Positive nuclear BAP1 immunostaining helps differentiate non-small cell lung carcinomas from malignant mesothelioma

PubMed Central

Carbone, Michele; Shimizu, David; Napolitano, Andrea; Tanji, Mika; Pass, Harvey I.; Yang, Haining; Pastorino, Sandra

2016-01-01

The differential diagnosis between pleural malignant mesothelioma (MM) and lung cancer is often challenging. Immunohistochemical (IHC) stains used to distinguish these malignancies include markers that are most often positive in MM and less frequently positive in carcinomas, and vice versa. However, in about 10–20% of the cases, the IHC results can be confusing and inconclusive, and novel markers are sought to increase the diagnostic accuracy. We stained 45 non-small cell lung cancer samples (32 adenocarcinomas and 13 squamous cell carcinomas) with a monoclonal antibody for BRCA1-associated protein 1 (BAP1) and also with an IHC panel we routinely use to help differentiate MM from carcinomas, which include, calretinin, Wilms Tumor 1, cytokeratin 5, podoplanin D2-40, pankeratin CAM5.2, thyroid transcription factor 1, Napsin-A, and p63. Nuclear BAP1 expression was also analyzed in 35 MM biopsies. All 45 non-small cell lung cancer biopsies stained positive for nuclear BAP1, whereas 22/35 (63%) MM biopsies lacked nuclear BAP1 staining, consistent with previous data. Lack of BAP1 nuclear staining was associated with MM (two-tailed Fisher's Exact Test, P = 5.4 × 10−11). Focal BAP1 staining was observed in a subset of samples, suggesting polyclonality. Diagnostic accuracy of other classical IHC markers was in agreement with previous studies. Our study indicated that absence of nuclear BAP1 stain helps differentiate MM from lung carcinomas. We suggest that BAP1 staining should be added to the IHC panel that is currently used to distinguish these malignancies. PMID:27447750

Repression of ESR1 transcription by MYOD potentiates letrozole-resistance in ERα-positive breast cancer cells.

PubMed

Zhang, Qiang; Liu, Xiao-Yan; Li, Shuang; Zhao, Zhao; Li, Juan; Cui, Ming-Ke; Wang, En-Hua

2017-10-21

Transcriptional silencing of estrogen receptor α (ERα) expression is an important etiology contributing to the letrozole-resistance in ERα-positive breast cancer (BCa) cells, but the transcription factors responsible for this transcriptional repression remain largely unidentified. Here we report that the expression of the basic helix-loop-helix myogenic regulatory factor MYOD was abnormally up-regulated in letrozole-resistant BCa tissues and in experimentally-induced letrozole-resistant BCa cells. Overexpression of the exogenous MYOD impaired ERα expression and potentiated letrozole-resistance in letrozole-sensitive MCF7 cells, whereas MYOD knockdown could effectively restore ERα expression and thereby promote letrozole-sensitivity in letrozole-resistant MCF-7/LR cells. Mechanistically, MYOD was shown to be a potent corepressor of ESR1 transcription, and this transcriptional repression was significantly enhanced in the presence of letrozole treatment. Thus, targeted inhibition of MYOD may restore ERα level and lead to resensitization to letrozole-based hormone therapy, providing a novel therapeutic strategy for relapsed ERα-positive BCa patients. Our data also underscore an unexpected chemotherapeutic facet of MYOD, which may operate as a novel regulator of BCa biology. Copyright © 2017 Elsevier Inc. All rights reserved.

Hypoxia-inducible factor-1 signalling promotes goblet cell hyperplasia in airway epithelium

PubMed Central

Polosukhin, Vasiliy V; Cates, Justin M; Lawson, William E; Milstone, Aaron P; Matafonov, Anton G; Massion, Pierre P; Lee, Jae Woo; Randell, Scott H; Blackwell, Timothy S

