Paepe, C; Sermon, K; Adriaenssens, T; Mackens, S; Tournaye, H; Brosens, J J; Van de, Velde H

2018-05-26

What are the changes in human embryos, in terms of morphology and gene expression, upon attachment to endometrial epithelial cells? Apposition and adhesion of human blastocysts to endometrial epithelial cells are predominantly initiated at the embryonic pole and these steps are associated with changes in expression of adhesion and extracellular matrix (ECM) genes in the embryo. Both human and murine embryos have been co-cultured with Ishikawa cells, although embryonic gene expression associated with attachment has not yet been investigated in an in-vitro implantation model. Vitrified human blastocysts were warmed and co-cultured for up to 48 h with Ishikawa cells, a model cell line for receptive endometrial epithelium. Six-days post fertilisation (6dpf) human embryos were co-cultured with Ishikawa cells for 12 h, 24 h (7dpf) or 48 h (8dpf) and attachment rate and morphological development investigated. Expression of 84 adhesion and ECM genes was analysed by quantitative PCR. Immunofluorescence microscopy was used to assess the expression of three informative genes at the protein level. Data are reported on 115 human embryos. Mann-Whitney U was used for statistical analysis between two groups, with P < 0.05 considered significant. The majority of embryos attached to Ishikawa cells at the level of the polar trophectoderm; 41% of co-cultured embryos were loosely attached after 12 h and 86% firmly attached after 24 h. Outgrowth of hCG-positive embryonic cells at 8dpf indicated differentiation of trophectoderm into invasive syncytiotrophoblast. Gene expression analysis was performed on loosely attached and unattached embryos co-cultured with Ishikawa cells for 12 h. In contrast to unattached embryos, loosely attached embryos expressed THBS1, TNC, COL12A1, CTNND2, ITGA3, ITGAV, and LAMA3 and had significantly higher CD44 and TIMP1 transcript levels (P = 0.014 and P = 0.029, respectively). LAMA3, THBS1 and TNC expression was validated at the protein level in firmly attached 7dpf embryos. Thrombospondin 1 (THBS1) resided in the cytoplasm of embryonic cells whereas laminin subunit alpha 3 (LAMA3) and tenascin C (TNC) were expressed on the cell surface of trophectoderm cells. Incubation with a neutralizing TNC antibody did not affect the rate of embryo attachment or hCG secretion. None. This in-vitro study made use of an endometrial adenocarcinoma cell line to mimic receptive luminal epithelium. Also, the number of embryos was limited. Contamination of recovered embryos with Ishikawa cells was unlikely based on their differential gene expression profiles. Taken together, we provide a 'proof of concept' that initiation of the implantation process coincides with the induction of specific embryonic genes. Genome-wide expression profiling of a larger sample set may provide insights into the molecular embryonic pathways underlying successful or failed implantation. A.A. was supported by a grant from the "Instituut voor Innovatie door Wetenschap en Technologie" (IWT, 121716, Flanders, Belgium). This work was supported by the "Wetenschappelijk Fonds Willy Gepts" (WFWG G142 and G170, Universitair Ziekenhuis Brussel). The authors declare no conflict of interest.

Allotopic expression of a mitochondrial alternative oxidase confers cyanide resistance to human cell respiration

PubMed Central

Hakkaart, Gerrit A J; Dassa, Emmanuel P; Jacobs, Howard T; Rustin, Pierre

2006-01-01

Human mitochondrial respiration is distinct from that of most plants, microorganisms and even some metazoans in that it reduces molecular oxygen only through the highly cyanide-sensitive enzyme cytochrome c oxidase. Here we show that expression of the cyanide-insensitive alternative oxidase (AOX), recently identified in the ascidian Ciona intestinalis, is well tolerated by cultured human cells and confers spectacular cyanide resistance to mitochondrial substrate oxidation. The expressed AOX seems to be confined to mitochondria. AOX involvement in electron flow is triggered by a highly reduced redox status of the respiratory chain (RC) and enhanced by pyruvate; otherwise, the enzyme remains essentially inactive. AOX expression promises to be a valuable tool to limit the deleterious consequences of RC deficiency in human cells and whole animals. PMID:16322757

Human osteocalcin and bone sialoprotein mediating osteomimicry of prostate cancer cells: role of cAMP-dependent protein kinase A signaling pathway.

PubMed

Huang, Wen-Chin; Xie, Zhihui; Konaka, Hiroyuki; Sodek, Jaro; Zhau, Haiyen E; Chung, Leland W K

2005-03-15

Osteocalcin and bone sialoprotein are the most abundant noncollagenous bone matrix proteins expressed by osteoblasts. Surprisingly, osteocalcin and bone sialoprotein are also expressed by malignant but not normal prostate epithelial cells. The purpose of this study is to investigate how osteocalcin and bone sialoprotein expression is regulated in prostate cancer cells. Our investigation revealed that (a) human osteocalcin and bone sialoprotein promoter activities in an androgen-independent prostate cancer cell line of LNCaP lineage, C4-2B, were markedly enhanced 7- to 12-fold in a concentration-dependent manner by conditioned medium collected from prostate cancer and bone stromal cells. (b) Deletion analysis of human osteocalcin and bone sialoprotein promoter regions identified cyclic AMP (cAMP)-responsive elements (CRE) as the critical determinants for conditioned medium-mediated osteocalcin and bone sialoprotein gene expression in prostate cancer cells. Consistent with these results, the protein kinase A (PKA) pathway activators forskolin and dibutyryl cAMP and the PKA pathway inhibitor H-89, respectively, increased or repressed human osteocalcin and bone sialoprotein promoter activities. (c) Electrophoretic mobility shift assay showed that conditioned medium-mediated stimulation of human osteocalcin and bone sialoprotein promoter activities occurs through increased interaction between CRE and CRE-binding protein. (d) Conditioned medium was found to induce human osteocalcin and bone sialoprotein promoter activities via increased CRE/CRE-binding protein interaction in a cell background-dependent manner, with marked stimulation in selected prostate cancer but not bone stromal cells. Collectively, these results suggest that osteocalcin and bone sialoprotein expression is coordinated and regulated through cAMP-dependent PKA signaling, which may define the molecular basis of the osteomimicry exhibited by prostate cancer cells.

Directing adult human periodontal ligament-derived stem cells to retinal fate.

PubMed

Huang, Li; Liang, Jiajian; Geng, Yiqun; Tsang, Wai-Ming; Yao, Xiaowu; Jhanji, Vishal; Zhang, Mingzhi; Cheung, Herman S; Pang, Chi Pui; Yam, Gary Hin-Fai

2013-06-06

To investigate the retinal fate competence of human postnatal periodontal ligament (PDL)-derived stem cells (PDLSC) through a directed differentiation mimicking mammalian retinogenesis. Human teeth were collected from healthy subjects younger than 35 years old. Primary PDLSC were isolated by collagenase digestion and cultivated. PDLSC at passage 3 were cultured in the induction media containing Noggin (antagonist of bone morphogenic protein) and Dkk-1 (antagonist of Wnt/β-catenin signaling). Gene expression of neural crest cells, retinal progenitors, and retinal neurons, including photoreceptors, was revealed by RNA analyses, immunofluorescence, and flow cytometry. The neuronal-like property of differentiated cells in response to excitatory glutamate was examined by fluo-4-acetoxymethyl calcium imaging assay. Primary human PDLSC stably expressed marker genes for neural crest (Notch1, BMP2, Slug, Snail, nestin, and Tuj1), mesenchymal stem cell (CD44, CD90, and vimentin), and embryonic stem cell (c-Myc, Klf4, Nanog, and SSEA4). Under low attachment culture, PDLSC generated neurospheres expressing nestin, p75/NGFR, Pax6, and Tuj1 (markers of neural progenitors). When neurospheres were plated on Matrigel-coated surface, they exhibited rosette-like outgrowth. They expressed eye field transcription factors (Pax6, Rx, Lhx, Otx2). By flow cytometry, 94% of cells were Pax6(nuclear)Rx(+), indicative of retinal progenitors. At prolonged induction, they expressed photoreceptor markers (Nrl, rhodopsin and its kinase) and showed significant responsiveness to excitatory glutamate. Primary human PDLSC could be directed to retinal progenitors with competence for photoreceptor differentiation. Human neural crest-derived PDL is readily accessible and can be an ample autologous source of undifferentiated cells for retinal cell regeneration.

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.

Regulation of lipid synthesis genes and milk fat production in human mammary epithelial cells during secretory activation

USDA-ARS?s Scientific Manuscript database

Expression of genes for lipid biosynthetic enzymes during initiation of lactation in humans is unknown. Our objective was to study mRNA expression of lipid metabolic enzymes in human mammary epithelial cell (MEC) in conjunction with the measurement of milk fatty acid (FA) composition during secretor...

The non-coding RNA landscape of human hematopoiesis and leukemia.

PubMed

Schwarzer, Adrian; Emmrich, Stephan; Schmidt, Franziska; Beck, Dominik; Ng, Michelle; Reimer, Christina; Adams, Felix Ferdinand; Grasedieck, Sarah; Witte, Damian; Käbler, Sebastian; Wong, Jason W H; Shah, Anushi; Huang, Yizhou; Jammal, Razan; Maroz, Aliaksandra; Jongen-Lavrencic, Mojca; Schambach, Axel; Kuchenbauer, Florian; Pimanda, John E; Reinhardt, Dirk; Heckl, Dirk; Klusmann, Jan-Henning

2017-08-09

Non-coding RNAs have emerged as crucial regulators of gene expression and cell fate decisions. However, their expression patterns and regulatory functions during normal and malignant human hematopoiesis are incompletely understood. Here we present a comprehensive resource defining the non-coding RNA landscape of the human hematopoietic system. Based on highly specific non-coding RNA expression portraits per blood cell population, we identify unique fingerprint non-coding RNAs-such as LINC00173 in granulocytes-and assign these to critical regulatory circuits involved in blood homeostasis. Following the incorporation of acute myeloid leukemia samples into the landscape, we further uncover prognostically relevant non-coding RNA stem cell signatures shared between acute myeloid leukemia blasts and healthy hematopoietic stem cells. Our findings highlight the importance of the non-coding transcriptome in the formation and maintenance of the human blood hierarchy.While micro-RNAs are known regulators of haematopoiesis and leukemogenesis, the role of long non-coding RNAs is less clear. Here the authors provide a non-coding RNA expression landscape of the human hematopoietic system, highlighting their role in the formation and maintenance of the human blood hierarchy.

Human X chromosome inactivation and reactivation: implications for cell reprogramming and disease.

PubMed

Cantone, Irene; Fisher, Amanda G

2017-11-05

X-chromosome inactivation (XCI) is an exemplar of epigenetic regulation that is set up as pluripotent cells differentiate. Once established, XCI is stably propagated, but can be reversed in vivo or by pluripotent reprogramming in vitro Although reprogramming provides a useful model for inactive X (Xi) reactivation in mouse, the relative instability and heterogeneity of human embryonic stem (ES) cells and induced pluripotent stem cells hampers comparable progress in human. Here we review studies aimed at reactivating the human Xi using different reprogramming strategies. We outline our recent results using mouse ES cells to reprogramme female human fibroblasts by cell-cell fusion. We show that pluripotent reprogramming induces widespread and rapid chromatin remodelling in which the human Xi loses XIST and H3K27m3 enrichment and selected Xi genes become reactivated, ahead of mitotic division. Using RNA sequencing to map the extent of human Xi reactivation, and chromatin-modifying drugs to potentiate reactivation, we outline how this approach could be used to better design strategies to re-express human X-linked loci. As cell fusion induces the expression of human pluripotency genes that represent both the 'primed' and 'naive' states, this approach may also offer a fresh opportunity to segregate human pluripotent states with distinct Xi expression profiles, using single-cell-based approaches.This article is part of the themed issue 'X-chromosome inactivation: a tribute to Mary Lyon'. © 2017 The Author(s).

Expression of the Grb2-related protein of the lymphoid system in B cell subsets enhances B cell antigen receptor signaling through mitogen-activated protein kinase pathways.

PubMed

Yankee, Thomas M; Solow, Sasha A; Draves, Kevin D; Clark, Edward A

2003-01-01

Adapter proteins play a critical role in regulating signals triggered by Ag receptor cross-linking. These small molecules link receptor proximal events with downstream signaling pathways. In this study, we explore the expression and function of the Grb2-related protein of the lymphoid system (GrpL)/Grb2-related adaptor downstream of Shc adapter protein in human B cells. GrpL is expressed in naive B cells and is down-regulated following B cell Ag receptor ligation. By contrast, germinal center and memory B cells express little or no GrpL. Using human B cell lines, we detected constitutive interactions between GrpL and B cell linker protein, Src homology (SH)2 domain-containing leukocyte protein of 76 kDa, hemopoietic progenitor kinase 1, and c-Cbl. The N-terminal SH3 domain of GrpL binds c-Cbl while the C-terminal SH3 domain binds B cell linker protein and SH2 domain-containing leukocyte protein of 76 kDa. Exogenous expression of GrpL in a GrpL-negative B cell line leads to enhanced Ag receptor-induced extracellular signal-related kinase and p38 mitogen-activated protein kinase phosphorylation. Thus, GrpL expression in human B cell subsets appears to regulate Ag receptor-mediated signaling events.

HPV infection and immunochemical detection of cell-cycle markers in verrucous carcinoma of the penis.

PubMed

Stankiewicz, Elzbieta; Kudahetti, Sakunthala C; Prowse, David M; Ktori, Elena; Cuzick, Jack; Ambroisine, Laurence; Zhang, Xiaoxi; Watkin, Nicholas; Corbishley, Catherine; Berney, Daniel M

2009-09-01

Penile verrucous carcinoma is a rare disease and little is known of its aetiology or pathogenesis. In this study we examined cell-cycle proteins expression and correlation with human papillomavirus infection in a series of 15 pure penile verrucous carcinomas from a single centre. Of 148 penile tumours, 15 (10%) were diagnosed as pure verrucous carcinomas. The expression of the cell-cycle-associated proteins p53, p21, RB, p16(INK4A) and Ki67 were examined by immunohistochemistry. Human papillomavirus infection was determined by polymerase chain reaction to identify a wide range of virus types. The expression of p16(INK4A) and Ki67 was significantly lower in verrucous carcinoma than in usual type squamous cell carcinoma, whereas the expression of p53, p21 and RB was not significantly different. p53 showed basal expression in contrast to usual type squamous cell carcinoma. Human papillomavirus infection was present in only 3 out of 13 verrucous carcinomas. Unique low-risk, high-risk and mixed viral infections were observed in each of the three cases. In conclusion, lower levels of p16(INK4A) and Ki67 expressions differentiate penile verrucous carcinoma from usual type squamous cell carcinoma. The low Ki67 index reflects the slow-growing nature of verrucous tumours. The low level of p16(INK4A) expression and human papillomavirus detection suggests that penile verrucous carcinoma pathogenesis is unrelated to human papillomavirus infection and the oncogenes and tumour suppressor genes classically altered by virus infection.

Neuropeptides activate human mast cell degranulation and chemokine production

PubMed Central

Kulka, Marianna; Sheen, Cecilia H; Tancowny, Brian P; Grammer, Leslie C; Schleimer, Robert P

2008-01-01

During neuronal-induced inflammation, mast cells may respond to stimuli such as neuropeptides in an FcεRI-independent manner. In this study, we characterized human mast cell responses to substance P (SP), nerve growth factor (NGF), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) and compared these responses to human mast cell responses to immunoglobulin E (IgE)/anti-IgE and compound 48/80. Primary cultured mast cells, generated from CD34+ progenitors in the presence of stem cell factor and interleukin-6 (IL-6), and human cultured mast cells (LAD2) were stimulated with these and other stimuli (gastrin, concanavalin A, radiocontrast media, and mannitol) and their degranulation and chemokine production was assessed. VIP and SP stimulated primary human mast cells and LAD cells to degranulate; gastrin, concanavalin A, radiocontrast media, mannitol, CGRP and NGF did not activate degranulation. While anti-IgE stimulation did not induce significant production of chemokines, stimulation with VIP, SP or compound 48/80 potently induced production of monocyte chemoattractant protein-1, inducible protein-10, monokine induced by interferon-γ (MIG), RANTES (regulated on activation, normal, T-cell expressed, and secreted) and IL-8. VIP, SP and compound 48/80 also activated release of tumour necrosis factor, IL-3 and granulocyte–macrophage colony-stimulating factor, but not IL-4, interferon-γ or eotaxin. Human mast cells expressed surface neurokinin 1 receptor (NK1R), NK2R, NK3R and VIP receptor type 2 (VPAC2) but not VPAC1 and activation of human mast cells by IgE/anti-IgE up-regulated expression of VPAC2, NK2R, and NK3R. These studies demonstrate the pattern of receptor expression and activation of mast cell by a host of G-protein coupled receptor ligands and suggest that SP and VIP activate a unique signalling pathway in human mast cells. These results are likely to have direct relevance to neuronally induced inflammatory diseases. PMID:17922833

IL-10 suppresses Th17 cells and promotes regulatory T cells in the CD4+ T cell population of rheumatoid arthritis patients.

PubMed

Heo, Yu-Jung; Joo, Young-Bin; Oh, Hye-Jwa; Park, Mi-Kyung; Heo, Yang-Mi; Cho, Mi-La; Kwok, Seung-Ki; Ju, Ji-Hyeon; Park, Kyung-Su; Cho, Seok Goo; Park, Sung-Hwan; Kim, Ho-Youn; Min, Jun-Ki

2010-01-04

Interleukin-17-producing CD4(+) T cells (Th17 cells) are the dominant pathogenic cellular component in autoimmune inflammatory diseases, including autoimmune arthritis. IL-10 promotes the generation of Foxp3(+) regulatory T cells via the IL-10 receptor signal. The objective of this study was to examine whether IL-10, which acts as an anti-inflammatory cytokine, has a suppressive effect on the activation of human Th17 cells. Expression of IL-17 and IL-10 was examined immunohistochemically in tissue obtained from rheumatoid arthritis patients. Human peripheral blood CD4(+) T cells were isolated and cultured under various stimulatory conditions. Th17 cells and regulatory T (Treg) cells were detected by flow cytometry. The gene expression of related cytokines and transcription factors were assessed by ELISA and RT-PCR. IL-17 was overexpressed in rheumatoid arthritis patients. IL-10 treatment significantly decreased the numbers of IL-17-producing and RORc-expressing cells among human CD4(+) T cells that had been activated in vitro by Th17-differentiating conditions in autoimmune arthritis patients. IL-10 induced Foxp3(+) regulatory T cells in the human CD4(+) T cell population. Our results demonstrate that IL-17 is overexpressed in autoimmune disease patients and that IL-10 suppresses IL-17 expression. IL-10 may be useful in the treatment of autoimmune diseases.

Notch signaling is a potent inducer of growth arrest and apoptosis in a wide range of B-cell malignancies

PubMed Central

Zweidler-McKay, Patrick A.; He, Yiping; Xu, Lanwei; Rodriguez, Carlos G.; Karnell, Fredrick G.; Carpenter, Andrea C.; Aster, Jon C.; Allman, David; Pear, Warren S.

2005-01-01

Although Notch receptor expression on malignant B cells is widespread, the effect of Notch signaling in these cells is poorly understood. To investigate Notch signaling in B-cell malignancy, we assayed the effect of Notch activation in multiple murine and human B-cell tumors, representing both immature and mature subtypes. Expression of constitutively active, truncated forms of the 4 mammalian Notch receptors (ICN1-4) inhibited growth and induced apoptosis in both murine and human B-cell lines but not T-cell lines. Similar results were obtained in human precursor B-cell acute lymphoblastic leukemia lines when Notch activation was achieved by coculture with fibroblasts expressing the Notch ligands Jagged1 or Jagged2. All 4 truncated Notch receptors, as well as the Jagged ligands, induced Hes1 transcription. Retroviral expression of Hairy/Enhancer of Split-1 (Hes1) recapitulated the Notch effects, suggesting that Hes1 is an important mediator of Notch-induced growth arrest and apoptosis in B cells. Among the B-cell malignancies that were susceptible to Notch-mediated growth inhibition/apoptosis were mature B-cell and therapy-resistant B-cell malignancies, including Hodgkin, myeloma, and mixed-lineage leukemia (MLL)–translocated cell lines. These results suggest that therapies capable of activating Notch/Hes1 signaling may have therapeutic potential in a wide range of human B-cell malignancies. PMID:16118316

Prostate cancer targeting motifs: expression of αν β3, neurotensin receptor 1, prostate specific membrane antigen, and prostate stem cell antigen in human prostate cancer cell lines and xenografts.

PubMed

Taylor, Robert M; Severns, Virginia; Brown, David C; Bisoffi, Marco; Sillerud, Laurel O

2012-04-01

Membrane receptors are frequent targets of cancer therapeutic and imaging agents. However, promising in vitro results often do not translate to in vivo clinical applications. To better understand this obstacle, we measured the expression differences in receptor signatures among several human prostate cancer cell lines and xenografts as a function of tumorigenicity. Messenger RNA and protein expression levels for integrin α(ν) β(3), neurotensin receptor 1 (NTSR1), prostate specific membrane antigen (PSMA), and prostate stem cell antigen (PSCA) were measured in LNCaP, C4-2, and PC-3 human prostate cancer cell lines and in murine xenografts using quantitative reverse transcriptase polymerase chain reaction, flow cytometry, and immunohistochemistry. Stable expression patterns were observed for integrin α(ν) and PSMA in all cells and corresponding xenografts. Integrin β(3) mRNA expression was greatly reduced in C4-2 xenografts and greatly elevated in PC-3 xenografts compared with the corresponding cultured cells. NTSR1 mRNA expression was greatly elevated in LNCaP and PC-3 xenografts. PSCA mRNA expression was elevated in C4-2 xenografts when compared with C4-2 cells cultured in vitro. Furthermore, at the protein level, PSCA was re-expressed in all xenografts compared with cells in culture. The regulation of mRNA and protein expression of the cell-surface target proteins α(ν) β(3), NTSR1, PSMA, and PSCA, in prostate cancer cells with different tumorigenic potential, was influenced by factors of the microenvironment, differing between cell cultures and murine xenotransplants. Integrin α(ν) β(3), NTRS1 and PSCA mRNA expression increased with tumorigenic potential, but mRNA expression levels for these proteins do not translate directly to equivalent expression levels of membrane bound protein. Copyright © 2011 Wiley Periodicals, Inc.

Expression of Pluripotency Markers in Nonpluripotent Human Neural Stem and Progenitor Cells.

PubMed

Vincent, Per Henrik; Benedikz, Eirikur; Uhlén, Per; Hovatta, Outi; Sundström, Erik

2017-06-15

Nonpluripotent neural progenitor cells (NPCs) derived from the human fetal central nervous system were found to express a number of messenger RNA (mRNA) species associated with pluripotency, such as NANOG, REX1, and OCT4. The expression was restricted to small subpopulations of NPCs. In contrast to pluripotent stem cells, there was no coexpression of the pluripotency-associated genes studied. Although the expression of these genes rapidly declined during the in vitro differentiation of NPCs, we found no evidence that the discrete expression was associated with the markers of multipotent neural stem cells (CD133 + /CD24 lo ), the capacity of sphere formation, or high cell proliferation rates. The rate of cell death among NPCs expressing pluripotency-associated genes was also similar to that of other NPCs. Live cell imaging showed that NANOG- and REX1-expressing NPCs continuously changed morphology, as did the nonexpressing cells. Depletion experiments showed that after the complete removal of the subpopulations of NANOG- and REX1-expressing NPCs, the expression of these genes appeared in other NPCs within a few days. The percentage of NANOG- and REX1-expressing cells returned to that observed before depletion. Our results are best explained by a model in which there is stochastic transient expression of pluripotency-associated genes in proliferating NPCs.

Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

PubMed

Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

1995-02-10

Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain.

Gasdermin (Gsdm) localizing to mouse Chromosome 11 is predominantly expressed in upper gastrointestinal tract but significantly suppressed in human gastric cancer cells.

PubMed

Saeki, N; Kuwahara, Y; Sasaki, H; Satoh, H; Shiroishi, T

2000-09-01

Amplification of proto-oncogenes associated with their over-expression is one of the critical carcinogenic events identified in human cancer cells. In many cases of human gastric cancer, a proto-oncogene ERBB-2 is co-amplified with CAB1 genes physically linked to ERBB-2, and both genes are over-expressed. The amplified region containing ERBB-2 and CAB1 was named 17q12 amplicon from its chromosomal location. The syntenic region corresponding to the 17q12 amplicon is well conserved in mouse. In this study we isolated and characterized a novel mouse gene that locates telomeric to the mouse syntenic region. Northern blot analysis using the mouse cDNA and a cloned partial cDNA of human homolog disclosed a unique expression pattern of the genes. They are expressed predominantly in the gastrointestinal (GI) tract and in the skin at a lower level. Moreover, in the GI tract, the expression is highly restricted to the esophagus and stomach. Thus, we named the mouse gene Gasdermin (Gsdm). This is the first report of a mammalian gene whose expression is restricted to both upper GI tract and skin. Interestingly, in spite of its expression in normal stomach, no transcript was detected by Northern blot analysis in human gastric cancer cells. These data suggest that the loss of the expression of the human homolog is required for the carcinogenesis of gastric tissue and that the gene has an activity adverse to malignant transformation of cells.

CXCR3 surface expression in human airway epithelial cells: cell cycle dependence and effect on cell proliferation.

PubMed

Aksoy, Mark O; Yang, Yi; Ji, Rong; Reddy, P J; Shahabuddin, Syed; Litvin, Judith; Rogers, Thomas J; Kelsen, Steven G

2006-05-01

We recently demonstrated that human bronchial epithelial cells (HBEC) constitutively express the CXC chemokine receptor CXCR3, which when activated, induces directed cell migration. The present study in HBEC examined the relative expression of the CXCR3 splice variants CXCR3-A and -B, cell cycle dependence of CXCR3 expression, and the effects of the CXCR3 ligand, the interferon-gamma-inducible CXC chemokine I-TAC/CXCL11, on DNA synthesis and cell proliferation. Both CXCR3-A and -B mRNA, assessed by real-time RT-PCR, were expressed in normal HBEC (NHBEC) and the HBEC line 16-HBE. However, CXCR3-B mRNA was 39- and 6-fold greater than CXCR3-A mRNA in NHBEC and 16-HBE, respectively. Although most HBEC (>80%) assessed by flow cytometry and immunofluorescence microscopy contained intracellular CXCR3, only a minority (<40%) expressed it on the cell surface. In this latter subset of cells, most (>75%) were in the S + G(2)/M phases of the cell cycle. Stimulation of CXCR3 with I-TAC enhanced thymidine incorporation and cell proliferation and increased p38 and ERK1/2 phosphorylation. These data indicate that 1) human airway epithelial cells primarily express CXCR3-B mRNA, 2) surface expression of CXCR3 is largely confined to the S + G(2)/M phases of the cell cycle, and 3) activation of CXCR3 induces DNA synthesis, cell proliferation, and activation of MAPK pathways. We speculate that activation of CXCR3 exerts a mitogenic effect in HBEC, which may be important during airway mucosal injury in obstructive airway diseases such as asthma and chronic obstructive pulmonary disease.

Ghrelin stimulates angiogenesis in human microvascular endothelial cells: Implications beyond GH release

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

Li Aihua; Cheng Guangli; Zhu Genghui

Ghrelin, a peptide hormone isolated from the stomach, releases growth hormone and stimulates appetite. Ghrelin is also expressed in pancreas, kidneys, cardiovascular system and in endothelial cells. The precise role of ghrelin in endothelial cell functions remains unknown. We examined the expression of ghrelin and its receptor (GHSR1) mRNAs and proteins in human microvascular endothelial cells (HMVEC) and determined whether ghrelin affects in these cells proliferation, migration and in vitro angiogenesis; and whether MAPK/ERK2 signaling is important for the latter action. We found that ghrelin and GHSR1 are constitutively expressed in HMVEC. Treatment of HMVEC with exogenous ghrelin significantly increasedmore » in these cells proliferation, migration, in vitro angiogenesis and ERK2 phosphorylation. MEK/ERK2 inhibitor, PD 98059 abolished ghrelin-induced in vitro angiogenesis. This is First demonstration that ghrelin and its receptor are expressed in human microvascular endothelial cells and that ghrelin stimulates HMVEC proliferation, migration, and angiogenesis through activation of ERK2 signaling.« less

MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin

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

Song, Yichen, E-mail: jeff200064017@163.com; Wang, Ping, E-mail: pingwang8000@163.com; Institute of Pathology and Pathophysiology, China Medical University, Shenyang 110001

MiR-17-92 cluster has recently been reported as an oncogene in some tumors. However, the association of miR-18a, an important member of this cluster, with glioblastoma remains unknown. Therefore, this study aims to investigate the expression of miR-18a in glioblastoma and its role in biological behavior of U87 and U251 human glioblastoma cell lines. Quantitative RT-PCR results showed that miR-18a was highly expressed in glioblastoma tissues and U87 and U251 cell lines compared with that in human brain tissues and primary normal human astrocytes, and the expression levels were increased along with the rising pathological grades of glioblastoma. Neogenin was identifiedmore » as the target gene of miR-18a by dual-luciferase reporter assays. RT-PCR and western blot results showed that its expression levels were decreased along with the rising pathological grades of glioblastoma. Inhibition of miR-18a expression was established by transfecting exogenous miR-18a inhibitor into U87 and U251 cells, and its effects on the biological behavior of glioblastoma cells were studied using CCK-8 assay, transwell assay and flow cytometry. Inhibition of miR-18a expression in U87 and U251 cells significantly up-regulated neogenin, and dramatically suppressed the abilities of cell proliferation, migration and invasion, induced cell cycle arrest and promoted cellular apoptosis. Collectively, these results suggest that miR-18a may regulate biological behavior of human glioblastoma cells by targeting neogenin, and miR-18a can serve as a potential target in the treatment of glioblastoma. - Highlights: • MiR-18a was highly expressed in glioblastoma tissues and U87 and U251 cell lines. • Neogenin was identified as the target gene of miR-18a. • Neogenin expressions were decreased along with the rising pathological grades of glioblastoma. • Inhibition of miR-18a suppressed biological behavior of glioma cells by up-regulating neogenin.« less

Galectin-1 influences trophoblast immune evasion and emerges as a predictive factor for the outcome of pregnancy.

PubMed

Tirado-González, Irene; Freitag, Nancy; Barrientos, Gabriela; Shaikly, Valerie; Nagaeva, Olga; Strand, Magnus; Kjellberg, Lennart; Klapp, Burghard F; Mincheva-Nilsson, Lucia; Cohen, Marie; Blois, Sandra M

2013-01-01

Galectin-1 (gal-1) is expressed at the feto-maternal interface and plays a role in regulating the maternal immune response against placental alloantigens, contributing to pregnancy maintenance. Both decidua and placenta contribute to gal-1 expression and may be important for the maternal immune regulation. The expression of gal-1 within the placenta is considered relevant to cell-adhesion and invasion of trophoblasts, but the role of gal-1 in the immune evasion machinery exhibited by trophoblast cells remains to be elucidated. In this study, we analyzed gal-1 expression in preimplantation human embryos and first-trimester decidua-placenta specimens and serum gal-1 levels to investigate the physiological role played by this lectin during pregnancy. The effect on human leukocyte antigen G (HLA-G) expression in response to stimulation or silencing of gal-1 was also determined in the human invasive, proliferative extravillous cytotrophoblast 65 (HIPEC65) cell line. Compared with normal pregnant women, circulating gal-1 levels were significantly decreased in patients who subsequently suffered a miscarriage. Human embryos undergoing preimplantation development expressed gal-1 on the trophectoderm and inner cell mass. Furthermore, our in vitro experiments showed that exogenous gal-1 positively regulated the membrane-bound HLA-G isoforms (HLA-G1 and G2) in HIPEC65 cells, whereas endogenous gal-1 also induced expression of the soluble isoforms (HLA-G5 and -G6). Our results suggest that gal-1 plays a key role in pregnancy maternal immune regulation by modulating HLA-G expression on trophoblast cells. Circulating gal-1 levels could serve as a predictive factor for pregnancy success in early human gestation.

Unique gene expression profiles of donor-matched human retinal and choroidal vascular endothelial cells.

PubMed

Smith, Justine R; Choi, Dongseok; Chipps, Timothy J; Pan, Yuzhen; Zamora, David O; Davies, Michael H; Babra, Bobby; Powers, Michael R; Planck, Stephen R; Rosenbaum, James T

2007-06-01

Consistent with clinical observations that posterior uveitis frequently involves the retinal vasculature and recent recognition of vascular heterogeneity, the hypothesis for this study was that retinal vascular endothelium was a cell population of unique molecular phenotype. Donor-matched cultures of primary retinal and choroidal endothelial cells from six human cadavers were incubated with either Toxoplasma gondii tachyzoites (10:1, parasites per cell) or Escherichia coli lipopolysaccharide (100 ng/mL); control cultures were simultaneously incubated with medium. Gene expression profiling of endothelial cells was performed using oligonucleotide arrays containing probes designed to detect 8746 human transcripts. After normalization, differential gene expression was assessed by the significance analysis of microarrays, with the false-discovery rate set at 5%. For selected genes, differences in the level of expression between retinal and choroidal cells were evaluated by real-time RT-PCR. Graphic descriptive analysis demonstrated a strong correlation between gene expression of unstimulated retinal and choroidal endothelial cells, but also highlighted distinctly different patterns of expression that were greater than differences noted between donors or between unstimulated and stimulated cells. Overall, 779 (8.9%) of 8746 transcripts were differentially represented. Of note, the 330 transcripts that were present at higher levels in retinal cells included a larger percentage of transcripts encoding molecules involved in the immune response. Differential gene expression was confirmed for 12 transcripts by RT-PCR. Retinal and choroidal vascular endothelial cells display distinctive gene expression profiles. The findings suggest the possibility of treating posterior uveitis by targeting specific interactions between the retinal endothelial cell and an infiltrating leukocyte.

Identification of stable reference genes in differentiating human pluripotent stem cells.

PubMed

Holmgren, Gustav; Ghosheh, Nidal; Zeng, Xianmin; Bogestål, Yalda; Sartipy, Peter; Synnergren, Jane

2015-06-01

Reference genes, often referred to as housekeeping genes (HKGs), are frequently used to normalize gene expression data based on the assumption that they are expressed at a constant level in the cells. However, several studies have shown that there may be a large variability in the gene expression levels of HKGs in various cell types. In a previous study, employing human embryonic stem cells (hESCs) subjected to spontaneous differentiation, we observed that the expression of commonly used HKG varied to a degree that rendered them inappropriate to use as reference genes under those experimental settings. Here we present a substantially extended study of the HKG signature in human pluripotent stem cells (hPSC), including nine global gene expression datasets from both hESC and human induced pluripotent stem cells, obtained during directed differentiation toward endoderm-, mesoderm-, and ectoderm derivatives. Sets of stably expressed genes were compiled, and a handful of genes (e.g., EID2, ZNF324B, CAPN10, and RABEP2) were identified as generally applicable reference genes in hPSCs across all cell lines and experimental conditions. The stability in gene expression profiles was confirmed by reverse transcription quantitative PCR analysis. Taken together, the current results suggest that differentiating hPSCs have a distinct HKG signature, which in some aspects is different from somatic cell types, and underscore the necessity to validate the stability of reference genes under the actual experimental setup used. In addition, the novel putative HKGs identified in this study can preferentially be used for normalization of gene expression data obtained from differentiating hPSCs. Copyright © 2015 the American Physiological Society.

Flow cytometric monitoring of hormone receptor expression in human solid tumors

NASA Astrophysics Data System (ADS)

Krishan, Awtar

2002-05-01

Hormone receptor expression in human breast and prostate tumors is of diagnostic and therapeutic importance. With the availability of anti-estrogen, androgen and progesterone antibodies, immunohistochemistry has become a standard tool for determination of receptor expression in human tumor biopsies. However, this method is dependent on examination of a small number of cells under a microscope and the data obtained in most cases is not quantitative. As most of the commercially used anti-hormone antibodies have nuclear specificity, we have developed methods for isolation and antigen unmasking of nuclei from formalin fixed/paraffin embedded archival human tumors. After immunostaining with the antibodies and propidium iodide (for DNA content and cell cycle analysis), nuclei are analyzed by multiparametric laser flow cytometry for hormone receptor expression, DNA content, aneuploidy and cell cycle determination. These multiparametric methods are especially important for retrospective studies seeking to correlate hormone receptor expression with clinical response to anti-hormonal therapy of human breast and prostate tumors.

Proteomic and cellular localisation studies suggest non-tight junction cytoplasmic and nuclear roles for occludin in astrocytes.

PubMed

Morgan, Sarah V; Garwood, Claire J; Jennings, Luke; Simpson, Julie E; Castelli, Lydia M; Heath, Paul R; Mihaylov, Simeon R; Vaquéz-Villaseñor, Irina; Minshull, Thomas C; Ince, Paul G; Dickman, Mark J; Hautbergue, Guillaume M; Wharton, Stephen B

2018-05-08

Occludin is a component of tight junctions, which are essential structural components of the blood-brain barrier. However, occludin is expressed in cells without tight junctions, implying additional functions. We determined the expression and localisation of occludin in astrocytes in cell culture and in human brain tissue, and sought novel binding partners using a proteomic approach. Expression was investigated by immunocytochemistry and immunoblotting in the 1321N1 astrocytoma cell line and ScienCell human primary astrocytes, and by immunohistochemistry in human autopsy brain tissue. Recombinant N- and C-terminal occludin was used to pull-down proteins from 1321N1 cell lysates and protein-binding partners identified by mass spectrometry analysis. Occludin was expressed in both the cytoplasm and nucleus of astrocytes in vitro and in vivo. Mass spectrometry identified binding to nuclear and cytoplasmic proteins, particularly those related to RNA metabolism and nuclear function. Occludin is expressed in several subcellular compartments of brain cell-types that do not form tight junctions and the expression patterns in cell culture reflect those in human brain tissue, indicating they are suitable model systems. Proteomic analysis suggests that occludin has novel functions in neuroepithelial cells that are unrelated to tight junction formation. Further research will establish the roles of these functions in both cellular physiology and in disease states. © 2018 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Downregulation of an Aim-1 Kinase Couples with Megakaryocytic Polyploidization of Human Hematopoietic Cells

PubMed Central

Kawasaki, Akira; Matsumura, Itaru; Miyagawa, Jun-ichiro; Ezoe, Sachiko; Tanaka, Hirokazu; Terada, Yasuhiko; Tatsuka, Masaaki; Machii, Takashi; Miyazaki, Hiroshi; Furukawa, Yusuke; Kanakura, Yuzuru

2001-01-01

During the late phase of megakaryopoiesis, megakaryocytes undergo polyploidization, which is characterized by DNA duplication without concomitant cell division. However, it remains unknown by which mechanisms this process occurs. AIM-1 and STK15 belong to the Aurora/increase-in-ploidy (Ipl)1 serine/threonine kinase family and play key roles in mitosis. In a human interleukin-3–dependent cell line, F-36P, the expressions of AIM-1 and STK15 mRNA were specifically observed at G2/M phase of the cell cycle during proliferation. In contrast, the expressions of AIM-1 and STK15 were continuously repressed during megakaryocytic polyploidization of human erythro/megakaryocytic cell lines (F-36P, K562, and CMK) treated with thrombopoietin, activated ras (H-rasG12V), or phorbol ester. Furthermore, their expressions were suppressed during thrombopoietin-induced polyploidization of normal human megakaryocytes. Activation of AIM-1 by the induced expression of AIM-1(wild-type) canceled TPA-induced polyploidization of K562 cells significantly, whereas that of STK15 did not. Moreover, suppression of AIM-1 by the induced expression of AIM-1 (K/R, dominant-negative type) led to polyploidization in 25% of K562 cells, whereas STK15(K/R) showed no effect. Also, the induced expression of AIM-1(K/R) in CMK cells provoked polyploidization up to 32N. These results suggested that downregulation of AIM-1 at M phase may be involved in abortive mitosis and polyploid formation of megakaryocytes. PMID:11266445

[The expression of Cyclin D1 modulated by somatotropin on human pancreas cancer cell lines Bxpc-3].

PubMed

Li, Fei; Liu, Da-chuan; Sun, Jia-bang

2004-04-07

To observe the growth effect of somatostapin on human pancreas cancer lines Bxpc-3. The Bxpc-3 pancreas cancer cells were treated with Somatotropin. The cells hyperplasia were detected by MTT and were observed apoptosis cells determinated quantitatively by TUNEL, quantify immune fluoresence double marked the proliferation cells and apoptosis cells, the expression of Cyclin D1 detected by immunohistochemical. The growth effect of pancrea cancer cells were limited by 10(-7) M, 10(-8) M, 10(-9) M Somatotropin on 2 day. The limited effect was decreased from 3 day. The cells proliferation were increased by somotostapin on 4day to 5day. The relationship between the expression of Cyclin D1 and apoptosis was negative correlation and the cells hyperplasia was positive correlation in Bxpc-3 cell line. From the cell study we knew the expression of Cyclin D1 reflected the prolefiration of pancreas cancer cells.

Transcriptional Inducers of Acetylcholinesterase Expression as Novel Antidotes for Protection Against Chemical Warfare Agents

DTIC Science & Technology

2005-10-01

neuroblastoma cell line , P19 and a human neuroblastoma cell line SH - SY5Y (data not shown). Effect of trichostatin A on...mouse neuroblastoma P19 cell line and a human neuroblastoma cell line SH - SY5Y . More experiments are needed to prove the potential of AChE expression in...treatment of nerve agent exposure. MATERIALS AND METHODS Neuronal cell lines and

The human Piwi protein Hiwi2 associates with tRNA-derived piRNAs in somatic cells

PubMed Central

Keam, Simon P.; Young, Paul E.; McCorkindale, Alexandra L.; Dang, Thurston H.Y.; Clancy, Jennifer L.; Humphreys, David T.; Preiss, Thomas; Hutvagner, Gyorgy; Martin, David I.K.; Cropley, Jennifer E.; Suter, Catherine M.

2014-01-01

The Piwi-piRNA pathway is active in animal germ cells where its functions are required for germ cell maintenance and gamete differentiation. Piwi proteins and piRNAs have been detected outside germline tissue in multiple phyla, but activity of the pathway in mammalian somatic cells has been little explored. In particular, Piwi expression has been observed in cancer cells, but nothing is known about the piRNA partners or the function of the system in these cells. We have surveyed the expression of the three human Piwi genes, Hiwi, Hili and Hiwi2, in multiple normal tissues and cancer cell lines. We find that Hiwi2 is ubiquitously expressed; in cancer cells the protein is largely restricted to the cytoplasm and is associated with translating ribosomes. Immunoprecipitation of Hiwi2 from MDAMB231 cancer cells enriches for piRNAs that are predominantly derived from processed tRNAs and expressed genes, species which can also be found in adult human testis. Our studies indicate that a Piwi-piRNA pathway is present in human somatic cells, with an uncharacterised function linked to translation. Taking this evidence together with evidence from primitive organisms, we propose that this somatic function of the pathway predates the germline functions of the pathway in modern animals. PMID:25038252

MicroRNA-23b mediates urokinase and c-met downmodulation and a decreased migration of human hepatocellular carcinoma cells.

PubMed

Salvi, Alessandro; Sabelli, Cristiano; Moncini, Silvia; Venturin, Marco; Arici, Bruna; Riva, Paola; Portolani, Nazario; Giulini, Stefano M; De Petro, Giuseppina; Barlati, Sergio

2009-06-01

Urokinase-type plasminogen activator (uPA) and c-met play a major role in cancer invasion and metastasis. Evidence has suggested that uPA and c-met overexpression may be coordinated in human hepatocellular carcinoma (HCC). In the present study, to understand whether the expression of these genes might be coregulated by specific microRNAs (miRs) in human cells, we predicted that Homo sapiens microRNA-23b could recognize two sites in the 3'-UTR of uPA and four sites in the c-met 3'-UTR by the algorithm pictar. The miR-23b expression analysis in human tumor and normal cells revealed an inverse trend with uPA and c-met expression, indicating that uPA and c-met negative regulation might depend on miR-23b expression. Transfection of miR-23b molecules in HCC cells (SKHep1C3) led to inhibition of protein expression of the target genes and caused a decrease in cell migration and proliferation capabilities. Furthermore, anti-miR-23b transfection in human normal AB2 dermal fibroblasts upregulated the expression of endogenous uPA and c-met. Cotransfection experiments in HCC cells of the miR-23b with pGL4.71 Renilla luciferase reporter gene constructs, containing the putative uPA and c-met 3'-UTR target sites, and with the pGL3 firefly luciferase-expressing vector showed a decrease in the relative luciferase activity. This would indicate that miR-23b can recognize target sites in the 3'-UTR of uPA and of c-met mRNAs and translationally repress the expression of uPA and c-met in HCC cells. The evidence obtained shows that overexpression of miR-23b leads to uPA and c-met downregulation and to decreased migration and proliferation abilities of HCC cells.

Effect of β-glucan on MUC4 and MUC5B expression in human airway epithelial cells.

PubMed

Kim, Yong-Dae; Bae, Chang Hoon; Song, Si-Youn; Choi, Yoon Seok

2015-08-01

β-Glucan is found in the cell walls of fungi, bacteria, and some plant tissues, and is detected by the innate immune system. Furthermore, this recognition is known to worsen respiratory symptoms in patients with allergic and inflammatory airway diseases. However, the means by which β-glucan affects the secretion of major mucins by human airway epithelial cells has not been elucidated. Therefore, in this study, the effect and signaling pathway of β-glucan on mucins MUC4 and MUC5B were investigated in human airway epithelial cells. In NCI-H292 cells and human normal nasal epithelial cells, the effect and signaling pathway of β-glucan on MUC4 and MUC5B expression were investigated using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with specific inhibitors and small interfering RNA (siRNA). β-Glucan increased MUC4 and MUC5B expression and activated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). SB203580 (a p38 MAPK inhibitor) and pyrrolidine dithiocarbamate (PDTC; a NF-κB inhibitor) inhibited β-glucan-induced MUC4 and MUC5B expression. In addition, siRNA knockdown of p38 MAPK blocked β-glucan-induced MUC4 and MUC5B mRNA expression and β-glucan-activated phosphorylation of NF-κB. Furthermore, Toll-like receptor 4 (TLR4) mRNA expression was increased by β-glucan, and siRNA knockdown of TLR4 blocked β-glucan-induced MUC4 and MUC5B mRNA expression and β-glucan-activated phosphorylation of p38 MAPK and NF-κB. These results demonstrate that in human airway epithelial cells β-glucan induces MUC4 and MUC5B expression via the TLR4-p38 MAPK-NF-κB signaling pathway. © 2015 ARS-AAOA, LLC.