2018-01-01

Goblet cell hyperplasia is a common feature of chronic obstructive pulmonary disease (COPD) airways, but the mechanisms that underlie this epithelial remodelling in COPD are not understood. Based on our previous finding of hypoxia-inducible factor-1α (HIF-1α) nuclear localization in large airways from patients with COPD, we investigated whether hypoxia-inducible signalling could influence the development of goblet cell hyperplasia. We evaluated large airway samples obtained from 18 lifelong non-smokers and 13 former smokers without COPD, and 45 former smokers with COPD. In these specimens, HIF-1α nuclear staining occurred almost exclusively in COPD patients in areas of airway remodelling. In COPD patients, 93.2 ± 3.9% (range 65 – 100%) of goblet cells were HIF-1α positive in areas of goblet cell hyperplasia, whereas nuclear HIF-1α was not detected in individuals without COPD or in normal-appearing pseudostratified epithelium from COPD patients. To determine the direct effects of hypoxia-inducible signalling on epithelial cell differentiation in vitro, human bronchial epithelial cells (HBECs) were grown in air-liquid interface cultures under hypoxia (1% O2) or following treatment with a selective HIF-1α stabilizer, (2R)-[(4-biphenylylsulphonyl)amino]-N-hydroxy-3-phenyl-propionamide (BiPS). HBECs grown in hypoxia or with BiPS treatment were characterized by HIF-1α activation, carbonic anhydrase IX expression, mucus-producing cell hyperplasia and increased expression of MUC5AC. Analysis of signal transduction pathways in cells with HIF-1α activation showed increased ERK1/2 phosphorylation without activation of epidermal growth factor receptor, Ras, PI3K-Akt or STAT6. These data indicate an important effect of hypoxia-inducible signalling on airway epithelial cell differentiation and identify a new potential target to limit mucus production in COPD. PMID:21557221

Positive feedback loop and synergistic effects between hypoxia-inducible factor-2α and stearoyl-CoA desaturase-1 promote tumorigenesis in clear cell renal cell carcinoma.

PubMed

Zhang, Yujian; Wang, Hui; Zhang, Jin; Lv, Jianwei; Huang, Yiran

2013-04-01

Adapting to hypoxic stress is pivotal in tumor progression and determining tumor malignancy. The transcriptional factor hypoxia-inducible factor (HIF) is crucial in modulating tumorous hypoxic responses through altering cell energy metabolism, which includes the modification of glucose and lipid metabolism-associated gene expression. Stearoyl-CoA desaturase-1 (SCD1) is the main isoform of SCDs, the rate-limiting enzymes in the biosynthesis of monounsaturated fatty acids from saturated fatty acids, which is extensively activated in cancer progression. In this study, we found that SCD1 and HIF-2α were overexpressed in human clear cell renal cell carcinoma (ccRCC) tissues and ccRCC cell lines, and were upregulated in the 786-0 ccRCC cell line under hypoxia. Knockdown of SCD1 or HIF-2α impacted the other's expression. Enhancing SCD1 resulted in HIF-2α upregulation, which could be blocked by inhibiting the PI3K/Akt pathway. Deficiency of SCD1 or HIF-2α in 768-0 cells led to apoptosis, less colony formation ability, and decreased cell migration. More obvious effects were observed in 786-0 cells with double SCD1 and HIF-2α knockdown. These results indicate a PI3K/Akt-mediated loop between SCD1 and HIF-2α that mutually enhances their protein levels. Both SCD1 and HIF-2α are critical to promoting tumorigenesis by synergistically acting on maintaining cell survival, triggering cell migration, and enhancing the colony formation ability of cancer cells. © 2013 Japanese Cancer Association.

Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy

NASA Technical Reports Server (NTRS)

Askari, Arman T.; Unzek, Samuel; Popovic, Zoran B.; Goldman, Corey K.; Forudi, Farhad; Kiedrowski, Matthew; Rovner, Aleksandr; Ellis, Stephen G.; Thomas, James D.; DiCorleto, Paul E.;

Tumor necrosis factor-{alpha} enhanced fusions between oral squamous cell carcinoma cells and endothelial cells via VCAM-1/VLA-4 pathway

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

Song, Kai; Zhu, Fei; Zhang, Han-zhong

Fusion between cancer cells and host cells, including endothelial cells, may strongly modulate the biological behavior of tumors. However, no one is sure about the driving factors and underlying mechanism involved in such fusion. We hypothesized in this study that inflammation, one of the main characteristics in tumor microenvironment, serves as a prominent catalyst for fusion events. Our results showed that oral cancer cells can fuse spontaneously with endothelial cells in co-culture and inflammatory cytokine tumor necrosis factor-{alpha} (TNF-{alpha}) increased fusion of human umbilical vein endothelium cells and oral cancer cells by up to 3-fold in vitro. Additionally, human oralmore » squamous cell carcinoma cell lines and 35 out of 50 (70%) oral squamous carcinoma specimens express VLA-4, an integrin, previously implicated in fusions between human peripheral blood CD34-positive cells and murine cardiomyocytes. Expression of VCAM-1, a ligand for VLA-4, was evident on vascular endothelium of oral squamous cell carcinoma. Moreover, immunocytochemistry and flow cytometry analysis revealed that expression of VCAM-1 increased obviously in TNF-{alpha}-stimulated endothelial cells. Anti-VLA-4 or anti-VCAM-1 treatment can decrease significantly cancer-endothelial adhesion and block such fusion. Collectively, our results suggested that TNF-{alpha} could enhance cancer-endothelial cell adhesion and fusion through VCAM-1/VLA-4 pathway. This study provides insights into regulatory mechanism of cancer-endothelial cell fusion, and has important implications for the development of novel therapeutic strategies for prevention of metastasis. -- Highlights: Black-Right-Pointing-Pointer Spontaneous oral cancer-endothelial cell fusion. Black-Right-Pointing-Pointer TNF-{alpha} enhanced cell fusions. Black-Right-Pointing-Pointer VCAM-1/VLA-4 expressed in oral cancer. Black-Right-Pointing-Pointer TNF-{alpha} increased expression of VCAM-1 on endothelial cells. Black

Adhesion of Epstein–Barr virus-positive natural killer cell lines to cultured endothelial cells stimulated with inflammatory cytokines

PubMed Central

Kanno, H; Watabe, D; Shimizu, N; Sawai, T

2008-01-01

Chronic active Epstein–Barr virus (EBV) infection (CAEBV) is characterized by chronic recurrent infectious mononucleosis-like symptoms. Approximately one-fourth of CAEBV patients develop vascular lesions with infiltration of EBV-positive lymphoid cells. Furthermore, EBV-positive natural killer (NK)/T cell lymphomas often exhibit angiocentric or angiodestructive lesions. These suggest an affinity of EBV-positive NK/T cells to vascular components. In this study, we evaluated the expression of adhesion molecules and cytokines in EBV-positive NK lymphoma cell lines, SNK1 and SNK6, and examined the role of cytokines in the interaction between NK cell lines and endothelial cells. SNKs expressed intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) at much higher levels than those in EBV-negative T cell lines. SNKs produced the larger amount of tumour necrosis factor (TNF)-α, which caused increased expression of ICAM-1 and VCAM-1 in cultured human endothelial cells, than that from EBV-negative T cell lines. Furthermore, SNKs exhibited increased adhesion to cultured endothelial cells stimulated with TNF-α or interleukin (IL)-1β, and the pretreatment of cytokine-stimulated endothelial cells with anti-VCAM-1-antibodies reduced cell adhesion. These indicate that the up-regulated expression of VCAM-1 on cytokine-stimulated endothelial cells would be important for the adhesion of EBV-positive NK cells and might initiate the vascular lesions. PMID:18190605

Nuclear Factor YY1 Inhibits Transforming Growth Factor β- and Bone Morphogenetic Protein-Induced Cell Differentiation

PubMed Central

Kurisaki, Keiko; Kurisaki, Akira; Valcourt, Ulrich; Terentiev, Alexei A.; Pardali, Katerina; ten Dijke, Peter; Heldin, Carl-Henrik; Ericsson, Johan; Moustakas, Aristidis

2003-01-01

Smad proteins transduce transforming growth factor β (TGF-β) and bone morphogenetic protein (BMP) signals that regulate cell growth and differentiation. We have identified YY1, a transcription factor that positively or negatively regulates transcription of many genes, as a novel Smad-interacting protein. YY1 represses the induction of immediate-early genes to TGF-β and BMP, such as the plasminogen activator inhibitor 1 gene (PAI-1) and the inhibitor of differentiation/inhibitor of DNA binding 1 gene (Id-1). YY1 inhibits binding of Smads to their cognate DNA elements in vitro and blocks Smad recruitment to the Smad-binding element-rich region of the PAI-1 promoter in vivo. YY1 interacts with the conserved N-terminal Mad homology 1 domain of Smad4 and to a lesser extent with Smad1, Smad2, and Smad3. The YY1 zinc finger domain mediates the association with Smads and is necessary for the repressive effect of YY1 on Smad transcriptional activity. Moreover, downregulation of endogenous YY1 by antisense and small interfering RNA strategies results in enhanced transcriptional responses to TGF-β or BMP. Ectopic expression of YY1 inhibits, while knockdown of endogenous YY1 enhances, TGF-β- and BMP-induced cell differentiation. In contrast, overexpression or knockdown of YY1 does not affect growth inhibition induced by TGF-β or BMP. Accordingly, YY1 does not interfere with the regulation of immediate-early genes involved in the TGF-β growth-inhibitory response, the cell cycle inhibitors p15 and p21, and the proto-oncogene c-myc. In conclusion, YY1 represses Smad transcriptional activities in a gene-specific manner and thus regulates cell differentiation induced by TGF-β superfamily pathways. PMID:12808092

Inhibition of ZEB1 by miR-200 characterizes Helicobacter pylori-positive gastric diffuse large B-cell lymphoma with a less aggressive behavior.