EphA2 signaling is impacted by carcinoembryonic antigen cell adhesion molecule 1-L expression in colorectal cancer liver metastasis in a cell context-dependent manner.

PubMed

Arabzadeh, Azadeh; McGregor, Kevin; Breton, Valérie; Van Der Kraak, Lauren; Akavia, Uri David; Greenwood, Celia M T; Beauchemin, Nicole

2017-11-28

We have shown that carcinoembryonic antigen cell adhesion molecule 1 long isoform (CEACAM1-L) expression in MC38 metastatic colorectal cancer (CRC) cells results in liver metastasis inhibition via CCL2 and STAT3 signaling. But other molecular mechanisms orchestrating CEACAM1-L-mediated metastasis inhibition remain to be defined. We screened a panel of mouse and human CRC cells and evaluated their metastatic outcome after CEACAM1 overexpression or downregulation. An unbiased transcript profiling and a phospho-receptor tyrosine kinase screen comparing MC38 CEACAM1-L-expressing and non-expressing (CT) CRC cells revealed reduced ephrin type-A receptor 2 (EPHA2) expression and activity. An EPHA2-specific inhibitor reduced EPHA2 downstream signaling in CT cells similar to that in CEACAM1-L cells with decreased proliferation and migration. Human CRC patients exhibiting high CEACAM1 in combination with low EPHA2 expression benefited from longer time to first recurrence/metastasis compared to those with high EPHA2 expression. With the added interaction of CEACAM6 , we denoted that CEACAM1 high- and EPHA2 low-expressing patient samples with lower CEACAM6 expression also exhibited a longer time to first recurrence/metastasis. In HT29 human CRC cells, down-regulation of CEACAM1 along with CEA and CEACAM6 up-regulation led to higher metastatic burden. Overall, CEACAM1-L expression in poorly differentiated CRC can inhibit liver metastasis through cell context-dependent EPHA2-mediated signaling. However, CEACAM1's role should be considered in the presence of other CEACAM family members.

HDM2 promotes WIP1-mediated medulloblastoma growth

PubMed Central

Buss, Meghan C.; Read, Tracy-Ann; Schniederjan, Matthew J.; Gandhi, Khanjan; Castellino, Robert C.

2012-01-01

Medulloblastoma is the most common malignant childhood brain tumor. The protein phosphatase and oncogene WIP1 is over-expressed or amplified in a significant number of primary human medulloblastomas and cell lines. In the present study, we examine an important mechanism by which WIP1 promotes medulloblastoma growth using in vitro and in vivo models. Human cell lines and intracerebellar xenografted animal models were used to study the role of WIP1 and the major TP53 regulator, HDM2, in medulloblastoma growth. Stable expression of WIP1 enhances growth of TP53 wild-type medulloblastoma cells, compared with cells with stable expression of an empty-vector or mutant WIP1. In an animal model, WIP1 enhances proliferation and reduces the survival of immunodeficient mice bearing intracerebellar xenografted human medulloblastoma cells. Cells with increased WIP1 expression also exhibit increased expression of HDM2. HDM2 knockdown or treatment with the HDM2 inhibitor Nutlin-3a, the active enantomer of Nutlin-3, specifically inhibits the growth of medulloblastoma cells with increased WIP1 expression. Nutlin-3a does not affect growth of medulloblastoma cells with stable expression of an empty vector or of mutant WIP1. Knockdown of WIP1 or treatment with the WIP1 inhibitor CCT007093 results in increased phosphorylation of known WIP1 targets, reduced HDM2 expression, and reduced growth specifically in WIP1 wild-type and high-expressing medulloblastoma cells. Combined WIP1 and HDM2 inhibition is more effective than WIP1 inhibition alone in blocking growth of WIP1 high-expressing medulloblastoma cells. Our preclinical study supports a role for therapies that target WIP1 and HDM2 in the treatment of medulloblastoma. PMID:22379189

Enhanced expression of G-protein coupled estrogen receptor (GPER/GPR30) in lung cancer

PubMed Central

2012-01-01

Background G-protein-coupled estrogen receptor (GPER/GPR30) was reported to bind 17β-estradiol (E2), tamoxifen, and ICI 182,780 (fulvestrant) and promotes activation of epidermal growth factor receptor (EGFR)-mediated signaling in breast, endometrial and thyroid cancer cells. Although lung adenocarcinomas express estrogen receptors α and β (ERα and ERβ), the expression of GPER in lung cancer has not been investigated. The purpose of this study was to examine the expression of GPER in lung cancer. Methods The expression patterns of GPER in various lung cancer lines and lung tumors were investigated using standard quantitative real time PCR (at mRNA levels), Western blot and immunohistochemistry (IHC) methods (at protein levels). The expression of GPER was scored and the pairwise comparisons (cancer vs adjacent tissues as well as cancer vs normal lung tissues) were performed. Results Analysis by real-time PCR and Western blotting revealed a significantly higher expression of GPER at both mRNA and protein levels in human non small cell lung cancer cell (NSCLC) lines relative to immortalized normal lung bronchial epithelial cells (HBECs). The virally immortalized human small airway epithelial cell line HPL1D showed higher expression than HBECs and similar expression to NSCLC cells. Immunohistochemical analysis of tissue sections of murine lung adenomas as well as human lung adenocarcinomas, squamous cell carcinomas and non-small cell lung carcinomas showed consistently higher expression of GPER in the tumor relative to the surrounding non-tumor tissue. Conclusion The results from this study demonstrate increased GPER expression in lung cancer cells and tumors compared to normal lung. Further evaluation of the function and regulation of GPER will be necessary to determine if GPER is a marker of lung cancer progression. PMID:23273253

Enhanced expression of G-protein coupled estrogen receptor (GPER/GPR30) in lung cancer.

PubMed

Jala, Venkatakrishna Rao; Radde, Brandie N; Haribabu, Bodduluri; Klinge, Carolyn M

2012-12-28

G-protein-coupled estrogen receptor (GPER/GPR30) was reported to bind 17β-estradiol (E2), tamoxifen, and ICI 182,780 (fulvestrant) and promotes activation of epidermal growth factor receptor (EGFR)-mediated signaling in breast, endometrial and thyroid cancer cells. Although lung adenocarcinomas express estrogen receptors α and β (ERα and ERβ), the expression of GPER in lung cancer has not been investigated. The purpose of this study was to examine the expression of GPER in lung cancer. The expression patterns of GPER in various lung cancer lines and lung tumors were investigated using standard quantitative real time PCR (at mRNA levels), Western blot and immunohistochemistry (IHC) methods (at protein levels). The expression of GPER was scored and the pairwise comparisons (cancer vs adjacent tissues as well as cancer vs normal lung tissues) were performed. Analysis by real-time PCR and Western blotting revealed a significantly higher expression of GPER at both mRNA and protein levels in human non small cell lung cancer cell (NSCLC) lines relative to immortalized normal lung bronchial epithelial cells (HBECs). The virally immortalized human small airway epithelial cell line HPL1D showed higher expression than HBECs and similar expression to NSCLC cells. Immunohistochemical analysis of tissue sections of murine lung adenomas as well as human lung adenocarcinomas, squamous cell carcinomas and non-small cell lung carcinomas showed consistently higher expression of GPER in the tumor relative to the surrounding non-tumor tissue. The results from this study demonstrate increased GPER expression in lung cancer cells and tumors compared to normal lung. Further evaluation of the function and regulation of GPER will be necessary to determine if GPER is a marker of lung cancer progression.

Expression of Fas ligand by human gastric adenocarcinomas: a potential mechanism of immune escape in stomach cancer.

PubMed

Bennett, M W; O'connell, J; O'sullivan, G C; Roche, D; Brady, C; Kelly, J; Collins, J K; Shanahan, F

1999-02-01

Despite being immunogenic, gastric cancers overcome antitumour immune responses by mechanisms that have yet to be fully elucidated. Fas ligand (FasL) is a molecule that induces Fas receptor mediated apoptosis of activated immunocytes, thereby mediating normal immune downregulatory roles including immune response termination, tolerance acquisition, and immune privilege. Colon cancer cell lines have previously been shown to express FasL and kill lymphoid cells by Fas mediated apoptosis in vitro. Many diverse tumours have since been found to express FasL suggesting that a "Fas counterattack" against antitumour immune effector cells may contribute to tumour immune escape. To ascertain if human gastric tumours express FasL in vivo, as a potential mediator of immune escape in stomach cancer. Thirty paraffin wax embedded human gastric adenocarcinomas. FasL protein was detected in gastric tumours using immunohistochemistry; FasL mRNA was detected in the tumours using in situ hybridisation. Cell death was detected in situ in tumour infiltrating lymphocytes using terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL). Prevalent expression of FasL was detected in all 30 resected gastric adenocarcinomas examined. In the tumours, FasL protein and mRNA were co-localised to neoplastic gastric epithelial cells, confirming expression by the tumour cells. FasL expression was independent of tumour stage, suggesting that it may be expressed throughout gastric cancer progression. TUNEL staining disclosed a high level of cell death among lymphocytes infiltrating FasL positive areas of tumour. Human gastric adenocarcinomas express the immune downregulatory molecule, FasL. The results suggest that FasL is a prevalent mediator of immune privilege in stomach cancer.

Human trabecular meshwork cells exhibit several characteristics of, but are distinct from, adipose-derived mesenchymal stem cells.

PubMed

Morgan, Joshua T; Wood, Joshua A; Walker, Naomi J; Raghunathan, Vijay Krishna; Borjesson, Dori L; Murphy, Christopher J; Russell, Paul

2014-01-01

To support the growing promise of regenerative medicine in glaucoma, we characterized the similarities and differences between human trabecular meshwork (HTM) cells and human mesenchymal stem cells (hMSCs). HTM cells and hMSCs were phenotypically characterized by flow cytometry. Using quantitative polymerase chain reaction, the expression of myoc, angptl7, sox2, pou5f1, and notch1 was determined in both cell types with and without dexamethasone (Dex). Immunosuppressive behavior of HTM cells and hMSCs was determined using T cells activated with phytohemagglutinin. T-cell proliferation was determined using BrdU incorporation and flow cytometry. Multipotency of HTM cells and hMSCs was determined using adipogenic and osteogenic differentiation media as well as aqueous humor (AH). Alpha-smooth muscle actin (αSMA) expression was determined in HTM cells, hMSCs, and HTM tissue. Phenotypically, HTM and hMSCs expressed CD73, CD90, CD105, and CD146 but not CD31, CD34, and CD45 and similar sox2, pou5f1, and notch1 expression. Both cell types suppressed T-cell proliferation. However, HTM cells, but not hMSCs, upregulated myoc and angptl7 in response to Dex. Additionally, HTM cells did not differentiate into adipocytes or osteocytes. Culture of hMSCs in 20%, but not 100%, AH potently induced alkaline phosphatase activity. HTM cells in culture possessed uniformly strong expression of αSMA, which contrasted with the limited expression in hMSCs and spatially discrete expression in HTM tissue. HTM cells possess a number of important similarities with hMSCs but lack multipotency, one of the defining characteristics of stem cells. Further work is needed to explore the molecular mechanisms and functional implications underlying the phenotypic similarities.

Human Trabecular Meshwork Cells Exhibit Several Characteristics of, but Are Distinct from, Adipose-Derived Mesenchymal Stem Cells

PubMed Central

Morgan, Joshua T.; Wood, Joshua A.; Walker, Naomi J.; Raghunathan, Vijay Krishna; Borjesson, Dori L.; Murphy, Christopher J.

2014-01-01

Abstract Purpose: To support the growing promise of regenerative medicine in glaucoma, we characterized the similarities and differences between human trabecular meshwork (HTM) cells and human mesenchymal stem cells (hMSCs). Methods: HTM cells and hMSCs were phenotypically characterized by flow cytometry. Using quantitative polymerase chain reaction, the expression of myoc, angptl7, sox2, pou5f1, and notch1 was determined in both cell types with and without dexamethasone (Dex). Immunosuppressive behavior of HTM cells and hMSCs was determined using T cells activated with phytohemagglutinin. T-cell proliferation was determined using BrdU incorporation and flow cytometry. Multipotency of HTM cells and hMSCs was determined using adipogenic and osteogenic differentiation media as well as aqueous humor (AH). Alpha-smooth muscle actin (αSMA) expression was determined in HTM cells, hMSCs, and HTM tissue. Results: Phenotypically, HTM and hMSCs expressed CD73, CD90, CD105, and CD146 but not CD31, CD34, and CD45 and similar sox2, pou5f1, and notch1 expression. Both cell types suppressed T-cell proliferation. However, HTM cells, but not hMSCs, upregulated myoc and angptl7 in response to Dex. Additionally, HTM cells did not differentiate into adipocytes or osteocytes. Culture of hMSCs in 20%, but not 100%, AH potently induced alkaline phosphatase activity. HTM cells in culture possessed uniformly strong expression of αSMA, which contrasted with the limited expression in hMSCs and spatially discrete expression in HTM tissue. Conclusions: HTM cells possess a number of important similarities with hMSCs but lack multipotency, one of the defining characteristics of stem cells. Further work is needed to explore the molecular mechanisms and functional implications underlying the phenotypic similarities. PMID:24456002

Generation of functional hepatocytes from human spermatogonial stem cells.

PubMed

Chen, Zheng; Sun, Min; Yuan, Qingqing; Niu, Minghui; Yao, Chencheng; Hou, Jingmei; Wang, Hong; Wen, Liping; Liu, Yun; Li, Zheng; He, Zuping

2016-02-23

To generate functional human hepatocytes from stem cells and/or extra-hepatic tissues could provide an important source of cells for treating liver diseases. Spermatogonial stem cells (SSCs) have an unlimited plasticity since they can dedifferentiate and transdifferentiate to other cell lineages. However, generation of mature and functional hepatocytes from human SSCs has not yet been achieved. Here we have for the first time reported direct transdifferentiation of human SSCs to mature and functional hepatocytes by three-step induction using the defined condition medium. Human SSCs were first transdifferentiated to hepatic stem cells, as evidenced by their morphology and biopotential nature of co-expressing hepatocyte and cholangiocyte markers but not hallmarks for embryonic stem cells. Hepatic stem cells were further induced to differentiate into mature hepatocytes identified by their morphological traits and strong expression of CK8, CK18, ALB, AAT, TF, TAT, and cytochrome enzymes rather than CK7 or CK19. Significantly, mature hepatocytes derived from human SSCs assumed functional attributes of human hepatocytes, because they could produce albumin, remove ammonia, and uptake and release indocyanine green. Moreover, expression of β-CATENIN, HNF4A, FOXA1 and GATA4 was upregulated during the transdifferentiation of human SSCs to mature hepatocytes. Collectively, human SSCs could directly transdifferentiate to mature and functional hepatocytes. This study could offer an invaluable source of human hepatocytes for curing liver disorders and drug toxicology screening and provide novel insights into mechanisms underlying human liver regeneration.

Generation of functional hepatocytes from human spermatogonial stem cells

PubMed Central

Chen, Zheng; Sun, Min; Yuan, Qingqing; Niu, Minghui; Yao, Chencheng; Hou, Jingmei; Wang, Hong; Wen, Liping; Liu, Yun; Li, Zheng; He, Zuping

2016-01-01

To generate functional human hepatocytes from stem cells and/or extra-hepatic tissues could provide an important source of cells for treating liver diseases. Spermatogonial stem cells (SSCs) have an unlimited plasticity since they can dedifferentiate and transdifferentiate to other cell lineages. However, generation of mature and functional hepatocytes from human SSCs has not yet been achieved. Here we have for the first time reported direct transdifferentiation of human SSCs to mature and functional hepatocytes by three-step induction using the defined condition medium. Human SSCs were first transdifferentiated to hepatic stem cells, as evidenced by their morphology and biopotential nature of co-expressing hepatocyte and cholangiocyte markers but not hallmarks for embryonic stem cells. Hepatic stem cells were further induced to differentiate into mature hepatocytes identified by their morphological traits and strong expression of CK8, CK18, ALB, AAT, TF, TAT, and cytochrome enzymes rather than CK7 or CK19. Significantly, mature hepatocytes derived from human SSCs assumed functional attributes of human hepatocytes, because they could produce albumin, remove ammonia, and uptake and release indocyanine green. Moreover, expression of β-CATENIN, HNF4A, FOXA1 and GATA4 was upregulated during the transdifferentiation of human SSCs to mature hepatocytes. Collectively, human SSCs could directly transdifferentiate to mature and functional hepatocytes. This study could offer an invaluable source of human hepatocytes for curing liver disorders and drug toxicology screening and provide novel insights into mechanisms underlying human liver regeneration. PMID:26840458

Gene Transfer and Molecular Cloning of the Human NGF Receptor

NASA Astrophysics Data System (ADS)

Chao, Moses V.; Bothwell, Mark A.; Ross, Alonzo H.; Koprowski, Hilary; Lanahan, Anthony A.; Buck, C. Randall; Sehgal, Amita

1986-04-01

Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

A global analysis of the complex landscape of isoforms and regulatory networks of p63 in human cells and tissues.

PubMed

Sethi, Isha; Romano, Rose-Anne; Gluck, Christian; Smalley, Kirsten; Vojtesek, Borivoj; Buck, Michael J; Sinha, Satrajit

2015-08-07

The transcription factor p63 belongs to the p53/p63/p73 family and plays key functional roles during normal epithelial development and differentiation and in pathological states such as squamous cell carcinomas. The human TP63 gene, located on chromosome 3q28 is driven by two promoters that generate the full-length transactivating (TA) and N-terminal truncated (ΔN) isoforms. Furthermore alternative splicing at the C-terminus gives rise to additional α, β, γ and likely several other minor variants. Teasing out the expression and biological function of each p63 variant has been both the focus of, and a cause for contention in the p63 field. Here we have taken advantage of a burgeoning RNA-Seq based genomic data-sets to examine the global expression profiles of p63 isoforms across commonly utilized human cell-lines and major tissues and organs. Consistent with earlier studies, we find ΔNp63 transcripts, primarily that of the ΔNp63α isoforms, to be expressed in most cells of epithelial origin such as those of skin and oral tissues, mammary glands and squamous cell carcinomas. In contrast, TAp63 is not expressed in the majority of normal cell-types and tissues; rather it is selectively expressed at moderate to high levels in a subset of Burkitt's and diffuse large B-cell lymphoma cell lines. We verify this differential expression pattern of p63 isoforms by Western blot analysis, using newly developed ΔN and TA specific antibodies. Furthermore using unsupervised clustering of human cell lines, tissues and organs, we show that ΔNp63 and TAp63 driven transcriptional networks involve very distinct sets of molecular players, which may underlie their different biological functions. In this study we report comprehensive and global expression profiles of p63 isoforms and their relationship to p53/p73 and other potential transcriptional co-regulators. We curate publicly available data generated in part by consortiums such as ENCODE, FANTOM and Human Protein Atlas to delineate the vastly different transcriptomic landscapes of ΔNp63 and TAp63. Our studies help not only in dispelling prevailing myths and controversies on p63 expression in commonly used human cell lines but also augur new isoform- and cell type-specific activities of p63.

Hepatic Leukemia Factor Promotes Resistance To Cell Death: Implications For Therapeutics and Chronotherapy

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

Waters, Katrina M.; Sontag, Ryan L.; Weber, Thomas J.

Physiological variation related to circadian rhythms and aberrant gene expression patterns are believed to modulate therapeutic efficacy, but the precise molecular determinants remain unclear. Here we examine the regulation of cell death by hepatic leukemia factor (HLF), which is an output regulator of circadian rhythms and is aberrantly expressed in human cancers, using an ectopic expression strategy in JB6 mouse epidermal cells and human keratinocytes. Ectopic HLF expression inhibited cell death in both JB6 cells and human keratinocytes, as induced by serum-starvation, tumor necrosis factor alpha and ionizing radiation. Microarray analysis indicates that HLF regulates a complex multi-gene transcriptional programmore » encompassing upregulation of anti-apoptotic genes, downregulation of pro-apoptotic genes, and many additional changes that are consistent with an anti-death program. Collectively, our results demonstrate that ectopic expression of HLF, an established transcription factor that cycles with circadian rhythms, can recapitulate many features associated with circadian-dependent physiological variation.« less

Excessive Cellular Proliferation Negatively Impacts Reprogramming Efficiency of Human Fibroblasts

PubMed Central

Gupta, Manoj K.; Teo, Adrian Kee Keong; Rao, Tata Nageswara; Bhatt, Shweta; Kleinridders, Andre; Shirakawa, Jun; Takatani, Tomozumi; Hu, Jiang; De Jesus, Dario F.; Windmueller, Rebecca; Wagers, Amy J.

2015-01-01

The impact of somatic cell proliferation rate on induction of pluripotent stem cells remains controversial. Herein, we report that rapid proliferation of human somatic fibroblasts is detrimental to reprogramming efficiency when reprogrammed using a lentiviral vector expressing OCT4, SOX2, KLF4, and cMYC in insulin-rich defined medium. Human fibroblasts grown in this medium showed higher proliferation, enhanced expression of insulin signaling and cell cycle genes, and a switch from glycolytic to oxidative phosphorylation metabolism, but they displayed poor reprogramming efficiency compared with cells grown in normal medium. Thus, in contrast to previous studies, our work reveals an inverse correlation between the proliferation rate of somatic cells and reprogramming efficiency, and also suggests that upregulation of proteins in the growth factor signaling pathway limits the ability to induce pluripotency in human somatic fibroblasts. Significance The efficiency with which human cells can be reprogrammed is of interest to stem cell biology. In this study, human fibroblasts cultured in media containing different concentrations of growth factors such as insulin and insulin-like growth factor-1 exhibited variable abilities to proliferate, with consequences on pluripotency. This occurred in part because of changes in the expression of proteins involved in the growth factor signaling pathway, glycolysis, and oxidative phosphorylation. These findings have implications for efficient reprogramming of human cells. PMID:26253715

Expression and distribution of the intermediate filament protein nestin and other stem cell related molecules in the human olfactory epithelium.

PubMed

Minovi, Amir; Witt, Martin; Prescher, Andreas; Gudziol, Volker; Dazert, Stefan; Hatt, Hanns; Benecke, Heike

2010-02-01

The olfactory epithelium (OE) is unique in regenerating throughout life and thus is an attractive target for examining neurogenesis. The nestin protein was shown to be expressed in the OE of rodents and is suggested to be essentially involved in the process of regeneration. Here we report the expression and distribution of nestin in the human OE at RNA and protein level. Moreover, we analysed the expression profiles in dependence on age and olfactory capacity. After sinus surgery, biopsies were taken from the olfactory epithelium of 16 patients aged 20-80 years with documented differences in their olfactory function. Our studies revealed that nestin is constantly detectable in the apical protuberances of sustentacular cells within the human OE of healthy adults. Its expression is not dependent on age, but rather appears to be related to the olfactory function, as a comparison with specimens obtained from patients suffering either from persistent anosmia or hyposmia suggests. Particularly, in the course of dystrophy, often accompanied with impaired olfaction, nestin expression was occasionally decreased. Contrarily, the expression of the p75-NGFR protein, a marker for human OE basal cells, was not altered, indicating that at least in the tested samples olfactory impairment is not connected with abnormalities at the basal cell level. These observations emphasize an essential role of nestin for the process of regeneration, and also highlight this factor as a candidate marker for sustentacular cells in the human olfactory epithelium.

Expression of peroxisome proliferator-activated receptor alpha messenger ribonucleic acid and protein in human and rat testis.

PubMed

Schultz, R; Yan, W; Toppari, J; Völkl, A; Gustafsson, J A; Pelto-Huikko, M

1999-07-01

Peroxisome proliferator-activated receptor a (PPARalpha), a member of the steroid hormone receptor superfamily, has been linked to lipid homeostasis and tumorigenesis in tissues with high expression of receptor protein. On the other hand, the role of PPARalpha in tissues with a lower expression is not well known. Here we demonstrate the localization of PPARalpha messenger RNA (mRNA) and protein in developing and adult rat testis. Additionally, we demonstrate the expression of PPARalpha protein in adult human testis. Our experiments with Northern analysis, in situ hybridization and immunocytochemistry reveal a complex distribution of PPARalpha in tubular and interstitial cells of both adult and developing rat testis. The overall expression is rather low but may be modified by exogenous or endogenous stimuli. An up-regulation of PPARalpha mRNA could be observed after stimulation with FSH. In the developing rat testis, a clear expression of PPARalpha mRNA was present from the first days after birth. Additionally, PPARalpha mRNA and protein increased toward adulthood. In adult human testis PPARalpha immunoreactivity (IR) was present in interstitial Leydig cells and tubular cells. In the seminiferous epithelium of adult human testis the expression of PPARalpha-IR could be seen in meiotic spermatocytes, spermatids and myoid peritubular cells. The findings of our study suggest that PPARalpha may be involved in the regulation of growth and differentiation of tubular and interstitial cells in rat and human testis.

ADAM28 is expressed by epithelial cells in human normal tissues and protects from C1q-induced cell death.

PubMed

Miyamae, Yuka; Mochizuki, Satsuki; Shimoda, Masayuki; Ohara, Kentaro; Abe, Hitoshi; Yamashita, Shuji; Kazuno, Saiko; Ohtsuka, Takashi; Ochiai, Hiroki; Kitagawa, Yuko; Okada, Yasunori

2016-05-01

ADAM28 (disintegrin and metalloproteinase 28), which was originally reported to be lymphocyte-specific, is over-expressed by carcinoma cells and plays a key role in cell proliferation and progression in human lung and breast carcinomas. We studied ADAM28 expression in human normal tissues and examined its biological function. By using antibodies specific to ADAM28, ADAM28 was immunolocalized mainly to epithelial cells in several tissues, including epididymis, bronchus and stomach, whereas lymphocytes in lymph nodes and spleen were negligibly immunostained. RT-PCR, immunoblotting and ELISA analyses confirmed the expression in these tissues, and low or negligible expression by lymphocytes was found in the lymph node and spleen. C1q was identified as a candidate ADAM28-binding protein from a human lung cDNA library by yeast two-hybrid system, and specific binding was demonstrated by binding assays, immunoprecipitation and surface plasmon resonance. C1q treatment of normal bronchial epithelial BEAS-2B and NHBE cells, both of which showed low-level expression of ADAM28, caused apoptosis through activation of p38 and caspase-3, and cell death with autophagy through accumulation of LC3-II and autophagosomes, respectively. C1q-induced cell death was attenuated by treatment of the cells with antibodies against the C1q receptor gC1qR/p33 or cC1qR/calreticulin. Treatment of C1q with recombinant ADAM28 prior to addition to culture media reduced C1q-induced cell death, and knockdown of ADAM28 using siRNAs increased cell death. These data demonstrate that ADAM28 is expressed by epithelial cells of several normal organs, and suggest that ADAM28 plays a role in cell survival by suppression of C1q-induced cytotoxicity in bronchial epithelial cells. © 2016 Federation of European Biochemical Societies.

Identification of markers for quiescent pancreatic stellate cells in the normal human pancreas.

PubMed

Nielsen, Michael Friberg Bruun; Mortensen, Michael Bau; Detlefsen, Sönke

2017-10-01

Pancreatic stellate cells (PSCs) play a central role as source of fibrogenic cells in pancreatic cancer and chronic pancreatitis. In contrast to quiescent hepatic stellate cells (qHSCs), a specific marker for quiescent PSCs (qPSCs) that can be used in formalin-fixed and paraffin embedded (FFPE) normal human pancreatic tissue has not been identified. The aim of this study was to identify a marker enabling the identification of qPSCs in normal human FFPE pancreatic tissue. Immunohistochemical (IHC), double-IHC, immunofluorescence (IF) and double-IF analyses were carried out using a tissue microarray consisting of cores with normal human pancreatic tissue. Cores with normal human liver served as control. Antibodies directed against adipophilin, α-SMA, CD146, CRBP-1, cytoglobin, desmin, GFAP, nestin, S100A4 and vinculin were examined, with special emphasis on their expression in periacinar cells in the normal human pancreas and perisinusoidal cells in the normal human liver. The immunolabelling capacity was evaluated according to a semiquantitative scoring system. Double-IF of the markers of interest together with markers for other periacinar cells was performed. Moreover, the utility of histochemical stains for the identification of human qPSCs was examined, and their ultrastructure was revisited by electron microscopy. Adipophilin, CRBP-1, cytoglobin and vinculin were expressed in qHSCs in the liver, whereas cytoglobin and adipophilin were expressed in qPSCs in the pancreas. Adipophilin immunohistochemistry was highly dependent on the preanalytical time interval (PATI) from removal of the tissue to formalin fixation. Cytoglobin, S100A4 and vinculin were expressed in periacinar fibroblasts (FBs). The other examined markers were negative in human qPSCs. Our data indicate that cytoglobin and adipophilin are markers of qPSCs in the normal human pancreas. However, the use of adipophilin as a qPSC marker may be limited due to its high dependence on optimal PATI. Cytoglobin, on the other hand, is a sensitive marker for qPSCs but is expressed in FBs as well.

Bergamot (Citrus bergamia Risso) fruit extracts as γ-globin gene expression inducers: phytochemical and functional perspectives.

PubMed

Guerrini, Alessandra; Lampronti, Ilaria; Bianchi, Nicoletta; Zuccato, Cristina; Breveglieri, Giulia; Salvatori, Francesca; Mancini, Irene; Rossi, Damiano; Potenza, Rocco; Chiavilli, Francesco; Sacchetti, Gianni; Gambari, Roberto; Borgatti, Monica

2009-05-27

Epicarps of Citrus bergamia fruits from organic farming were extracted with the objective of obtaining derived products differently rich in coumarins and psoralens. The extracts were chemically characterized by (1)H nuclear magnetic resonance (NMR), gas chromatography-flame ionization detection (GC-FID), gas chromatography-mass spectrometry (GC-MS), and high-pressure liquid chromatography (HPLC) for detecting and quantifying the main constituents. Both bergamot extracts and chemical standards corresponding to the main constituents detected were then assayed for their capacity to increase erythroid differentiation of K562 cells and expression of γ-globin genes in human erythroid precursor cells. Three experimental cell systems were employed: (a) the human leukemic K562 cell line, (b) K562 cell clones stably transfected with a pCCL construct carrying green-enhanced green fluorescence protein (EGFP) under the γ-globin gene promoter, and (c) the two-phase liquid culture of human erythroid progenitors isolated from healthy donors. The results suggest that citropten and bergapten are powerful inducers of differentiation and γ-globin gene expression in human erythroid cells. These data could have practical relevance, because pharmacologically mediated regulation of human γ-globin gene expression, with the consequent induction of fetal hemoglobin, is considered to be a potential therapeutic approach in hematological disorders, including β-thalassemia and sickle cell anemia.

A lincRNA connected to cell mortality and epigenetically-silenced in most common human cancers

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

Vrba, Lukas; Garbe, James C.; Stampfer, Martha R.

Immortality is an essential characteristic of human carcinoma cells. We recently developed an efficient, reproducible method that immortalizes human mammary epithelial cells (HMEC) in the absence of gross genomic changes by targeting 2 critical senescence barriers. Consistent transcriptomic changes associated with immortality were identified using microarray analysis of isogenic normal finite pre-stasis, abnormal finite post-stasis, and immortal HMECs from 4 individuals. A total of 277 genes consistently changed in cells that transitioned from post-stasis to immortal. Gene ontology analysis of affected genes revealed biological processes significantly altered in the immortalization process. These immortalization-associated changes showed striking similarity to the genemore » expression changes seen in The Cancer Genome Atlas (TCGA) clinical breast cancer data. The most dramatic change in gene expression seen during the immortalization step was the downregulation of an unnamed, incompletely annotated transcript that we called MORT, for mortality, since its expression was closely associated with the mortal, finite lifespan phenotype. We show here that MORT (ZNF667-AS1) is expressed in all normal finite lifespan human cells examined to date and is lost in immortalized HMEC. MORT gene silencing at the mortal/immortal boundary was due to DNA hypermethylation of its CpG island promoter. This epigenetic silencing is also seen in human breast cancer cell lines and in a majority of human breast tumor tissues. The functional importance of DNA hypermethylation in MORT gene silencing is supported by the ability of 5-aza-2'- deoxycytidine to reactivate MORT expression. Analysis of TCGA data revealed deregulation of MORT expression due to DNA hypermethylation in 15 out of the 17 most common human cancers. In conclusion, the epigenetic silencing of MORT in a large majority of the common human cancers suggests a potential fundamental role in cellular immortalization during human carcinogenesis.« less

A lincRNA connected to cell mortality and epigenetically-silenced in most common human cancers

DOE PAGES

Vrba, Lukas; Garbe, James C.; Stampfer, Martha R.; ...

2015-10-19

Immortality is an essential characteristic of human carcinoma cells. We recently developed an efficient, reproducible method that immortalizes human mammary epithelial cells (HMEC) in the absence of gross genomic changes by targeting 2 critical senescence barriers. Consistent transcriptomic changes associated with immortality were identified using microarray analysis of isogenic normal finite pre-stasis, abnormal finite post-stasis, and immortal HMECs from 4 individuals. A total of 277 genes consistently changed in cells that transitioned from post-stasis to immortal. Gene ontology analysis of affected genes revealed biological processes significantly altered in the immortalization process. These immortalization-associated changes showed striking similarity to the genemore » expression changes seen in The Cancer Genome Atlas (TCGA) clinical breast cancer data. The most dramatic change in gene expression seen during the immortalization step was the downregulation of an unnamed, incompletely annotated transcript that we called MORT, for mortality, since its expression was closely associated with the mortal, finite lifespan phenotype. We show here that MORT (ZNF667-AS1) is expressed in all normal finite lifespan human cells examined to date and is lost in immortalized HMEC. MORT gene silencing at the mortal/immortal boundary was due to DNA hypermethylation of its CpG island promoter. This epigenetic silencing is also seen in human breast cancer cell lines and in a majority of human breast tumor tissues. The functional importance of DNA hypermethylation in MORT gene silencing is supported by the ability of 5-aza-2'- deoxycytidine to reactivate MORT expression. Analysis of TCGA data revealed deregulation of MORT expression due to DNA hypermethylation in 15 out of the 17 most common human cancers. In conclusion, the epigenetic silencing of MORT in a large majority of the common human cancers suggests a potential fundamental role in cellular immortalization during human carcinogenesis.« less

Human population-specific gene expression and transcriptional network modification with polymorphic transposable elements

PubMed Central

Wang, Lu; Mariño-Ramírez, Leonardo

2017-01-01

Abstract Transposable element (TE) derived sequences are known to contribute to the regulation of the human genome. The majority of known TE-derived regulatory sequences correspond to relatively ancient insertions, which are fixed across human populations. The extent to which human genetic variation caused by recent TE activity leads to regulatory polymorphisms among populations has yet to be thoroughly explored. In this study, we searched for associations between polymorphic TE (polyTE) loci and human gene expression levels using an expression quantitative trait loci (eQTL) approach. We compared locus-specific polyTE insertion genotypes to B cell gene expression levels among 445 individuals from 5 human populations. Numerous human polyTE loci correspond to both cis and trans eQTL, and their regulatory effects are directly related to cell type-specific function in the immune system. PolyTE loci are associated with differences in expression between European and African population groups, and a single polyTE loci is indirectly associated with the expression of numerous genes via the regulation of the B cell-specific transcription factor PAX5. The polyTE-gene expression associations we found indicate that human TE genetic variation can have important phenotypic consequences. Our results reveal that TE-eQTL are involved in population-specific gene regulation as well as transcriptional network modification. PMID:27998931

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

Zhong, Yiming; Program in Molecular, Cellular, and Developmental Biology, The Ohio State University, Columbus, OH; Sullenbarger, Brent

Research highlights: {yields} HoxB4 overexpression in human TF1 cells increased the expression of CD61 and CD41a. {yields} HoxB4 fusion protein enhanced megakaryocytic development of CD34{sup +} cord blood cells. {yields} Ectopic HoxB4 increased Tpo receptor expression and decreased c-Myb expression. {yields} HoxB4 RNA silencing increased c-Myb expression and decreased Fli-1 expression. -- Abstract: In order to produce clinically useful quantities of platelets ex vivo we may need to firstly enhance early self-renewal of hematopoietic stem cells (HSCs) and/or megakaryocyte (Mk) progenitors. The homeodomain transcription factor HoxB4 has been shown to be an important regulator of stem cell renewal and hematopoiesis;more » however, its effect on megakaryopoiesis is unclear. In this study, we investigated the effect of HoxB4 overexpression or RNA silencing on megakaryocytic development in the human TF1 progenitor cell line; we then used recombinant tPTD-HoxB4 fusion protein to study the effect of exogenous HoxB4 on megakaryocytic development of human CD34 positively-selected cord blood cells. We found that ectopic HoxB4 in TF1 cells increased the antigen expression of CD61and CD41a, increased the gene expression of thrombopoietin receptor (TpoR), Scl-1, Cyclin D1, Fog-1 and Fli-1 while it decreased c-Myb expression. HoxB4 RNA silencing in TF1 cells decreased the expression of CD61 and CD41a and decreased Fli-1 expression while it increased the expression of c-Myb. Recombinant tPTD-HoxB4 fusion protein increased the percentages and absolute numbers of CD41a and CD61 positive cells during megakaryocytic differentiation of CD34 positively-selected cord blood cells and increased the numbers of colony-forming unit-megakaryocyte (CFU-Mk). Adding tPTD-HoxB4 fusion protein increased the gene expression of TpoR, Cyclin D1, Fog-1 and Fli-1 while it inhibited c-Myb expression. Our data suggest that increased HoxB4 enhanced early megakaryocytic development in human TF1 cells and CD34 positively-selected cord blood cells primarily by upregulating TpoR and Fli-1 expression and downregulating c-Myb expression. Increasing HoxB4 expression or adding recombinant HoxB4 protein might be a way to expand Mks for the production of platelets for use in transfusion medicine.« less

CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

PubMed Central

Alonso-Camino, Vanesa; Sánchez-Martín, David; Compte, Marta; Nuñez-Prado, Natalia; Diaz, Rosa M; Vile, Richard; Alvarez-Vallina, Luis

2013-01-01

A human single-chain variable fragment (scFv) antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs). The repertoire was fused to a first-generation T cell receptor ζ (TCRζ)-based chimeric antigen receptor (CAR). We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2) bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR) and the selection context (cell synapse), which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells. PMID:23695536

Epithelial Membrane Protein-2 is a Novel Therapeutic Target in Ovarian Cancer

PubMed Central

Fu, Maoyong; Maresh, Erin L.; Soslow, Robert A.; Alavi, Mohammad; Mah, Vei; Zhou, Qin; Iasonos, Alexia; Goodglick, Lee; Gordon, Lynn K.; Braun, Jonathan; Wadehra, Madhuri

2010-01-01

Purpose The tetraspan protein epithelial membrane protein-2 (EMP2) has been shown to regulate the surface display and signaling from select integrin pairs, and it was recently identified as a prognostic biomarker in human endometrial cancer. In this study, we assessed the role of EMP2 in human ovarian cancer. Experimental Design We examined the expression of EMP2 within a population of women with ovarian cancer using tissue microarray assay technology. We evaluated the efficacy of EMP2-directed antibody therapy using a fully human recombinant bivalent antibody fragment (diabody) in vitro and ovarian cancer xenograft models in vivo. Results EMP2 was found to be highly expressed in over 70% of serous and endometrioid ovarian tumors compared to non-malignant ovarian epithelium using a human ovarian cancer tissue microarray. Using anti-EMP2 diabody, we evaluated the in vitro response of 9 human ovarian cancer cell lines with detectable EMP2 expression. Treatment of human ovarian cancer cell lines with anti-EMP2 diabodies induced cell death and retarded cell growth, and these response rates correlated with cellular EMP2 expression. We next assessed the effects of anti-EMP2 diabodies in mice bearing xenografts from the ovarian endometrioid carcinoma cell line OVCAR5. Anti-EMP2 diabodies significantly suppressed tumor growth and induced cell death in OVCAR5 xenografts. Conclusions These findings indicate that EMP2 is expressed in the majority of ovarian tumors and it may be a feasible target in vivo. PMID:20670949

Arctigenin from Arctium lappa inhibits interleukin-2 and interferon gene expression in primary human T lymphocytes.

PubMed

Tsai, Wei-Jern; Chang, Chu-Ting; Wang, Guei-Jane; Lee, Tzong-Huei; Chang, Shwu-Fen; Lu, Shao-Chun; Kuo, Yuh-Chi

2011-03-25

Arctium lappa (Niubang), a Chinese herbal medicine, is used to treat tissue inflammation. This study investigates the effects of arctigenin (AC), isolated from A. lappa, on anti-CD3/CD28 Ab-stimulated cell proliferation and cytokine gene expression in primary human T lymphocytes. Cell proliferation was determined with enzyme immunoassays and the tritiated thymidine uptake method. Cytokine production and gene expression were analyzed with reverse transcription-polymerase chain reaction. AC inhibited primary human T lymphocytes proliferation activated by anti-CD3/CD28 Ab. Cell viability test indicated that the inhibitory effects of AC on primary human T lymphocyte proliferation were not due to direct cytotoxicity. AC suppressed interleukin-2 (IL-2) and interferon-γ (IFN-γ) production in a concentration-dependent manner. Furthermore, AC decreased the IL-2 and IFN-γ gene expression in primary human T lymphocytes induced by anti-CD3/CD28 Ab. Reporter gene analyses revealed that AC decreased NF-AT-mediated reporter gene expression. AC inhibited T lymphocyte proliferation and decreased the gene expression of IL-2, IFN-γ and NF-AT.

BMP6 down-regulates GDNF expression through SMAD1/5 and ERK1/2 signaling pathways in human granulosa-lutein cells.

PubMed

Zhang, Xin-Yue; Chang, Hsun-Ming; Taylor, Elizabeth L; Leung, Peter C K; Liu, Rui-Zhi

2018-05-09

Bone morphogenetic protein 6 (BMP6) is a critical regulator of follicular development that is expressed in mammalian oocytes and granulosa cells. Glial cell line-derived neurotrophic factor (GDNF) is an intraovarian neurotrophic factor that plays an essential role in regulating mammalian oocyte maturation. The aim of this study was to investigate the effect of BMP6 on the regulation of GDNF expression and the potential underlying mechanisms. We used an established immortalized human granulosa cell line (SVOG cells) and primary human granulosa-lutein cells as in vitro cell models. Our results showed that BMP6 significantly down-regulated the expression of GDNF in both SVOG and primary human granulosa-lutein cells. Using dual inhibition approaches (kinase receptor inhibitor and small interfering RNA knockdown), our results showed that both ALK2 and ALK3 are involved in BMP6-induced down-regulation of GDNF. In addition, BMP6 induced the phosphorylation of SMAD1/5/8 and ERK1/2 but not AKT or p38. Among three downstream mediators, both SMAD1 and SMAD5 are involved in BMP6-induced down-regulation of GDNF. Moreover, concomitant knockdown of endogenous SMAD4 and inhibition of ERK1/2 activity completely reversed BMP6-induced down-regulation of GDNF, indicating that both SMAD and ERK1/2 signaling pathways are required for the regulatory effect of BMP6 on GDNF expression. Our findings suggest an additional role for an intrafollicular growth factor in regulating follicular function through their paracrine interactions in human granulosa cells.

Rho GTPases and Regulation of Cell Migration and Polarization in Human Corneal Epithelial Cells

PubMed Central

Hou, Aihua; Toh, Li Xian; Gan, Kah Hui; Lee, Khee Jin Ryan; Manser, Edward; Tong, Louis

2013-01-01

Purpose Epithelial cell migration is required for regeneration of tissues and can be defective in a number of ocular surface diseases. This study aimed to determine the expression pattern of Rho family small G-proteins in human corneal epithelial cells to test their requirement in directional cell migration. Methods Rho family small G-protein expression was assessed by reverse transcription-polymerase chain reaction. Dominant-inhibitory constructs encoding Rho proteins or Rho protein targeting small interfering RNA were transfected into human corneal epithelial large T antigen cells, and wound closure rate were evaluated by scratch wounding assay, and a complementary non-traumatic cell migration assay. Immunofluorescence staining was performed to study cell polarization and to assess Cdc42 downstream effector. Results Cdc42, Chp, Rac1, RhoA, TC10 and TCL were expressed in human corneal epithelial cells. Among them, Cdc42 and TCL were found to significantly affect cell migration in monolayer scratch assays. These results were confirmed through the use of validated siRNAs directed to Cdc42 and TCL. Scramble siRNA transfected cells had high percentage of polarized cells than Cdc42 or TCL siRNA transfected cells at the wound edge. We showed that the Cdc42-specific effector p21-activated kinase 4 localized predominantly to cell-cell junctions in cell monolayers, but failed to translocate to the leading edge in Cdc42 siRNA transfected cells after monolayer wounding. Conclusion Rho proteins expressed in cultured human corneal epithelial cells, and Cdc42, TCL facilitate two-dimensional cell migration in-vitro. Although silencing of Cdc42 and TCL did not noticeably affect the appearance of cell adhesions at the leading edge, the slower migration of these cells indicates both GTP-binding proteins play important roles in promoting cell movement of human corneal epithelial cells. PMID:24130842

PD-1 ligand expression by human colonic myofibroblasts/fibroblasts regulates CD4+ T-cell activity.

PubMed

Pinchuk, Irina V; Saada, Jamal I; Beswick, Ellen J; Boya, Gushyalatha; Qiu, Sumin M; Mifflin, Randy C; Raju, Gottumukkala S; Reyes, Victor E; Powell, Don W

2008-10-01

A prominent role for inhibitory molecules PD-L1 and PD-L2 in peripheral tolerance has been proposed. However, the phenotype and function of PD-L-expressing cells in human gut remains unclear. Recent studies suggest that colonic myofibroblasts (CMFs) and fibroblasts are important in the switch from acute inflammation to adaptive immunity. In the normal human colon, CMFs represent a distinct population of major histocompatibility complex class II(+) cells involved in the regulation of mucosal CD4(+) T-cell responses. PD-L1 and PD-L2 expression on human CMFs was determined using Western blot, fluorescence-activated cell sorter analysis and confocal microscopy. Lymphoproliferation assays and cytokine enzyme-linked immunosorbent assays were used to evaluate the role of B7 costimulators expressed by CMFs with regard to the regulation of preactivated T-helper cell responses. We demonstrate here the expression of PD-L1/2 molecules by normal human CMF and fibroblasts in situ and in culture. Both molecules support suppressive functions of CMFs in the regulation of activated CD4(+) T-helper cell proliferative responses; blocking this interaction reverses the suppressive effect of CMFs on T-cell proliferation and leads to increased production of the major T-cell growth factor, interleukin (IL)-2. PD-L1/2-mediated CMF suppressive functions are mainly due to the inhibition of IL-2 production, because supplementation of the coculture media with exogenous IL-2 led to partial recovery of activated T-cell proliferation. Our data suggest that stromal myofibroblasts and fibroblasts may limit T-helper cell proliferative activity in the gut and, thus, might play a prominent role in mucosal intestinal tolerance.

Effect of cadmium on the expression levels of interleukin-1α and interleukin-10 cytokines in human lung cells.

PubMed

Odewumi, Caroline; Latinwo, Lekan M; Sinclair, Andre; Badisa, Veera L D; Abdullah, Ahkinyala; Badisa, Ramesh B

2015-11-01

Cadmium is an environmentally hazardous metal, which causes toxicity in humans. Inhalation of cigarette smoke and industrial fumes containing cadmium are sources of cadmium exposure. It is responsible for the malfunction of various organs, leading to disease particularly in the lungs, liver and kidneys. In the present study, the effect of cadmium chloride (CdCl2) on cell viability, and the expression levels of interleukin (IL)‑1α and IL‑10 cytokines at various concentrations and incubation durations were assessed in MRC‑9 human normal lung and A549 human lung cancer cells to elucidate the mechanism of cadmium toxicity. Cell viability was measured using a crystal violet dye binding assay. The expression levels of the cytokines were measured by cytokine specific enzyme‑linked immunosorbent assay kits. The viability assay results revealed higher sensitivity of the A549 lung cancer cells to CdCl2 compared with the normal MRC‑9 lung cells. In the normal MRC‑9 lung cells, higher expression levels of the cytokines were observed at the lowest CdCl2 concentration at a shorter exposure time compared with the lung cancer cells. Higher levels of the cytokines were observed in the A549 lung cancer cells at all other times and concentrations compared with the MRC‑9 cells, indicating higher levels of inflammation. The cytokine levels were reduced at higher CdCl2 concentrations and longer exposure durations, demonstrating the toxic effect of cadmium. The results indicated that CdCl2 affected the expression levels of the cytokines and led to cytotoxicity in human lung cells, and suggested that compounds which reduce inflammation may prevent cadmium toxicity.

Drug-loaded nanoparticles induce gene expression in human pluripotent stem cell derivatives

NASA Astrophysics Data System (ADS)

Gajbhiye, Virendra; Escalante, Leah; Chen, Guojun; Laperle, Alex; Zheng, Qifeng; Steyer, Benjamin; Gong, Shaoqin; Saha, Krishanu

2013-12-01

Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained higher fibroblast proliferation levels and MMP activity. The results demonstrate that the PEG-H40-DXC nanoparticle system provides an effective tool to controlling gene expression in human stem cell derivatives.Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained higher fibroblast proliferation levels and MMP activity. The results demonstrate that the PEG-H40-DXC nanoparticle system provides an effective tool to controlling gene expression in human stem cell derivatives. Electronic supplementary information (ESI) available: ESI containing 1H NMR spectra and additional fibroblast characterization data. See DOI: 10.1039/c3nr04794f

Heat Shock Enhances the Expression of the Human T Cell Leukemia Virus Type-I (HTLV-I) Trans-Activator (Tax) Antigen in Human HTLV-I Infected Primary and Cultured T Cells.

PubMed

Kunihiro, Marie; Fujii, Hideki; Miyagi, Takuya; Takahashi, Yoshiaki; Tanaka, Reiko; Fukushima, Takuya; Ansari, Aftab A; Tanaka, Yuetsu

2016-07-11

The environmental factors that lead to the reactivation of human T cell leukemia virus type-1 (HTLV-I) in latently infected T cells in vivo remain unknown. It has been previously shown that heat shock (HS) is a potent inducer of HTLV-I viral protein expression in long-term cultured cell lines. However, the precise HTLV-I protein(s) and mechanisms by which HS induces its effect remain ill-defined. We initiated these studies by first monitoring the levels of the trans-activator (Tax) protein induced by exposure of the HTLV-I infected cell line to HS. HS treatment at 43 °C for 30 min for 24 h led to marked increases in the level of Tax antigen expression in all HTLV-I-infected T cell lines tested including a number of HTLV-I-naturally infected T cell lines. HS also increased the expression of functional HTLV-I envelope gp46 antigen, as shown by increased syncytium formation activity. Interestingly, the enhancing effect of HS was partially inhibited by the addition of the heat shock protein 70 (HSP70)-inhibitor pifithlin-μ (PFT). In contrast, the HSP 70-inducer zerumbone (ZER) enhanced Tax expression in the absence of HS. These data suggest that HSP 70 is at least partially involved in HS-mediated stimulation of Tax expression. As expected, HS resulted in enhanced expression of the Tax-inducible host antigens, such as CD83 and OX40. Finally, we confirmed that HS enhanced the levels of Tax and gp46 antigen expression in primary human CD4⁺ T cells isolated from HTLV-I-infected humanized NOD/SCID/γc null (NOG) mice and HTLV-I carriers. In summary, the data presented herein indicate that HS is one of the environmental factors involved in the reactivation of HTLV-I in vivo via enhanced Tax expression, which may favor HTLV-I expansion in vivo.

Facilitative glucose transporter gene expression in human lymphocytes, monocytes, and macrophages: a role for GLUT isoforms 1, 3, and 5 in the immune response and foam cell formation.

PubMed

Fu, Yuchang; Maianu, Lidia; Melbert, Barry R; Garvey, W Timothy

2004-01-01

Cellular glucose uptake is mediated by a family of facilitative glucose transporters (GLUT) exhibiting differences in kinetics, substrate specificity, and tissue-specific expression. GLUT isoform expression has not been comprehensively studied in human leukocytes, which participate in immune and inflammatory responses and are critical for host defense. Therefore, we studied the regulated expression of GLUT 1-5 mRNA and protein in isolated human lymphocytes and monocytes and in human THP-1 macrophages and foam cells. Lymphocytes expressed GLUT 1 and GLUT 3 proteins, and cellular levels of both isoforms were augmented 3.5- to 6-fold following activation by phytohemagglutinin (PHA). Monocytes expressed 8.4-fold more GLUT 3 protein and 88% less GLUT 1 than lymphocytes, and activation by lipopolysaccharide (LPS) led to a 1.9-fold increase in GLUT 1. At the level of mRNA expression, GLUT 3 mRNA was the most prevalent GLUT mRNA species in monocytes, while lymphocytes expressed equal numbers of GLUT 1 and GLUT 3 transcripts. Differentiation of THP-1 monocytes into macrophages was associated with marked induction of GLUT 3 and GLUT 5 protein expression, and high levels of GLUT 1, GLUT 3, and GLUT 5 were maintained after transformation to foam cells. GLUT 5 mRNA was expressed in 2-fold greater abundance in macrophages and foam cells than that observed for GLUT 1 mRNA, while the level of GLUT 3 mRNA was intermediate. This facilitative glucose transporters are differentially expressed and regulated in human leukocytes in a pattern that could facilitate cellular functions. Speculatively, high GLUT 1 and GLUT 3 expression could provide cellular fuel for the immune response, and high levels of high-affinity GLUT 3 in macrophages might allow the cell to compete with pathogens for hexoses, even in the presence of low interstitial glucose concentrations. Ample expression of GLUT 1 and GLUT 3 in foam cells could also provide hexose substrates and promote lipid loading. The role for high levels of the fructose transporter GLUT 5 in macrophages and foam cells is unknown since interstitial and circulating fructose concentrations are low in these cells.

Persistence of hAQP1 expression in human salivary gland cells following AdhAQP1 transduction is associated with a lack of methylation of hCMV promoter

PubMed Central

Zheng, C; Baum, BJ; Liu, X; Goldsmith, CM; Perez, P; Jang, S-I; Cotrim, AP; McCullagh, L; Ambudkar, IS; Alevizos, I

2017-01-01

In 2012, we reported that 5 out of 11 subjects in a clinical trial (NCT00372320) administering AdhAQP1 to radiation-damaged parotid glands showed increased saliva flow rates and decreased symptoms over the initial 42 days. AdhAQP1 is a first-generation, E1-deleted, replication-defective, serotype 5 adenoviral vector encoding human aquaporin-1 (hAQP1). This vector uses the human cytomegalovirus enhancer/promoter (hCMVp). As subject peak responses were at times much longer (7–42 days) than expected, we hypothesized that the hCMVp may not be methylated in human salivary gland cells to the extent previously observed in rodent salivary gland cells. This hypothesis was supported in human salivary gland primary cultures and human salivary gland cell lines after transduction with AdhAQP1. Importantly, hAQP1 maintained its function in those cells. Conversely, when we transduced mouse and rat cell lines in vitro and submandibular glands in vivo with AdhAQP1, the hCMVp was gradually methylated over time and associated with decreased hAQP1 expression and function in vitro and decreased hAQP1 expression in vivo. These data suggest that the hCMVp in AdhAQP1was probably not methylated in transduced human salivary gland cells of responding subjects, resulting in an unexpectedly longer functional expression of hAQP1. PMID:26177970

Expression of NK cell receptors on decidual T cells in human pregnancy.

PubMed

Tilburgs, Tamara; van der Mast, Barbara J; Nagtzaam, Nicole M A; Roelen, Dave L; Scherjon, Sicco A; Claas, Frans H J

2009-06-01

Specific receptors enable NK cells to discriminate between cells with normal expression of MHC class I and cells that have low or absent expression of MHC class I molecules. In addition to NK cells, these receptors can be expressed on T cell subsets, mainly on CD8+ T cells but also on gammadeltaTCR+ T cells and CD4+ T cells. Although the function of NK cell receptor expression on T cells is not completely understood, various studies have shown that they are involved in down regulation of T cell receptor (TCR)-mediated activation and influence effector functions, like cytotoxicity and cytokine production. The aim of this study was to analyze expression of NK cell receptors on peripheral blood and decidual T cells during human pregnancy using flow cytometry. We demonstrate that a proportion of decidual T cells express HLA-C specific killer immunoglobulin-like receptors (KIRs). Furthermore, a small proportion of decidual T cells express the HLA-E specific CD94-NKG2A inhibitory and CD94-NKG2C activating receptors. Decidual KIR+ and CD94-NKG2+ T cells mainly display a CD3+CD4-CD8- phenotype. However, decidual tissue also contains higher percentages of KIR and CD94-NKG2 expressing CD4+ and CD8+ T cells compared to peripheral blood. So far, the functional capacities of decidual T cells expressing the NK cell receptors are unknown but NK cell receptor expression on decidual T cells may provide an alternative means by which decidual T cells distinguish self (maternal) cells from allogeneic fetal cells, and act to modulate the decidual immune response.

Interleukin-9 enhances interleukin-5 receptor expression, differentiation, and survival of human eosinophils.

PubMed

Gounni, A S; Gregory, B; Nutku, E; Aris, F; Latifa, K; Minshall, E; North, J; Tavernier, J; Levit, R; Nicolaides, N; Robinson, D; Hamid, Q

2000-09-15

Interleukin-9 (IL-9) has been implicated in the pathogenesis of allergic disorders. To examine the interaction between IL-9 and eosinophils, we evaluated mature peripheral blood eosinophils for their expression of the specific alpha-subunit of the IL-9 receptor (IL-9R-alpha). The expression of IL-9R-alpha by human eosinophils was detected at the messenger RNA (mRNA) and protein levels by reverse transcriptase-polymerase chain reaction (RT-PCR), flow cytometry, and immunocytochemical analysis, respectively. Functional analyses demonstrated that recombinant human (rh)IL-9 inhibited in vitro peripheral blood human eosinophil apoptosis in a concentration-dependent manner. We then examined the role of IL-9 in eosinophil differentiation using the human cord blood CD34(+) cells and human promyelocytic leukemia cells (HL-60). The addition of IL-9 to CD34(+) cells cultured in IL-3 and IL-5 enhanced eosinophil development, and IL-9 alone induced the expression of IL-5R-alpha. IL-9 also up-regulated the IL-5R-alpha chain cell surface expression during terminal eosinophil differentiation of the HL-60 cell line. Our findings suggest that IL-9 may potentiate in vivo eosinophil function by increasing their survival and IL-5-mediated differentiation and maturation. Taken together, these results suggest a mechanism by which IL-9 potentiates airway and tissue eosinophilia.

Human Lymphoid Tissues Harbor a Distinct CD69+CXCR6+ NK Cell Population.

PubMed

Lugthart, Gertjan; Melsen, Janine E; Vervat, Carly; van Ostaijen-Ten Dam, Monique M; Corver, Willem E; Roelen, Dave L; van Bergen, Jeroen; van Tol, Maarten J D; Lankester, Arjan C; Schilham, Marco W

2016-07-01

Knowledge of human NK cells is based primarily on conventional CD56(bright) and CD56(dim) NK cells from blood. However, most cellular immune interactions occur in lymphoid organs. Based on the coexpression of CD69 and CXCR6, we identified a third major NK cell subset in lymphoid tissues. This population represents 30-60% of NK cells in marrow, spleen, and lymph node but is absent from blood. CD69(+)CXCR6(+) lymphoid tissue NK cells have an intermediate expression of CD56 and high expression of NKp46 and ICAM-1. In contrast to circulating NK cells, they have a bimodal expression of the activating receptor DNAX accessory molecule 1. CD69(+)CXCR6(+) NK cells do not express the early markers c-kit and IL-7Rα, nor killer cell Ig-like receptors or other late-differentiation markers. After cytokine stimulation, CD69(+)CXCR6(+) NK cells produce IFN-γ at levels comparable to CD56(dim) NK cells. They constitutively express perforin but require preactivation to express granzyme B and exert cytotoxicity. After hematopoietic stem cell transplantation, CD69(+)CXCR6(+) lymphoid tissue NK cells do not exhibit the hyperexpansion observed for both conventional NK cell populations. CD69(+)CXCR6(+) NK cells constitute a separate NK cell population with a distinct phenotype and function. The identification of this NK cell population in lymphoid tissues provides tools to further evaluate the cellular interactions and role of NK cells in human immunity. Copyright © 2016 by The American Association of Immunologists, Inc.

L-Dopa decarboxylase expression profile in human cancer cells.

PubMed

Chalatsa, Ioanna; Nikolouzou, Eleftheria; Fragoulis, Emmanuel G; Vassilacopoulou, Dido

2011-02-01

L-Dopa decarboxylase (DDC) catalyses the decarboxylation of L-Dopa. It has been shown that the DDC gene undergoes alternative splicing within its 5'-untranslated region (UTR), in a tissue-specific manner, generating identical protein products. The employment of two alternative 5'UTRs is thought to be responsible for tissue-specific expression of the human DDC mRNA. In this study, we focused on the investigation of the nature of the mRNA expression in human cell lines of neural and non-neural origin. Our results show the expression of a neural-type DDC mRNA splice variant, lacking exon 3 in all cell lines studied. Co-expression of the full length non-neural DDC mRNA and the neural-type DDC splice variant lacking exon 3 was detected in all cell lines. The alternative DDC protein isoform, Alt-DDC, was detected in SH-SY5Y and HeLa cells. Our findings suggest that the human DDC gene undergoes complex processing, leading to the formation of multiple mRNA isoforms. The study of the significance of this phenomenon of multiple DDC mRNA isoforms could provide us with new information leading to the elucidation of the complex biological pathways that the human enzyme is involved in.

Effect of increased HoxB4 on human megakaryocytic development

PubMed Central

Zhong, Yiming; Sullenbarger, Brent; Lasky, Larry C.

2010-01-01

In order to ex vivo produce clinically useful quantity of platelets, we may need to firstly enhance early self-renewal of hematopoietic stem cells (HSCs) and/or megakaryocyte (Mk) progenitors. The homeodomain transcription factor HoxB4 has been shown to be an important regulator of stem cell renewal and hematopoiesis; however, its effect on megakaryopoiesis is unclear. In this study, we investigated the effect of HoxB4 overexpression or RNA silencing on megakaryocytic development in the human TF1 progenitor cell line; we then used recombinant tPTD-HoxB4 fusion protein to study the effect of exogenous HoxB4 on megakaryocytic development of human CD34 positively-selected cord blood cells. We found that ectopic HoxB4 in TF1 cells increased the antigen expression of CD61and CD41a, increased the gene expression of thrombopoietin receptor (TpoR), Scl-1, Cyclin D1, Fog-1 and Fli-1 while it decreased c-Myb expression. HoxB4 RNA silencing in TF1 cells decreased the expression of CD61 and CD41a and decreased Fli-1 expression while it increased the expression of c-Myb. Recombinant tPTD-HoxB4 fusion protein increased the percentages and absolute numbers of CD41a and CD61 positive cells during megakaryocytic differentiation of CD34 positively-selected cord blood cells and increased the numbers of colony forming unit-megakaryocyte (CFU-Mk). Adding tPTD-HoxB4 fusion protein increased the gene expression of TpoR, Cyclin D1, Fog-1 and Fli-1 while it inhibited c-Myb expression. Our data indicate that increased HoxB4 enhanced early megakaryocytic development in human TF1 cells and CD34 positively-selected cord blood cells primarily by upregulating Tpo R and Fli-1 expression and downregulating c-Myb expression. Increasing HoxB4 expression or adding recombinant HoxB4 protein might be a way to expand Mks for the production of platelets for use in transfusion medicine. PMID:20599537

Requirement of the Listeria monocytogenes broad-range phospholipase PC-PLC during infection of human epithelial cells.

PubMed

Gründling, Angelika; Gonzalez, Mark D; Higgins, Darren E

2003-11-01

In this study, we investigated the requirement of the Listeria monocytogenes broad-range phospholipase C (PC-PLC) during infection of human epithelial cells. L. monocytogenes is a facultative intracellular bacterial pathogen of humans and a variety of animal species. After entering a host cell, L. monocytogenes is initially surrounded by a membrane-bound vacuole. Bacteria promote their escape from this vacuole, grow within the host cell cytosol, and spread from cell to cell via actin-based motility. Most infection studies with L. monocytogenes have been performed with mouse cells or an in vivo mouse model of infection. In all mouse-derived cells tested, the pore-forming cytolysin listeriolysin O (LLO) is absolutely required for lysis of primary vacuoles formed during host cell entry. However, L. monocytogenes can escape from primary vacuoles in the absence of LLO during infection of human epithelial cell lines Henle 407, HEp-2, and HeLa. Previous studies have shown that the broad-range phospholipase C, PC-PLC, promotes lysis of Henle 407 cell primary vacuoles in the absence of LLO. Here, we have shown that PC-PLC is also required for lysis of HEp-2 and HeLa cell primary vacuoles in the absence of LLO expression. Furthermore, our results indicated that the amount of PC-PLC activity is critical for the efficiency of vacuolar lysis. In an LLO-negative derivative of L. monocytogenes strain 10403S, expression of PC-PLC has to increase before or upon entry into human epithelial cells, compared to expression in broth culture, to allow bacterial escape from primary vacuoles. Using a system for inducible PC-PLC expression in L. monocytogenes, we provide evidence that phospholipase activity can be increased by elevated expression of PC-PLC or Mpl, the enzyme required for proteolytic activation of PC-PLC. Lastly, by using the inducible PC-PLC expression system, we demonstrate that, in the absence of LLO, PC-PLC activity is not only required for lysis of primary vacuoles in human epithelial cells but is also necessary for efficient cell-to-cell spread. We speculate that the additional requirement for PC-PLC activity is for lysis of secondary double-membrane vacuoles formed during cell-to-cell spread.

Rebamipide increases the mucin-like glycoprotein production in corneal epithelial cells.

PubMed

Takeji, Yasuhiro; Urashima, Hiroki; Aoki, Akihiro; Shinohara, Hisashi

2012-06-01

Dry eye is a multifactorial disease of tears and the ocular surface due to tear deficiency or excessive tear evaporation. Tear film instability is due to a disturbance in ocular surface mucin leading to a dysfunction of mucin, resulting in dry eye. In this study, we examined the effect of rebamipide, an anti-ulcer agent, on glycoconjugate production, as an indicator of mucin-like glycoprotein in cultured corneal epithelial cells. Further, we investigated the effect of rebamipide on the gene expression of membrane-associated mucins. Confluent cultured human corneal epithelial cells were incubated with rebamipide for 24 h. The glycoconjugate content in the supernatant and the cell extracts was measured by wheat germ agglutinin-enzyme-linked lectin assay combined gel-filtration method. In the experiment on mucin gene expression, cultured human corneal epithelial cells were collected at 0, 3, 6, and 12 h after administration of rebamipide. Real-time quantitative polymerase chain reaction was used to analyze the quantity of MUC1, MUC 4, and MUC16 gene expression. Rebamipide significantly increased the glycoconjugate contents in the supernatant and cell extract. In the mucin gene expression in the cells, rebamipide increased MUC1 and MUC4 gene expression, but did not increase MUC16 gene expression. Rebamipide promoted glycoconjugate, which has a property as a mucin-like glycoprotein, in human corneal epithelial cells. The increased production was mediated by MUC1 and MUC4 gene expression.

BMP signalling in human fetal ovary somatic cells is modulated in a gene-specific fashion by GREM1 and GREM2.

PubMed

Bayne, Rosemary A; Donnachie, Douglas J; Kinnell, Hazel L; Childs, Andrew J; Anderson, Richard A

2016-09-01

Do changes in the expression of bone morphogenetic proteins (BMPs) 2 and 4, and their antagonists Gremlin1 (GREM1) and Gremlin2 (GREM2) during human fetal ovarian development impact on BMP pathway activity and lead to changes in gene expression that may influence the fate and/or function of ovarian somatic cells? BMPs 2 and 4 differentially regulate gene expression in cultured human fetal ovarian somatic cells. Expression of some, but not all BMP target genes is antagonised by GREM1 and GREM2, indicating the existence of a mechanism to fine-tune BMP signal intensity in the ovary. Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), a marker of immature ovarian somatic cells, is identified as a novel transcriptional target of BMP4. Extensive re-organisation of the germ and somatic cell populations in the feto-neonatal ovary culminates in the formation of primordial follicles, which provide the basis for a female's future fertility. BMP growth factors play important roles at many stages of ovarian development and function. GREM1, an extracellular antagonist of BMP signalling, regulates the timing of primordial follicle formation in the mouse ovary, and mRNA levels of BMP4 decrease while those of BMP2 increase prior to follicle formation in the human fetal ovary. Expression of genes encoding BMP pathway components, BMP antagonists and markers of ovarian somatic cells were determined by quantitative (q)RT-PCR in human fetal ovaries (from 8 to 21 weeks gestation) and fetal ovary-derived somatic cell cultures. Ovarian expression of GREM1 protein was confirmed by immunoblotting. Primary human fetal ovarian somatic cell cultures were derived from disaggregated ovaries by differential adhesion and cultured in the presence of recombinant human BMP2 or BMP4, with or without the addition of GREM1 or GREM2. We demonstrate that the expression of BMP antagonists GREM1, GREM2 and CHRD  increases in the lead-up to primordial follicle formation in the human fetal ovary, and that the BMP pathway is active in cultured ovarian somatic cells. This leads to differential changes in the expression of a number of genes, some of which are further modulated by GREM1 and/or GREM2. The positive transcriptional regulation of LGR5 (a marker of less differentiated somatic cells) by BMP4 in vitro suggests that increasing levels of GREM1 and reduced levels of BMP4 as the ovary develops in vivo may act to reduce LGR5 levels and allow pre-granulosa cell differentiation. While we have demonstrated that markers of different somatic cell types are expressed in the cultured ovarian somatic cells, their proportions may not represent the same cells in the intact ovary which also contains germ cells. This study extends previous work identifying germ cells as targets of ovarian BMP signalling, and suggests BMPs may regulate the development of both germ and somatic cells in the developing ovary around the time of follicle formation. Not applicable. This work was supported by The UK Medical Research Council (Grant No.: G1100357 to RAA), and Medical Research Scotland (Grant No. 345FRG to AJC). The authors have no competing interests to declare. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

Mouse monoclonal antibodies against human c-Mpl and characterization for flow cytometry applications.

PubMed

Abbott, Christina; Huang, Guo; Ellison, Aaron R; Chen, Ching; Arora, Taruna; Szilvassy, Stephen J; Wei, Ping

2010-04-01

Mouse monoclonal antibodies (MAbs) against human c-Mpl, the cognate receptor for thrombopoietin (TPO), were generated using hybridoma technology and characterized by various assays to demonstrate their specificity and affinity. Two such MAbs, 1.6 and 1.75, were determined to be superior for flow cytometry studies and exhibited double-digit picomolar (pM) affinities to soluble human c-Mpl protein. Both MAbs specifically bound to cells engineered to overexpress human c-Mpl protein, immortalized human hematopoietic cell lines that express endogenous c-Mpl, primary human bone marrow and peripheral blood-derived CD34(+) cells, and purified human platelets. No binding was detected on cell lines that did not express c-Mpl. Receptor competition and siRNA knock-down studies further confirmed the specificity of antibodies 1.6 and 1.75 for human c-Mpl. In contrast to these newly generated MAbs, none of eight commercially available anti-c-Mpl antibodies tested were found to bind specifically to human c-Mpl and were thus shown to be unsuitable for flow cytometry studies. Monoclonal antibodies 1.6 and 1.75 will therefore be useful flow cytometry reagents to detect cell surface c-Mpl expression.

RANKL/RANK interaction promotes the growth of cervical cancer cells by strengthening the dialogue between cervical cancer cells and regulation of IL-8 secretion.

PubMed

Shang, Wen-Qing; Li, Hui; Liu, Li-Bing; Chang, Kai-Kai; Yu, Jia-Jun; Xie, Feng; Li, Ming-Qing; Yu, Jin-Jin

2015-12-01

Receptor activator for nuclear factor κB ligand (RANKL) is a member of the tumor necrosis factor (TNF) family. The interaction between RANKL and its receptor RANK plays an important role in the development and function of diverse tissues. However, the expression and role of RANKL in cervical cancer are still unknown. In the present study, we found that RANKL and RANK were highly co-expressed in cervical cancer. HeLa and SiHa cells secreted soluble RANKL (sRANKL), expressed member RANKL (mRANKL) and RANK. Recombinant human RANKL protein had no effect on the viability of HeLa and SiHa cells. Yet, blocking RANKL with an anti-human RANKL neutralizing antibody (α-RANKL) or recombinant human osteoprotegrin (OPG) protein resulted in the downregulation of Ki-67 and B-cell lymphoma 2 (Bcl-2) expression and an increase in Fas and Fas ligand (FasL) expression, as well as a high level of viability and a low level of apoptosis in the HeLa and SiHa cells. In addition, α-RANKL led to a decrease in IL-8 secretion. Recombinant human IL-8 protein reversed the effect of α-RANKL on the expression of proliferation- and apoptosis‑related molecules, and proliferation and apoptosis in the HeLa and SiHa cells. The present study suggests that a high level of mRANKL/RANK expression in cervical cancer lesions plays an important role in the rapid growth of cervical cancer cells possibly through strengthening the dialogue between cervical cancer cells and regulation of IL-8 secretion, which may be a possible target for cervical cancer therapy.

76 FR 51374 - Direct Discovery of HLA Associated Influenza Epitopes Isolated From Human Cells for Vaccine and...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-08-18

...] Direct Discovery of HLA Associated Influenza Epitopes Isolated From Human Cells for Vaccine and... (human leucocyte antigen) expressed by human cells. Initial studies will examine and characterize... human lung cell lines. There is a growing interest in developing universal vaccines for influenza by...

Examining FKBP5 mRNA expression in human iPSC-derived neural cells

PubMed Central

Lieberman, Richard; Kranzler, Henry R.; Levine, Eric S.; Covault, Jonathan

2016-01-01

In peripheral blood leukocytes, FKBP5 mRNA expression is upregulated following glucocorticoid receptor activation. The single nucleotide polymorphism rs1360780 in FKBP5 is associated with psychiatric illness and has functional molecular effects. However, examination of FKBP5 regulation has largely been limited to peripheral cells, which may not reflect regulation in neural cells. We used 27 human induced pluripotent stem cell lines (iPSCs) derived from 20 subjects to examine FKBP5 mRNA expression following GR activation. Following differentiation into forebrain-lineage neural cultures, cells were exposed to 1μM dexamethasone and mRNA expression of FKBP5 and NR3C1 analyzed. Results from the iPSC-derived neural cells were compared with those from 15 donor matched fibroblast lines. Following dexamethasone treatment, there was a 670% increase in FKBP5 expression in fibroblasts, mimicking findings in peripheral blood-derived cells, but only a 23% increase in iPSC-derived neural cultures. FKBP5 rs1360780 genotype did not affect the induction of FKBP5 mRNA in either fibroblasts or neural cells. These results suggest that iPSC-derived forebrain-lineage neurons may not be an optimal neural cell type in which to examine relationships between GR activation, FKBP5 expression, and genetic variation in human subjects. Further, FKBP5 induction following GR activation may differ between cell types derived from the same individual. PMID:27915167

Expression and regulation of the tumor suppressor, SEF, during folliculogenesis in humans and mice.

PubMed

Lutwak, Ela; Price, Christopher A; Abramovich, Sagit-Sela; Rabinovitz, Shiri; Granot, Irit; Dekel, Nava; Ron, Dina

2014-11-01

Similar expression to FGF (Sef or IL17-RD), is a tumor suppressor and an inhibitor of growth factors as well as of pro-inflammatory cytokine signaling. In this study, we examined the regulation of Sef expression by gonadotropins during ovarian folliculogenesis. In sexually immature mice, in situ hybridization (ISH) localized Sef gene expression to early developing oocytes and granulosa cells (GC) but not to theca cells. Sef was also expressed in mouse ovarian endothelial cells, in the fallopian tube epithelium as well as in adipose tissue venules. SEF protein expression, determined by immunohistochemistry (IHC), correlated well with Sef mRNA expression in GC, while differential expression was noticed in oocytes. High Sef mRNA but undetectable SEF protein levels were observed in the oocytes of primary/secondary follicles, while an inverse correlation was found in the oocytes of preantral and small antral follicles. Sef mRNA expression dropped after pregnant mare's serum gonadotropin (PMSG) administration, peaked at 6-8 h after human chorionic gonadotropin (hCG) treatment, and declined by 12 h after this treatment. ISH and IHC localized the changes to oocytes and mural GC following PMSG treatment, whereas Sef expression increased in mural GC and declined in granulosa-lutein cells upon hCG treatment. The ovarian expression of SEF was confirmed using human samples. ISH localized SEF transcripts to human GC of antral follicles but not to corpora lutea. Furthermore, SEF mRNA was detected in human GC recovered from preovulatory follicles. These results are the first to demonstrate SEF expression in a healthy ovary during folliculogenesis. Hormonal regulation of its expression suggests that SEF may be an important factor involved in intra-ovarian control mechanisms. © 2014 Society for Reproduction and Fertility.

Expression of bovine non-classical major histocompatibility complex class I proteins in mouse P815 and human K562 cells.

PubMed

Parasar, Parveen; Wilhelm, Amanda; Rutigliano, Heloisa M; Thomas, Aaron J; Teng, Lihong; Shi, Bi; Davis, William C; Suarez, Carlos E; New, Daniel D; White, Kenneth L; Davies, Christopher J

2016-08-01

Major histocompatibility complex class I (MHC-I) proteins can be expressed as cell surface or secreted proteins. To investigate whether bovine non-classical MHC-I proteins are expressed as cell surface or secreted proteins, and to assess the reactivity pattern of monoclonal antibodies with non-classical MHC-I isoforms, we expressed the MHC proteins in murine P815 and human K562 (MHC-I deficient) cells. Following antibiotic selection, stably transfected cell lines were stained with H1A or W6/32 antibodies to detect expression of the MHC-I proteins by flow cytometry. Two non-classical proteins (BoLA-NC1*00501 and BoLA-NC3*00101) were expressed on the cell surface in both cell lines. Surprisingly, the BoLA-NC4*00201 protein was expressed on the cell membrane of human K562 but not mouse P815 cells. Two non-classical proteins (BoLA-NC1*00401, which lacks a transmembrane domain, and BoLA-NC2*00102) did not exhibit cell surface expression. Nevertheless, Western blot analyses demonstrated expression of the MHC-I heavy chain in all transfected cell lines. Ammonium-sulfate precipitation of proteins from culture supernatants showed that BoLA-NC1*00401 was secreted and that all surface expressed proteins where shed from the cell membrane by the transfected cells. Interestingly, the surface expressed MHC-I proteins were present in culture supernatants at a much higher concentration than BoLA-NC1*00401. This comprehensive study shows that bovine non-classical MHC-I proteins BoLA-NC1*00501, BoLA-NC3*00101, and BoLA-NC4*00201 are expressed as surface isoforms with the latter reaching the cell membrane only in K562 cells. Furthermore, it demonstrated that BoLA-NC1*00401 is a secreted isoform and that significant quantities of membrane associated MHC-I proteins can be shed from the cell membrane. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

AMP-activated protein kinase activation mediates CCL3-induced cell migration and matrix metalloproteinase-2 expression in human chondrosarcoma

PubMed Central

2013-01-01

Chemokine (C-C motif) ligand 3 (CCL3), also known as macrophage inflammatory protein-1α, is a cytokine involved in inflammation and activation of polymorphonuclear leukocytes. CCL3 has been detected in infiltrating cells and tumor cells. Chondrosarcoma is a highly malignant tumor that causes distant metastasis. However, the effect of CCL3 on human chondrosarcoma metastasis is still unknown. Here, we found that CCL3 increased cellular migration and expression of matrix metalloproteinase (MMP)-2 in human chondrosarcoma cells. Pre-treatment of cells with the MMP-2 inhibitor or transfection with MMP-2 specific siRNA abolished CCL3-induced cell migration. CCL3 has been reported to exert its effects through activation of its specific receptor, CC chemokine receptor 5 (CCR5). The CCR5 and AMP-activated protein kinase (AMPK) inhibitor or siRNA also attenuated CCL3-upregulated cell motility and MMP-2 expression. CCL3-induced expression of MMP-2 and migration were also inhibited by specific inhibitors, and inactive mutants of AMPK, p38 mitogen activated protein kinase (p38 or p38-MAPK), and nuclear factor κB (NF-κB) cascades. On the other hand, CCL3 treatment demonstrably activated AMPK, p38, and NF-κB signaling pathways. Furthermore, the expression levels of CCL3, CCR5, and MMP-2 were correlated in human chondrosarcoma specimens. Taken together, our results indicate that CCL3 enhances the migratory ability of human chondrosarcoma cells by increasing MMP-2 expression via the CCR5, AMPK, p38, and NF-κB pathways. PMID:24047437

Vitex rotundifolia Fruit Extract Induces Apoptosis Through the Downregulation of ATF3-Mediated Bcl-2 Expression in Human Colorectal Cancer Cells.

PubMed

Song, Hun Min; Park, Gwang Hun; Koo, Jin Suk; Jeong, Hyung Jin; Jeong, Jin Boo

2017-01-01

Fruit from Vitex rotundifolia L. (VF) has been reported to initiate apoptosis in human colorectal cancer cells through the accumulation of reactive oxygen species. Since various regulatory factors are involved in the apoptotic pathway, further study of the potential mechanisms of VF associated with the induction of apoptosis may be important despite the fact that the molecular target of VF for apoptosis has already been elucidated. In this study, we showed a new potential mechanism for the relationship between VF-mediated ATF3 expression and apoptosis to better understand the apoptotic mechanism of VF in human colorectal cancer cells. VF reduced the cell viability and induced apoptosis in human colorectal cancer cells. VF treatment increased both the protein and mRNA level of ATF3 and upregulated ATF3 promoter activity. The cis-element responsible for ATF3 transcriptional activation by VF was CREB which is located between [Formula: see text]147 to [Formula: see text]85 of ATF3 promoter. Inhibitions of ERK1/2, p38, JNK and GSK3[Formula: see text] blocked VF-mediated ATF3 expression. ATF3 knockdown by ATF3 siRNA attenuated the cleavage of PARP by VF, while ATF3 overexpression increased VF-mediated cleaved PARP. ATF3 knockdown also attenuated VF-mediated cell viability and cell death. In addition, VF downregulated Bcl-2 expression at both protein and mRNA level. ATF3 knockdown by ATF3 siRNA blocked VF-mediated downregulation of Bcl-2. In conclusion, VF may activate ATF3 expression through transcriptional regulation and subsequently suppress Bcl-2 expression as an anti-apoptotic protein, which may result in the induction of apoptosis in human colorectal cancer cells.

Genetic engineering of human NK cells to express CXCR2 improves migration to renal cell carcinoma.

PubMed

Kremer, Veronika; Ligtenberg, Maarten A; Zendehdel, Rosa; Seitz, Christina; Duivenvoorden, Annet; Wennerberg, Erik; Colón, Eugenia; Scherman-Plogell, Ann-Helén; Lundqvist, Andreas

2017-09-19

Adoptive natural killer (NK) cell transfer is being increasingly used as cancer treatment. However, clinical responses have so far been limited to patients with hematological malignancies. A potential limiting factor in patients with solid tumors is defective homing of the infused NK cells to the tumor site. Chemokines regulate the migration of leukocytes expressing corresponding chemokine receptors. Various solid tumors, including renal cell carcinoma (RCC), readily secrete ligands for the chemokine receptor CXCR2. We hypothesize that infusion of NK cells expressing high levels of the CXCR2 chemokine receptor will result in increased influx of the transferred NK cells into tumors, and improved clinical outcome in patients with cancer. Blood and tumor biopsies from 14 primary RCC patients were assessed by flow cytometry and chemokine analysis. Primary NK cells were transduced with human CXCR2 using a retroviral system. CXCR2 receptor functionality was determined by Calcium flux and NK cell migration was evaluated in transwell assays. We detected higher concentrations of CXCR2 ligands in tumors compared with plasma of RCC patients. In addition, CXCL5 levels correlated with the intratumoral infiltration of CXCR2-positive NK cells. However, tumor-infiltrating NK cells from RCC patients expressed lower CXCR2 compared with peripheral blood NK cells. Moreover, healthy donor NK cells rapidly lost their CXCR2 expression upon in vitro culture and expansion. Genetic modification of human primary NK cells to re-express CXCR2 improved their ability to specifically migrate along a chemokine gradient of recombinant CXCR2 ligands or RCC tumor supernatants compared with controls. The enhanced trafficking resulted in increased killing of target cells. In addition, while their functionality remained unchanged compared with control NK cells, CXCR2-transduced NK cells obtained increased adhesion properties and formed more conjugates with target cells. To increase the success of NK cell-based therapies of solid tumors, it is of great importance to promote their homing to the tumor site. In this study, we show that stable engineering of human primary NK cells to express a chemokine receptor thereby enhancing their migration is a promising strategy to improve anti-tumor responses following adoptive transfer of NK cells.

Occupational levels of radiation exposure induce surface expression of interleukin-2 receptors in stimulated human peripheral blood lymphocytes.

PubMed

Xu, Y; Greenstock, C L; Trivedi, A; Mitchel, R E

1996-05-01

Interleukin-2 (IL-2) is a cytokine responsible for a variety of immune and non-immune stimulatory and regulatory functions, including the activation and stimulation of cytotoxic cells able to recognize and kill human tumour cells and T-cell proliferation and differentiation. We show that low doses of radiation, in the range commonly received by atomic radiation workers or as a result of minor medical diagnostic procedures (0.25 to 10 mGy), stimulate the expression of IL-2 receptors (IL-2R) on the surface of peripheral blood lymphocytes (PBL) taken from normal human donors. This stimulated surface expression after in vitro irradiation is an indirect effect, resulting from the secretion into the medium of a soluble factor from the irradiated cells. This factor can also stimulate IL-2R surface expression in unirradiated cells. Consequently, radiation stimulation of IL-2R expression in a large population of PBL shows a triggered-type response rather than being proportional to dose. These results demonstrate that normal human cells can respond to doses of radiation in the range of common occupational or medical exposures. The data also demonstrate a possible defence mechanism against environmental stress by which a radiation-exposed cell can use an indirect signalling mechanism to communicate with and influence the biological processes in an unexposed cell.

Expression and functional roles of estrogen receptor GPR30 in human intervertebral disc.

PubMed

Wei, Aiqun; Shen, Bojiang; Williams, Lisa A; Bhargav, Divya; Yan, Feng; Chong, Beng H; Diwan, Ashish D

2016-04-01

Estrogen withdrawal, a characteristic of female aging, is associated with age-related intervertebral disc (IVD) degeneration. The function of estrogen is mediated by two classic nuclear receptors, estrogen receptor (ER)-α and -β, and a membrane bound G-protein-coupled receptor 30 (GPR30). To date, the expression and function of GPR30 in human spine is poorly understood. This study aimed to evaluate GPR30 expression in IVD, and its role in estrogen-related regulation of proliferation and apoptosis of disc nucleus pulposus (NP) cells. GPR30 expression was examined in 30 human adult NP and 9 fetal IVD. Results showed that GPR30 was expressed in NP cells at both mRNA and protein levels. In human fetal IVD, GPR30 protein was expressed in the NP at 12-14 weeks gestation, but was undetectable at 8-11 weeks. The effect of 17β-estradiol (E2) on GPR30-mediated proliferation and interleukin-1β (IL-1β)-induced apoptosis of NP cells was investigated. Cultured NP cells were treated with or without E2, GPR30 antagonist G36, and ER antagonist ICI 182,780. NP cell viability was tested by MTS assay. Apoptosis was determined by flow cytometry using fluorescence labeled annexin-V, TUNEL assay and immumnocytochemical staining of activated caspase-3. E2 enhanced cell proliferation and prevented IL-1β-induced cell death, but the effect was partially blocked by G36 and completely abrogated by a combination of ICI 182,780 and G36. This study demonstrates that GPR30 is expressed in human IVD to transmit signals triggering E2-induced NP cell proliferation and protecting against IL-1β-induced apoptosis. The effects of E2 on NP cells require both GPR30 and classic estrogen receptors. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Expression of stanniocalcin 1 in thyroid side population cells and thyroid cancer cells.

PubMed

Hayase, Suguru; Sasaki, Yoshihito; Matsubara, Tsutomu; Seo, Daekwan; Miyakoshi, Masaaki; Murata, Tsubasa; Ozaki, Takashi; Kakudo, Kennichi; Kumamoto, Kensuke; Ylaya, Kris; Cheng, Sheue-yann; Thorgeirsson, Snorri S; Hewitt, Stephen M; Ward, Jerrold M; Kimura, Shioko

2015-04-01

Mouse thyroid side population (SP) cells consist of a minor population of mouse thyroid cells that may have multipotent thyroid stem cell characteristics. However the nature of thyroid SP cells remains elusive, particularly in relation to thyroid cancer. Stanniocalcin (STC) 1 and 2 are secreted glycoproteins known to regulate serum calcium and phosphate homeostasis. In recent years, the relationship of STC1/2 expression to cancer has been described in various tissues. Microarray analysis was carried out to determine genes up- and down-regulated in thyroid SP cells as compared with non-SP cells. Among genes up-regulated, stanniocalcin 1 (STC1) was chosen for study because of its expression in various thyroid cells by Western blotting and immunohistochemistry. Gene expression analysis revealed that genes known to be highly expressed in cancer cells and/or involved in cancer invasion/metastasis were markedly up-regulated in SP cells from both intact as well as partial thyroidectomized thyroids. Among these genes, expression of STC1 was found in five human thyroid carcinoma-derived cell lines as revealed by analysis of mRNA and protein, and its expression was inversely correlated with the differentiation status of the cells. Immunohistochemical analysis demonstrated higher expression of STC1 in the thyroid tumor cell line and thyroid tumor tissues from humans and mice. These results suggest that SP cells contain a population of cells that express genes also highly expressed in cancer cells including Stc1, which warrants further study on the role of SP cells and/or STC1 expression in thyroid cancer.

Expression of Stanniocalcin 1 in Thyroid Side Population Cells and Thyroid Cancer Cells

PubMed Central

Hayase, Suguru; Sasaki, Yoshihito; Matsubara, Tsutomu; Seo, Daekwan; Miyakoshi, Masaaki; Murata, Tsubasa; Ozaki, Takashi; Kakudo, Kennichi; Kumamoto, Kensuke; Ylaya, Kris; Cheng, Sheue-yann; Thorgeirsson, Snorri S.; Hewitt, Stephen M.; Ward, Jerrold M.

2015-01-01

Background: Mouse thyroid side population (SP) cells consist of a minor population of mouse thyroid cells that may have multipotent thyroid stem cell characteristics. However the nature of thyroid SP cells remains elusive, particularly in relation to thyroid cancer. Stanniocalcin (STC) 1 and 2 are secreted glycoproteins known to regulate serum calcium and phosphate homeostasis. In recent years, the relationship of STC1/2 expression to cancer has been described in various tissues. Method: Microarray analysis was carried out to determine genes up- and down-regulated in thyroid SP cells as compared with non-SP cells. Among genes up-regulated, stanniocalcin 1 (STC1) was chosen for study because of its expression in various thyroid cells by Western blotting and immunohistochemistry. Results: Gene expression analysis revealed that genes known to be highly expressed in cancer cells and/or involved in cancer invasion/metastasis were markedly up-regulated in SP cells from both intact as well as partial thyroidectomized thyroids. Among these genes, expression of STC1 was found in five human thyroid carcinoma–derived cell lines as revealed by analysis of mRNA and protein, and its expression was inversely correlated with the differentiation status of the cells. Immunohistochemical analysis demonstrated higher expression of STC1 in the thyroid tumor cell line and thyroid tumor tissues from humans and mice. Conclusion: These results suggest that SP cells contain a population of cells that express genes also highly expressed in cancer cells including Stc1, which warrants further study on the role of SP cells and/or STC1 expression in thyroid cancer. PMID:25647164

Visualisation and quantification of intracellular interactions of Neisseria meningitidis and human α-actinin by confocal imaging.

PubMed

Murillo, Isabel; Virji, Mumtaz

2010-10-24

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an effective invasin for human epithelial and endothelial cells. We have identified endothelial surface-located integrins as major receptors for Opc, a process which requires Opc to first bind to integrin ligands such as vitronectin and via these to the cell-expressed receptors(1). This process leads to bacterial invasion of endothelial cells(2). More recently, we observed an interaction of Opc with a 100 kDa protein found in whole cell lysates of human cells(3). We initially observed this interaction when host cell proteins separated by electrophoresis and blotted on to nitrocellulose were overlaid with Opc-expressing Nm. The interaction was direct and did not involve intermediate molecules. By mass spectrometry, we established the identity of the protein as α-actinin. As no surface expressed α-actinin was found on any of the eight cell lines examined, and as Opc interactions with endothelial cells in the presence of serum lead to bacterial entry into the target cells, we examined the possibility of the two proteins interacting intracellularly. For this, cultured human brain microvascular endothelial cells (HBMECs) were infected with Opc-expressing Nm for extended periods and the locations of internalised bacteria and α-actinin were examined by confocal microscopy. We observed time-dependent increase in colocalisation of Nm with the cytoskeletal protein, which was considerable after an eight hour period of bacterial internalisation. In addition, the use of quantitative imaging software enabled us to obtain a relative measure of the colocalisation of Nm with α-actinin and other cytoskeletal proteins. Here we present a protocol for visualisation and quantification of the colocalisation of the bacterium with intracellular proteins after bacterial entry into human endothelial cells, although the procedure is also applicable to human epithelial cells.

Functional TRPV2 and TRPV4 channels in human cardiac c-kit(+) progenitor cells.

PubMed

Che, Hui; Xiao, Guo-Sheng; Sun, Hai-Ying; Wang, Yan; Li, Gui-Rong

2016-06-01

The cellular physiology and biology of human cardiac c-kit(+) progenitor cells has not been extensively characterized and remains an area of active research. This study investigates the functional expression of transient receptor potential vanilloid (TRPV) and possible roles for this ion channel in regulating proliferation and migration of human cardiac c-kit(+) progenitor cells. We found that genes coding for TRPV2 and TRPV4 channels and their proteins are significantly expressed in human c-kit(+) cardiac stem cells. Probenecid, an activator of TRPV2, induced an increase in intracellular Ca(2+) (Ca(2+) i ), an effect that may be attenuated or abolished by the TRPV2 blocker ruthenium red. The TRPV4 channel activator 4α-phorbol 12-13-dicaprinate induced Ca(2+) i oscillations, which can be inhibited by the TRPV4 blocker RN-1734. The alteration of Ca(2+) i by probenecid or 4α-phorbol 12-13-dicprinate was dramatically inhibited in cells infected with TRPV2 short hairpin RNA (shRNA) or TRPV4 shRNA. Silencing TRPV2, but not TRPV4, significantly reduced cell proliferation by arresting cells at the G0/G1 boundary of the cell cycle. Cell migration was reduced by silencing TRPV2 or TRPV4. Western blot revealed that silencing TRPV2 decreased expression of cyclin D1, cyclin E, pERK1/2 and pAkt, whereas silencing TRPV4 only reduced pAkt expression. Our results demonstrate for the first time that functional TRPV2 and TRPV4 channels are abundantly expressed in human cardiac c-kit(+) progenitor cells. TRPV2 channels, but not TRPV4 channels, participate in regulating cell cycle progression; moreover, both TRPV2 and TRPV4 are involved in migration of human cardiac c-kit(+) progenitor cells. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

The production of glial cell line-derived neurotrophic factor by human sertoli cells is substantially reduced in sertoli cell-only testes.

PubMed

Singh, D; Paduch, D A; Schlegel, P N; Orwig, K E; Mielnik, A; Bolyakov, A; Wright, W W

2017-05-01

Do human Sertoli cells in testes that exhibit the Sertoli cell-only (SCO) phenotype produce substantially less glial cell line-derived neurotrophic factor (GDNF) than Sertoli cells in normal testes? In human SCO testes, both the amounts of GDNF mRNA per testis and the concentration of GDNF protein per Sertoli cell are markedly reduced as compared to normal testes. In vivo, GDNF is required to sustain the numbers and function of mouse spermatogonial stem cells (SSCs) and their immediate progeny, transit-amplifying progenitor spermatogonia. GDNF is expressed in the human testis, and the ligand-binding domain of the GDNF receptor, GFRA1, has been detected on human SSCs. The numbers and/or function of these stem cells are markedly reduced in some infertile men, resulting in the SCO histological phenotype. We determined the numbers of human spermatogonia per mm2 of seminiferous tubule surface that express GFRA1 and/or UCHL1, another marker of human SSCs. We measured GFRA1 mRNA expression in order to document the reduced numbers and/or function of SSCs in SCO testes. We quantified GDNF mRNA in testes of humans and mice, a species with GDNF-dependent SSCs. We also compared GDNF mRNA expression in human testes with normal spermatogenesis to that in testes exhibiting the SCO phenotype. As controls, we also measured transcripts encoding two other Sertoli cell products, kit ligand (KITL) and clusterin (CLU). Finally, we compared the amounts of GDNF per Sertoli cell in normal and SCO testes. Normal human testes were obtained from beating heart organ donors. Biopsies of testes from men who were infertile due to maturation arrest or the SCO phenotype were obtained as part of standard care during micro-testicular surgical sperm extraction. Cells expressing GFRA1, UCHL1 or both on whole mounts of normal human seminiferous tubules were identified by immunohistochemistry and confocal microscopy and their numbers were determined by image analysis. Human GDNF mRNA and GFRA1 mRNA were quantified by use of digital PCR and Taqman primers. Transcripts encoding mouse GDNF and human KITL, CLU and 18 S rRNA, used for normalization of data, were quantified by use of real-time PCR and Taqman primers. Finally, we used two independent methods, flow cytometric analysis of single cells and ELISA assays of homogenates of whole testis biopsies, to compare amounts of GDNF per Sertoli cell in normal and SCO testes. Normal human testes contain a large population of SSCs that express GFRA1, the ligand-binding domain of the GDNF receptor. In human SCO testes, GFRA1 mRNA was detected but at markedly reduced levels. Expression of GDNF mRNA and the amount of GDNF protein per Sertoli cell were also significantly reduced in SCO testes. These results were observed in multiple, independent samples, and the reduced amount of GDNF in Sertoli cells of SCO testes was demonstrated using two different analytical approaches. N/A. There currently are no approved protocols for the in vivo manipulation of human testis GDNF concentrations. Thus, while our data suggest that insufficient GDNF may be the proximal cause of some cases of human male infertility, our results are correlative in nature. We propose that insufficient GDNF expression may contribute to the infertility of some men with an SCO testicular phenotype. If their testes contain some SSCs, an approach that increases their testicular GDNF concentrations might expand stem cell numbers and possibly sperm production. This research was funded by the Eunice Kennedy Shriver National Institute for Child Health and Human Development, National Centers for Translational Research in Reproduction and Infertility Program (NCTRI) Grant 1R01HD074542-04, as well as grants R01 HD076412-02 and P01 HD075795-02 and the U.S.-Israel Binational Science Foundation. Support for this research was also provided by NIH P50 HD076210, the Robert Dow Foundation, the Frederick & Theresa Dow Wallace Fund of the New York Community Trust and the Brady Urological Foundation. There are no competing interests. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Microarray analysis of human milk cells: persistent high expression of osteopontin during the lactation period

PubMed Central

NAGATOMO, T; OHGA, S; TAKADA, H; NOMURA, A; HIKINO, S; IMURA, M; OHSHIMA, K; HARA, T

2004-01-01

To continue the search for immunological roles of breast milk, cDNA microarray analysis on cytokines and growth factors was performed for human milk cells. Among the 240 cytokine-related genes, osteopontin (OPN) gene ranked top of the expression. Real-time PCR revealed that the OPN mRNA levels in colostrum cells were approximately 100 times higher than those in PHA-stimulated peripheral blood mononuclear cells (PBMNCs), and 10 000 times higher than those in PB CD14+ cells. The median levels of OPN mRNA in early milk or mature milk cells were more than three times higher than those in colostrum cells. Western blot analysis of human milk showed appreciable expression of full-length and short form proteins of OPN. The concentrations of full-length OPN in early milk or mature milk whey continued to be higher than those in colostrum whey and plasma as assessed by ELISA. The early milk (3–7 days postpartum) contained the highest concentrations of OPN protein, while the late mature milk cells (1 years postpartum) had the highest expression of OPN mRNA of all the lactating periods. The results of immunohistochemical and immunocytochemical staining indicated that OPN-producing epithelial cells and macrophages are found in actively lactating mammary glands. These results suggest that the persistently and extraordinarily high expression of OPN in human milk cells plays a potential role in the immunological development of breast-fed infants. PMID:15373904

Upregulation of Glucose Uptake and Hexokinase Activity of Primary Human CD4+ T Cells in Response to Infection with HIV-1

PubMed Central

Kavanagh Williamson, Maia; Coombes, Naomi; Juszczak, Florian; Athanasopoulos, Marios; Khan, Mariam B.; Eykyn, Thomas R.; Srenathan, Ushani; Dias Zeidler, Julianna; Huthoff, Hendrik

2018-01-01

Infection of primary CD4+ T cells with HIV-1 coincides with an increase in glycolysis. We investigated the expression of glucose transporters (GLUT) and glycolytic enzymes in human CD4+ T cells in response to infection with HIV-1. We demonstrate the co-expression of GLUT1, GLUT3, GLUT4, and GLUT6 in human CD4+ T cells after activation, and their concerted overexpression in HIV-1 infected cells. The investigation of glycolytic enzymes demonstrated activation-dependent expression of hexokinases HK1 and HK2 in human CD4+ T cells, and a highly significant increase in cellular hexokinase enzyme activity in response to infection with HIV-1. HIV-1 infected CD4+ T cells showed a marked increase in expression of HK1, as well as the functionally related voltage-dependent anion channel (VDAC) protein, but not HK2. The elevation of GLUT, HK1, and VDAC expression in HIV-1 infected cells mirrored replication kinetics and was dependent on virus replication, as evidenced by the use of reverse transcription inhibitors. Finally, we demonstrated that the upregulation of HK1 in HIV-1 infected CD4+ T cells is independent of the viral accessory proteins Vpu, Vif, Nef, and Vpr. Though these data are consistent with HIV-1 dependency on CD4+ T cell glucose metabolism, a cellular response mechanism to infection cannot be ruled out. PMID:29518929

Expression and localization of c-ros oncogene along the human excurrent duct.

PubMed

Légaré, Christine; Sullivan, Robert

2004-09-01

Compared to other animals, the anatomy of the human epididymis appears unusual. The caput epididymis is composed mostly of efferent ducts with an undefined initial segment. The present study investigates the regionalization of c-ros in human epididymis by real-time quantitative RT-PCR, in situ hybridization and immunohistochemistry studies. C-ros gene encodes a receptor-type protein tyrosine kinase that is expressed in adult mice exclusively in the epithelial cells of the initial segment of the epididymis. Transgenic mice targeted for the c-ros gene lack the initial segment of the epididymis and are infertile. Real-time PCR analysis showed that c-ros mRNA is expressed all along the human epididymis with the exception of the proximal caput epididymidis, where c-ros transcript was undetectable. In situ hydridization revealed that c-ros transcript was strongly expressed by principal cells and to a lower level by basal cells. Immunohistochemical studies showed that c-ros protein distribution was similar to the transcript. These results showed that c-ros expression in the human epididymis differs from that in mice suggesting that the unusual morphology of the human epididymis may reflect species differences in gene expression along the excurrent duct.

Expression and regulation of neurotrophins in the nondegenerate and degenerate human intervertebral disc

PubMed Central

Purmessur, Devina; Freemont, Anthony J; Hoyland, Judith A

2008-01-01

Introduction The neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) have been identified in the human intervertebral disc (IVD) and have been implicated in the mechanisms associated with nerve ingrowth and nociception in degeneration of the IVD. The aim of the current study was to investigate an association between neurotrophin expression in the IVD and the severity of disc degeneration, including the effect of disc-related proinflammatory cytokines on neurotrophin and neuropeptide expression in cells derived from the human IVD. Methods Immunohistochemical analysis was performed to examine the expression of NGF, BDNF and their high-affinity receptors Trk-A and Trk-B in human IVD samples, divided into three categories: non-degenerate, moderate degeneration and severe degeneration. In order to study the effect of disc-related cytokines on neurotrophin/neuropeptide gene expression, nucleus pulposus cells derived from non-degenerate and degenerate IVD samples were seeded in alginate and were stimulated with either IL-1β or TNFα for 48 hours. RNA was extracted, cDNA was synthesised and quantitative real-time PCR was performed to examine the expression of NGF, BDNF and substance P. Results Immunohistochemistry showed expression of NGF and BDNF in the native chondrocyte-like cells in all regions of the IVD and in all grades of degeneration. Interestingly only BDNF significantly increased with the severity of degeneration (P < 0.05). Similar expression was observed for Trk-A and Trk-B, although no association with disease severity was demonstrated. In cultured human nucleus pulposus cells, stimulation with IL-1β led to significant increases in NGF and BDNF gene expression (P < 0.05). Treatment with TNFα was associated with an upregulation of substance P expression only. Conclusion Our findings show that both the annulus fibrosus and nucleus pulposus cells of the IVD express the neurotrophins NGF and BDNF, factors that may influence and enhance innervation and pain in the degenerate IVD. Expression of Trk-A and Trk-B by cells of the nondegenerate and degenerate IVD suggests an autocrine role for neurotrophins in regulation of disc cell biology. Furthermore, modulation of neurotrophin expression by IL-1β and modulation of substance P expression by TNFα, coupled with their increased expression in the degenerate IVD, highlights novel roles for these cytokines in regulating nerve ingrowth in the degenerate IVD and associated back pain. PMID:18727839

Loss of intercellular adhesion activates a transition from low- to high-grade human squamous cell carcinoma.

PubMed

Margulis, Alexander; Zhang, Weitian; Alt-Holland, Addy; Pawagi, Sujata; Prabhu, Padmaja; Cao, Jian; Zucker, Stanley; Pfeiffer, Laurence; Garfield, Jacqueline; Fusenig, Norbert E; Garlick, Jonathan A

2006-02-15

The relationship between loss of intercellular adhesion and the biologic properties of human squamous cell carcinoma is not well understood. We investigated how abrogation of E-cadherin-mediated adhesion influenced the behavior and phenotype of squamous cell carcinoma in 3D human tissues. Cell-cell adhesion was disrupted in early-stage epithelial tumor cells (HaCaT-II-4) through expression of a dominant-negative form of E-cadherin (H-2Kd-Ecad). Three-dimensional human tissue constructs harboring either H-2Kd-Ecad-expressing or control II-4 cells (pBabe, H-2Kd-EcadDeltaC25) were cultured at an air-liquid interface for 8 days and transplanted to nude mice; tumor phenotype was analyzed 2 days and 2 and 4 weeks later. H-2Kd-Ecad-expressing tumors demonstrated a switch to a high-grade aggressive tumor phenotype characterized by poorly differentiated tumor cells that infiltrated throughout the stroma. This high-grade carcinoma revealed elevated cell proliferation in a random pattern, loss of keratin 1 and diffuse deposition of laminin 5 gamma2 chain. When II-4 cell variants were seeded into type I collagen gels as an in vitro assay for cell migration, we found that only E-cadherin-deficient cells detached, migrated as single cells and expressed N-cadherin. Function-blocking studies demonstrated that this migration was matrix metalloproteinase-dependent, as GM-6001 and TIMP-2, but not TIMP-1, could block migration. Gene expression profiles revealed that E-cadherin-deficient II-4 cells demonstrated increased expression of proteases and cell-cell and cell-matrix proteins. These findings showed that loss of E-cadherin-mediated adhesion plays a causal role in the transition from low- to high-grade squamous cell carcinomas and that the absence of E-cadherin is an important prognostic marker in the progression of this disease.

[Lentivirus-mediated shRNA silencing of LAMP2A inhibits the proliferation of multiple myeloma cells].

PubMed

Li, Lixuan; Li, Jia

2015-05-01

To study the effects of lentivirus-mediated short hairpin RNA (shRNA) silencing of lysosome-associated membrane protein type 2A (LAMP2A) expression on the proliferation of multiple myeloma cells. The constructed shRNA lentiviral vector was applied to infect human multiple myeloma cell line MM.1S, and stable expression cell line was obtained by puromycin screening. Western blotting was used to verify the inhibitory effect on LAMP2A protein expression. MTT assay was conducted to detect the effect of knocked-down LAMP2A on MM.1S cell proliferation, and the anti-tumor potency of suberoylanilide hydroxamic acid (SAHA) against the obtained MM.1S LAMP2A(shRNA) stable cell line. Lactate assay was performed to observe the impact of low LAMP2A expression on cell glycolysis. The stable cell line with low LAMP2A expression were obtained with the constructed human LAMP2A-shRNA lentiviral vector. Down-regulation of LAMP2A expression significantly inhibited MM.1S cell proliferation and enhanced the anti-tumor activity of SAHA. Interestingly, decreased LAMP2A expression also inhibited MM.1S cell lactic acid secretion. Down-regulation of LAMP2A expression could inhibit cell proliferation in multiple myeloma cells.

Establishment and characterization of five immortalized human scalp dermal papilla cell lines.

PubMed

Kwack, Mi Hee; Yang, Jung Min; Won, Gong Hee; Kim, Moon Kyu; Kim, Jung Chul; Sung, Young Kwan

2018-02-05

Dermal papilla (DP) regulates the growth and cycling of hair follicles. Cultured DP cells are useful for the study of their role in relation to hair growth and regeneration. However, cultivation of human DP cells is tedious and difficult. In addition, cultured DP cells possess a relatively short replicative life span, requiring immortalized human DP cell lines. We previously established an immortalized human DP cell line, SV40T-hTERT-DPC, by introducing human telomerase reverse transcriptase (hTERT) gene into the transformed cell line, SV40T-DPC. In this study, we co-transfected the simian virus 40 large T antigen (SV40T-Ag) and hTERT into DP cells from scalp hair follicles from a male with androgenetic alopecia and established five immortalized DP cell lines and named KNU-101, KNU-102, KNU-103, KNU-201 and KNU-202. We then evaluated tumorigenicity, expression of DP markers, responses to androgen, Wnt3a and BMP4, and expression of DP signature genes. These cell lines displayed early passage morphology and maintained responses to androgen, Wnt and BMP. Furthermore, these cell lines expressed DP markers and DP signature genes. KNU cell lines established in this study are potentially useful sources for hair research. Copyright © 2018 Elsevier Inc. All rights reserved.

Human somatic cells subjected to genetic induction with six germ line-related factors display meiotic germ cell-like features

PubMed Central

Medrano, Jose V.; Martínez-Arroyo, Ana M.; Míguez, Jose M.; Moreno, Inmaculada; Martínez, Sebastián; Quiñonero, Alicia; Díaz-Gimeno, Patricia; Marqués-Marí, Ana I.; Pellicer, Antonio; Remohí, Jose; Simón, Carlos

2016-01-01

The in vitro derivation of human germ cells has attracted interest in the last years, but their direct conversion from human somatic cells has not yet been reported. Here we tested the ability of human male somatic cells to directly convert into a meiotic germ cell-like phenotype by inducing them with a combination of selected key germ cell developmental factors. We started with a pool of 12 candidates that were reduced to 6, demonstrating that ectopic expression of the germ line-related genes PRDM1, PRDM14, LIN28A, DAZL, VASA and SYCP3 induced direct conversion of somatic cells (hFSK (46, XY), and hMSC (46, XY)) into a germ cell-like phenotype in vitro. Induced germ cell-like cells showed a marked switch in their transcriptomic profile and expressed several post-meiotic germ line related markers, showed meiotic progression, evidence of epigenetic reprogramming, and approximately 1% were able to complete meiosis as demonstrated by their haploid status and the expression of several post-meiotic markers. Furthermore, xenotransplantation assays demonstrated that a subset of induced cells properly colonize the spermatogonial niche. Knowledge obtained from this work can be used to create in vitro models to study gamete-related diseases in humans. PMID:27112843

Characterization of human myoblast differentiation for tissue-engineering purposes by quantitative gene expression analysis.

PubMed

Stern-Straeter, Jens; Bonaterra, Gabriel Alejandro; Kassner, Stefan S; Zügel, Stefanie; Hörmann, Karl; Kinscherf, Ralf; Goessler, Ulrich Reinhart

2011-08-01

Tissue engineering of skeletal muscle is an encouraging possibility for the treatment of muscle loss through the creation of functional muscle tissue in vitro from human stem cells. Currently, the preferred stem cells are primary, non-immunogenic satellite cells ( = myoblasts). The objective of this study was to determine the expression patterns of myogenic markers within the human satellite cell population during their differentiation into multinucleated myotubes for an accurate characterization of stem cell behaviour. Satellite cells were incubated (for 1, 4, 8, 12 or 16 days) with a culture medium containing either a low [ = differentiation medium (DM)] or high [ = growth medium (GM)] concentration of growth factors. Furthermore, we performed a quantitative gene expression analysis of well-defined differentiation makers: myogenic factor 5 (MYF5), myogenin (MYOG), skeletal muscle αactin1 (ACTA1), embryonic (MYH3), perinatal (MYH8) and adult skeletal muscle myosin heavy chain (MYH1). Additionally, the fusion indices of forming myotubes of MYH1, MYH8 and ACTA1 were calculated. We show that satellite cells incubated with DM expressed multiple characteriztic features of mature skeletal muscles, verified by time-dependent upregulation of MYOG, MYH1, MYH3, MYH8 and ACTA1. However, satellite cells incubated with GM did not reveal all morphological aspects of muscle differentiation. Immunocytochemical investigations with antibodies directed against the differentiation markers showed correlations between the gene expression and differentiation. Our data provide information about time-dependent gene expression of differentiation markers in human satellite cells, which can be used for maturation analyses in skeletal muscle tissue-engineering applications. Copyright © 2011 John Wiley & Sons, Ltd.

YKL-40 is differentially expressed in human embryonic stem cells and in cell progeny of the three germ layers.

PubMed

Brøchner, Christian B; Johansen, Julia S; Larsen, Lars A; Bak, Mads; Mikkelsen, Hanne B; Byskov, Anne Grete; Andersen, Claus Yding; Møllgård, Kjeld

2012-03-01

The secreted glycoprotein YKL-40 participates in cell differentiation, inflammation, and cancer progression. High YKL-40 expression is reported during early human development, but its functions are unknown. Six human embryonic stem cell (hESC) lines were cultured in an atmosphere of low or high oxygen tension, in culture medium with or without basic fibroblast growth factor, and on feeder layers comprising mouse embryonic fibroblasts or human foreskin fibroblasts to evaluate whether hESCs and their progeny produced YKL-40 and to characterize YKL-40 expression during differentiation. Secreted YKL-40 protein and YKL-40 mRNA expression were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative RT-PCR. Serial-sectioned colonies were stained for YKL-40 protein and for pluripotent hESC (OCT4, NANOG) and germ layer (HNF-3β, PDX1, CD34, p63, nestin, PAX6) markers. Double-labeling showed YKL-40 expression in OCT4-positive hESCs, PAX6-positive neuroectodermal cells, and HNF-3β-positive endodermal cells. The differentiating progeny showed strong YKL-40 expression. Abrupt transition between YKL-40 and OCT4-positive hESCs and YKL-40-positive ecto- and neuroectodermal lineages was observed within the same epithelial-like layer. YKL-40-positive cells within deeper layers lacked contact with OCT4-positive cells. YKL-40 may be important in initial cell differentiation from hESCs toward ectoderm and neuroectoderm, with retained epithelial morphology, whereas later differentiation into endoderm and mesoderm involves a transition into the deeper layers of the colony.

KIT Signaling Promotes Growth of Colon Xenograft Tumors in Mice and Is Up-Regulated in a Subset of Human Colon Cancers.

PubMed

Chen, Evan C; Karl, Taylor A; Kalisky, Tomer; Gupta, Santosh K; O'Brien, Catherine A; Longacre, Teri A; van de Rijn, Matt; Quake, Stephen R; Clarke, Michael F; Rothenberg, Michael E

2015-09-01

Receptor tyrosine kinase (RTK) inhibitors have advanced colon cancer treatment. We investigated the role of the RTK KIT in development of human colon cancer. An array of 137 patient-derived colon tumors and their associated xenografts were analyzed by immunohistochemistry to measure levels of KIT and its ligand KITLG. KIT and/or KITLG was stably knocked down by expression of small hairpin RNAs from lentiviral vectors in DLD1, HT29, LS174T, and COLO320 DM colon cancer cell lines, and in UM-COLON#8 and POP77 xenografts; cells transduced with only vector were used as controls. Cells were analyzed by real-time quantitative reverse transcription polymerase chain reaction, single-cell gene expression analysis, flow cytometry, and immunohistochemical, immunoblot, and functional assays. Xenograft tumors were grown from control and KIT-knockdown DLD1 and UM-COLON#8 cells in immunocompromised mice and compared. Some mice were given the RTK inhibitor imatinib after injection of cancer cells; tumor growth was measured based on bioluminescence. We assessed tumorigenicity using limiting dilution analysis. KIT and KITLG were expressed heterogeneously by a subset of human colon tumors. Knockdown of KIT decreased proliferation of colon cancer cell lines and growth of xenograft tumors in mice compared with control cells. KIT knockdown cells had increased expression of enterocyte markers, decreased expression of cycling genes, and, unexpectedly, increased expression of LGR5 associated genes. No activating mutations in KIT were detected in DLD1, POP77, or UM-COLON#8 cells. However, KITLG-knockdown DLD1 cells formed smaller xenograft tumors than control cells. Gene expression analysis of single CD44(+) cells indicated that KIT can promote growth via KITLG autocrine and/or paracrine signaling. Imatinib inhibited growth of KIT(+) colon cancer organoids in culture and growth of xenograft tumors in mice. Cancer cells with endogenous KIT expression were more tumorigenic in mice. KIT and KITLG are expressed by a subset of human colon tumors. KIT signaling promotes growth of colon cancer cells and organoids in culture and xenograft tumors in mice via its ligand, KITLG, in an autocrine or paracrine manner. Patients with KIT-expressing colon tumors can benefit from KIT RTK inhibitors. Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.

KIT Signaling Promotes Growth of Colon Xenograft Tumors in Mice and is Upregulated in a Subset of Human Colon Cancers

PubMed Central

Chen, Evan C.; Karl, Taylor A.; Kalisky, Tomer; Gupta, Santosh K.; O’Brien, Catherine A.; Longacre, Teri A.; van de Rijn, Matt; Quake, Stephen R.; Clarke, Michael F.; Rothenberg, Michael E.

2015-01-01

Background & Aims Receptor tyrosine kinase (RTK) inhibitors have advanced colon cancer treatment. We investigated the role of the RTK KIT in development of human colon cancer. Methods An array of 137 patient-derived colon tumors and their associated xenografts were analyzed by immunohistochemistry to measure levels of KIT and its ligand KITLG. KIT and/or KITLG was stably knocked down by expression of small hairpin RNAs from lentiviral vectors in DLD1, HT29, LS174T, and COLO320 colon cancer cell lines, and in UM-COLON#8 and POP77 xenografts; cells transduced with only vector were used as controls. Cells were analyzed by real-time quantitative reverse transcription PCR, single-cell gene expression analysis, flow cytometry, and immunohistochemical, immunoblot, and functional assays. Xenograft tumors were grown from control and KIT-knockdown DLD1 and UM-COLON#8 cells in immunocompromised mice and compared. Some mice were given the RTK inhibitor imatinib following injection of cancer cells; tumor growth was measured based on bioluminescence. We assessed tumorigenicity using limiting dilution analysis. Results KIT and KITLG were expressed heterogeneously by a subset of human colon tumors. Knockdown of KIT decreased proliferation of colon cancer cell lines and growth of xenograft tumors in mice, compared with control cells. KIT knockdown cells had increased expression of enterocyte markers, decreased expression of cycling genes, and, unexpectedly, increased expression of LGR5-associated genes. No activating mutations in KIT were detected in DLD1, POP77, or UM-COLON#8 cell lines. However, KITLG-knockdown DLD1 cells formed smaller xenograft tumors than control cells. Gene expression analysis of single CD44+ cells indicated that KIT may promote growth via KITLG autocrine and/or paracrine signaling. Imatinib inhibited growth of KIT+ colon cancer organoids in culture and growth of xenograft tumors in mice. Cancer cells with endogenous KIT expression were more tumorigenic in mice. Conclusions KIT and KITLG are expressed by a subset of human colon tumors. KIT signaling promotes growth of colon cancer cells and organoids in culture and xenograft tumors in mice via its ligand, KITLG, in an autocrine or paracrine manner. Patients with KIT-expressing colon tumors may benefit from KIT RTK inhibitors. PMID:26026391

Expression of a suicidal gene under control of the human secreted protein acidic and rich in cysteine (SPARC) promoter in tumor or stromal cells led to the inhibition of tumor cell growth

PubMed Central

Lopez, María V.; Blanco, Patricia; Viale, Diego L.; Cafferata, Eduardo G.; Carbone, Cecilia; Gould, David; Chernajovsky, Yuti; Podhajcer, Osvaldo L.

2009-01-01

The successful use of transcriptional targeting for cancer therapy depends on the activity of a given promoter inside the malignant cell. Because solid human tumors evolve as a “cross-talk” between the different cell types within the tumor, we hypothesized that targeting the entire tumor mass might have better therapeutic effect. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein overexpressed in different human cancers malignant melanomas both in the malignant cells compartment as in the stromal one (fibroblasts and endothelial cells). We have shown that expression of the herpes simplex virus-thymidine kinase (TK) gene driven by the SPARC promoter in combination with ganciclovir inhibited human melanoma cell growth in monolayer as well as in multicellular spheroids. This inhibitory effect was observed both in homotypic spheroids composed of melanoma cells alone as well as in spheroids made of melanoma cells and stromal cells. Expression of the TK gene was also efficient to inhibit the in vivo tumor growth of established melanomas when TK was expressed either by the malignant cells themselves or by coadministered endothelial cells. Our data suggest that the use of therapeutic genes driven by SPARC promoter could be a valuable strategy for cancer therapy aiming to target all the cellular components of the tumor mass. PMID:17041094

Human T-cell leukemia virus type 1 Tax oncoprotein represses the expression of the BCL11B tumor suppressor in T-cells.

PubMed

Takachi, Takayuki; Takahashi, Masahiko; Takahashi-Yoshita, Manami; Higuchi, Masaya; Obata, Miki; Mishima, Yukio; Okuda, Shujiro; Tanaka, Yuetsu; Matsuoka, Masao; Saitoh, Akihiko; Green, Patrick L; Fujii, Masahiro

2015-04-01

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia (ATL), which is an aggressive form of T-cell malignancy. HTLV-1 oncoproteins, Tax and HBZ, play crucial roles in the immortalization of T-cells and/or leukemogenesis by dysregulating the cellular functions in the host. Recent studies show that HTLV-1-infected T-cells have reduced expression of the BCL11B tumor suppressor protein. In the present study, we explored whether Tax and/or HBZ play a role in downregulating BCL11B in HTLV-1-infected T-cells. Lentiviral transduction of Tax in a human T-cell line repressed the expression of BCL11B at both the protein and mRNA levels, whereas the transduction of HBZ had little effect on the expression. Tax mutants with a decreased activity for the NF-κB, CREB or PDZ protein pathways still showed a reduced expression of the BCL11B protein, thereby implicating a different function of Tax in BCL11B downregulation. In addition, the HTLV-2 Tax2 protein reduced the BCL11B protein expression in T-cells. Seven HTLV-1-infected T-cell lines, including three ATL-derived cell lines, showed reduced BCL11B mRNA and protein expression relative to an uninfected T-cell line, and the greatest reductions were in the cells expressing Tax. Collectively, these results indicate that Tax is responsible for suppressing BCL11B protein expression in HTLV-1-infected T-cells; Tax-mediated repression of BCL11B is another mechanism that Tax uses to promote oncogenesis of HTLV-1-infected T-cells. © 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Human Plasmacytoid Dendritic Cells Display and Shed B Cell Maturation Antigen upon TLR Engagement.

PubMed

Schuh, Elisabeth; Musumeci, Andrea; Thaler, Franziska S; Laurent, Sarah; Ellwart, Joachim W; Hohlfeld, Reinhard; Krug, Anne; Meinl, Edgar

2017-04-15

The BAFF-APRIL system is best known for its control of B cell homeostasis, and it is a target of therapeutic intervention in autoimmune diseases and lymphoma. By analyzing the expression of the three receptors of this system, B cell maturation Ag (BCMA), transmembrane activator and CAML interactor, and BAFF receptor, in sorted human immune cell subsets, we found that BCMA was transcribed in plasmacytoid dendritic cells (pDCs) in both blood and lymphoid tissue. Circulating human pDCs contained BCMA protein without displaying it on the cell surface. After engagement of TLR7/8 or TLR9, BCMA was detected also on the cell surface of pDCs. The display of BCMA on the surface of human pDCs was accompanied by release of soluble BCMA (sBCMA); inhibition of γ-secretase enhanced surface expression of BCMA and reduced the release of sBCMA by pDCs. In contrast with human pDCs, murine pDCs did not express BCMA, not even after TLR9 activation. In this study, we extend the spectrum of BCMA expression to human pDCs. sBCMA derived from pDCs might determine local availability of its high-affinity ligand APRIL, because sBCMA has been shown to function as an APRIL-specific decoy. Further, therapeutic trials targeting BCMA in patients with multiple myeloma should consider possible effects on pDCs. Copyright © 2017 by The American Association of Immunologists, Inc.

Prostate stem cell antigen is overexpressed in human transitional cell carcinoma.

PubMed

Amara, N; Palapattu, G S; Schrage, M; Gu, Z; Thomas, G V; Dorey, F; Said, J; Reiter, R E

2001-06-15

Prostate stem cell antigen (PSCA), a homologue of the Ly-6/Thy-1 family of cell surface antigens, is expressed by a majority of human prostate cancers and is a promising target for prostate cancer immunotherapy. In addition to its expression in normal and malignant prostate, we recently reported that PSCA is expressed at low levels in the transitional epithelium of normal bladder. In the present study, we compared the expression of PSCA in normal and malignant urothelial tissues to assess its potential as an immunotherapeutic target in transitional cell carcinoma (TCC). Immunohistochemical analysis of PSCA protein expression was performed on tissue sections from 32 normal bladder specimens, as well as 11 cases of low-grade transitional cell dysplasia, 21 cases of carcinoma in situ (CIS), 38 superficial transitional cell tumors (STCC, stages T(a)-T(1)), 65 muscle-invasive TCCs (ITCCs, stages T(2)-T(4)), and 7 bladder cancer metastases. The level of PSCA protein expression was scored semiquantitatively by assessing both the intensity and frequency (i.e., percentage of positive tumor cells) of staining. We also examined PSCA mRNA expression in a representative sample of normal and malignant human transitional cell tissues. In normal bladder, PSCA immunostaining was weak and confined almost exclusively to the superficial umbrella cell layer. Staining in CIS and STCC was more intense and uniform than that seen in normal bladder epithelium (P < 0.001), with staining detected in 21 (100%) of 21 cases of CIS and 37 (97%) of 38 superficial tumors. PSCA protein was also detected in 42 (65%) of 65 of muscle-invasive and 4 (57%) of 7 metastatic cancers, with the highest levels of PSCA expression (i.e., moderate-strong staining in >50% of tumor cells) seen in 32% of invasive and 43% of metastatic samples. Higher levels of PSCA expression correlated with increasing tumor grade for both STCCs and ITCCs (P < 0.001). Northern blot analysis confirmed the immunohistochemical data, showing a dramatic increase in PSCA mRNA expression in two of five muscle-invasive transitional cell tumors when compared with normal samples. Confocal microscopy demonstrated that PSCA expression in TCC is confined to the cell surface. These data demonstrate that PSCA is overexpressed in a majority of human TCCs, particularly CIS and superficial tumors, and may be a useful target for bladder cancer diagnosis and therapy.

Regulation of CD93 cell surface expression by protein kinase C isoenzymes.

PubMed

Ikewaki, Nobunao; Kulski, Jerzy K; Inoko, Hidetoshi

2006-01-01

Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by cells with a myeloid lineage, endothelial cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)-mediated enhancement of phagocytosis. The intracellular molecular events responsible for the regulation of its expression on the cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the cell surface of a human monocyte-like cell line (U937), a human NK-like cell line (KHYG-1), and a human umbilical vein endothelial cell line (HUV-EC-C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde-fixed cellular enzyme-linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI-11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down-regulated CD93 expression on the U937 cells in a dose-dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down-regulated by Go6976, but not by Rottlerin, in the KHYG-1 cells and by both Rottlerin and Go6976 in the HUV-EC-C cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up-regulated CD93 expression on the cell surface of all three cell-lines and induced interleukin-8 (IL-8) production by the U937 cells and interferon-gamma (IFN-gamma) production by the KHYG-1 cells. In addition, both Go6976 and Rottlerin inhibited the up-regulation of CD93 expression induced by PMA and IL-8 or IFN-gamma production in the respective cell-lines. Whereas recombinant tumor necrosis factor-alpha (rTNF-alpha) slightly up-regulated CD93 expression on the U937 cells, recombinant interleukin-1beta (rIL-1beta), recombinant interleukin-2 (rIL-2), recombinant interferon-gamma (rIFN-gamma) and lipopolysaccharide (LPS) had no effect. Taken together, these findings indicate that the regulation of CD93 expression on these cells involves the PKC isoenzymes.

Human Embryonic and Induced Pluripotent Stem Cells Express TRAIL Receptors and Can Be Sensitized to TRAIL-Induced Apoptosis

PubMed Central

Vinarsky, Vladimir; Krivanek, Jan; Rankel, Liina; Nahacka, Zuzana; Barta, Tomas; Jaros, Josef; Andera, Ladislav

2013-01-01

Death ligands and their tumor necrosis factor receptor (TNFR) family receptors are the best-characterized and most efficient inducers of apoptotic signaling in somatic cells. In this study, we analyzed whether these prototypic activators of apoptosis are also expressed and able to be activated in human pluripotent stem cells. We examined human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC) and found that both cell types express primarily TNF-related apoptosis-inducing ligand (TRAIL) receptors and TNFR1, but very low levels of Fas/CD95. We also found that although hESC and hiPSC contain all the proteins required for efficient induction and progression of extrinsic apoptotic signaling, they are resistant to TRAIL-induced apoptosis. However, both hESC and hiPSC can be sensitized to TRAIL-induced apoptosis by co-treatment with protein synthesis inhibitors such as the anti-leukemia drug homoharringtonine (HHT). HHT treatment led to suppression of cellular FLICE inhibitory protein (cFLIP) and Mcl-1 expression and, in combination with TRAIL, enhanced processing of caspase-8 and full activation of caspase-3. cFLIP likely represents an important regulatory node, as its shRNA-mediated down-regulation significantly sensitized hESC to TRAIL-induced apoptosis. Thus, we provide the first evidence that, irrespective of their origin, human pluripotent stem cells express canonical components of the extrinsic apoptotic system and on stress can activate death receptor-mediated apoptosis. PMID:23806100

Novel scalable 3D cell based model for in vitro neurotoxicity testing: Combining human differentiated neurospheres with gene expression and functional endpoints.

PubMed

Terrasso, Ana Paula; Pinto, Catarina; Serra, Margarida; Filipe, Augusto; Almeida, Susana; Ferreira, Ana Lúcia; Pedroso, Pedro; Brito, Catarina; Alves, Paula Marques

2015-07-10

There is an urgent need for new in vitro strategies to identify neurotoxic agents with speed, reliability and respect for animal welfare. Cell models should include distinct brain cell types and represent brain microenvironment to attain higher relevance. The main goal of this study was to develop and validate a human 3D neural model containing both neurons and glial cells, applicable for toxicity testing in high-throughput platforms. To achieve this, a scalable bioprocess for neural differentiation of human NTera2/cl.D1 cells in stirred culture systems was developed. Endpoints based on neuronal- and astrocytic-specific gene expression and functionality in 3D were implemented in multi-well format and used for toxicity assessment. The prototypical neurotoxicant acrylamide affected primarily neurons, impairing synaptic function; our results suggest that gene expression of the presynaptic marker synaptophysin can be used as sensitive endpoint. Chloramphenicol, described as neurotoxicant affected both cell types, with cytoskeleton markers' expression significantly reduced, particularly in astrocytes. In conclusion, a scalable and reproducible process for production of differentiated neurospheres enriched in mature neurons and functional astrocytes was obtained. This 3D approach allowed efficient production of large numbers of human differentiated neurospheres, which in combination with gene expression and functional endpoints are a powerful cell model to evaluate human neuronal and astrocytic toxicity. Copyright © 2014 Elsevier B.V. All rights reserved.

House dust mite induces expression of intercellular adhesion molecule-1 in EoL-1 human eosinophilic leukemic cells.

PubMed

Kwon, Byoung Chul; Sohn, Myung Hyun; Kim, Kyung Won; Kim, Eun Soo; Kim, Kyu-Earn; Shin, Myeong Heon

2007-10-01

The house dust mite (HDM) is considered to be the most common indoor allergen associated with bronchial asthma. In this study, we investigated whether crude extract of the HDM Dermatophagoides farinae could activate human eosinophilic leukemic cells (EoL-1) to induce upregulation of cell-surface adhesion molecules. When EoL-1 cells were incubated with D. farinae extract, expression of intercellular adhesion molecule-1 (ICAM-1) significantly increased on the cell surfaces compared to cells incubated with medium alone. In contrast, surface expression of CD11b and CD49d in EoL-1 cells was not affected by D. farinae extract. In addition, pretreatment of cells with NF-kappaB inhibitor (MG-132) or JNK inhibitor (SP600125) significantly inhibited ICAM-1 expression promoted by HDM extract. However, neither p38 MAP kinase inhibitor nor MEK inhibitor prevented HDM-induced ICAM-1 expression in EoL-1 cells. These results suggest that crude extract of D. farinae induces ICAM-1 expression in EoL-1 cells through signaling pathways involving both NF-kappaB and JNK.

House Dust Mite Induces Expression of Intercellular Adhesion Molecule-1 in EoL-1 Human Eosinophilic Leukemic Cells

PubMed Central

Kwon, Byoung Chul; Sohn, Myung Hyun; Kim, Kyung Won; Kim, Eun Soo; Kim, Kyu-Earn

2007-01-01

The house dust mite (HDM) is considered to be the most common indoor allergen associated with bronchial asthma. In this study, we investigated whether crude extract of the HDM Dermatophagoides farinae could activate human eosinophilic leukemic cells (EoL-1) to induce upregulation of cell-surface adhesion molecules. When EoL-1 cells were incubated with D. farinae extract, expression of intercellular adhesion molecule-1 (ICAM-1) significantly increased on the cell surfaces compared to cells incubated with medium alone. In contrast, surface expression of CD11b and CD49d in EoL-1 cells was not affected by D. farinae extract. In addition, pretreatment of cells with NF-κB inhibitor (MG-132) or JNK inhibitor (SP600125) significantly inhibited ICAM-1 expression promoted by HDM extract. However, neither p38 MAP kinase inhibitor nor MEK inhibitor prevented HDM-induced ICAM-1 expression in EoL-1 cells. These results suggest that crude extract of D. farinae induces ICAM-1 expression in EoL-1 cells through signaling pathways involving both NF-κB and JNK. PMID:17982228

Aberrant, ectopic expression of VEGF and VEGF receptors 1 and 2 in malignant colonic epithelial cells. Implications for these cells growth via an autocrine mechanism

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

Ahluwalia, Amrita; Jones, Michael K.; Department of Medicine, University of California, Irvine, CA

2013-08-09

Highlights: •Malignant colonic epithelial cells express VEGF and its receptors. •Cultured colon cancer cells secrete VEGF into the medium. •Inhibition of VEGF receptor significantly decreases colon cancer cell proliferation. •VEGF is critical for colon cancer cell growth. -- Abstract: Vascular endothelial growth factor A (referred to as VEGF) is implicated in colon cancer growth. Currently, the main accepted mechanism by which VEGF promotes colon cancer growth is via the stimulation of angiogenesis, which was originally postulated by late Judah Folkman. However, the cellular source of VEGF in colon cancer tissue; and, the expression of VEGF and its receptors VEGF-R1 andmore » VEGF-R2 in colon cancer cells are not fully known and are subjects of controversy. Material and methods: We examined and quantified expression of VEGF, VEGF-R1 and VEGF-R2 in three different human colonic tissue arrays containing sections of adenocarcinoma (n = 43) and normal mucosa (n = 41). In human colon cancer cell lines HCT116 and HT29 and normal colon cell lines NCM356 and NCM460, we examined expression of VEGF, VEGF-R1 and VEGF-R2 mRNA and protein, VEGF production and secretion into the culture medium; and, the effect of a potent, selective inhibitor of VEGF receptors, AL-993, on cell proliferation. Results: Human colorectal cancer specimens had strong expression of VEGF in cancer cells and also expressed VEGF-R1 and VEGF-R2.In vitro studies showed that human colon cancer cell lines, HCT116 and HT29, but not normal colonic cell lines, express VEGF, VEGF-R1 and VEGF-R2 and secrete VEGF into the medium up to a concentration 2000 pg/ml within 48 h. Furthermore, we showed that inhibition of VEGF receptors using a specific VEGF-R inhibitor significantly reduced proliferation (by >50%) of cultured colon cancer cell lines. Conclusions: Our findings support the contention that VEGF generated by colon cancer cells stimulates their growth directly through an autocrine mechanism that is independent of its primary function in the induction of angiogenesis.« less

Design of retrovirus vectors for transfer and expression of the human. beta. -globin gene

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

Miller, A.D.; Bender, M.A.; Harris, E.A.S.

1988-11-01

Regulated expression of the human ..beta..-globin gene has been demonstrated in cultured murine erythroleukemia cells and in mice after retrovirus-mediated gene transfer. However, the low titer of recombinant viruses described to date results in relatively inefficient gene transfer, which limits their usefulness for animal studies and for potential gene therapy in humans for diseases involving defective ..beta..-globin genes. The authors found regions that interfered with virus production within intron 2 of the ..beta..-globin gene and on both sides of the gene. The flanking regions could be removed, but intron 2 was required for ..beta..-globin expression. Inclusion of ..beta..-globin introns necessitatesmore » an antisense orientation of the gene within the retrovirus vector. However, they found no effect of the antisense ..beta..-globin transcription on virus production. A region downstream of the ..beta..-globin gene that stimulates expression of the gene in transgenic mice was included in the viruses without detrimental effects on virus titer. Virus titers of over 10/sup 6/ CFU/ml were obtained with the final vector design, which retained the ability to direct regulated expression of human ..beta..-globin in murine erythroleukemia cells. The vector also allowed transfer and expression of the human ..beta..-globin gene in hematopoietic cells (CFU-S cells) in mice.« less