PubMed

Huang, Wei-Ting; Kuo, Sung-Hsin; Cheng, Ann-Lii; Lin, Chung-Wu

2014-08-01

Primary gastric diffuse large B-cell lymphomas may or may not have a concurrent component of mucosa-associated lymphoid tissue lymphoma. Diffuse large B-cell lymphoma/mucosa-associated lymphoid tissue lymphomas are often associated with Helicobacter pylori (H. pylori) infection, suggesting that the large cells are transformed from mucosa-associated lymphoid tissue lymphomas. In contrast, only limited data are available on the clinical and molecular features of pure gastric diffuse large B-cell lymphomas. In 102 pure gastric diffuse large B-cell lymphomas, we found H. pylori infection in 53% of the cases. H. pylori-positive gastric diffuse large B-cell lymphomas were more likely to present at an earlier stage (73% vs 52% at stage I/II, P=0.03), to achieve complete remission (75% vs 43%, P=0.001), and had a better 5-year disease-free survival rate (73% vs 29%, P<0.001) than H. pylori-negative gastric diffuse large B-cell lymphomas. Through genome-wide expression profiles of both miRNAs and mRNAs in nine H. pylori-positive and nine H. pylori-negative gastric diffuse large B-cell lymphomas, we identified inhibition of ZEB1 (zinc-finger E-box-binding homeobox 1) by miR-200 in H. pylori-positive gastric diffuse large B-cell lymphomas. ZEB1, a transcription factor for marginal zone B cells, can suppress BCL6, the master transcription factor for germinal center B cells. In 30 H. pylori-positive and 30 H. pylori-negative gastric diffuse large B-cell lymphomas, we confirmed that H. pylori-positive gastric diffuse large B-cell lymphomas had higher levels of miR-200 by qRT-PCR, and lower levels of ZEB1 and higher levels of BCL6 using immunohistochemistry. As BCL6 is a known predictor of a better prognosis in gastric diffuse large B-cell lymphomas, our data demonstrate that inhibition of ZEB1 by miR-200, with secondary increase in BCL6, is a molecular event that characterizes H. pylori-positive gastric diffuse large B-cell lymphomas with a less aggressive behavior.

Inhibitor of differentiation 1 transcription factor promotes metabolic reprogramming in hepatocellular carcinoma cells

PubMed Central

Sharma, Bal Krishan; Kolhe, Ravindra; Black, Stephen M.; Keller, Jonathan R.; Mivechi, Nahid F.; Satyanarayana, Ande

2016-01-01

Reprograming of metabolism is one of the central hallmarks of cancer. The majority of cancer cells depend on high rates of glycolysis and glutaminolysis for their growth and survival. A number of oncogenes and tumor suppressors have been connected to the regulation of altered glucose and glutamine metabolism in cancer cells. For example, the oncogene c-Myc plays vital roles in cancer cell metabolic adaptation by directly regulating various genes that participate in aerobic glycolysis and glutaminolysis. Inhibitor of differentiation 1 (Id1) is a helix-loop-helix transcription factor that plays important roles in cell proliferation, differentiation, and cell fate determination. Overexpression of Id1 causes intestinal adenomas and thymic lymphomas in mice, suggesting that Id1 could function as an oncogene. Despite it being an oncogene, whether Id1 plays any prominent role in cancer cell metabolic reprograming is unknown. Here, we demonstrate that Id1 is strongly expressed in human and mouse liver tumors and in hepatocellular carcinoma (HCC) cell lines, whereas its expression is very low or undetectable in normal liver tissues. In HCC cells, Id1 expression is regulated by the MAPK/ERK pathway at the transcriptional level. Knockdown of Id1 suppressed aerobic glycolysis and glutaminolysis, suggesting that Id1 promotes a metabolic shift toward aerobic glycolysis. At the molecular level, Id1 mediates its metabolic effects by regulating the expression levels of c-Myc. Knockdown of Id1 resulted in down-regulation (∼75%) of c-Myc, whereas overexpression of Id1 strongly induced (3-fold) c-Myc levels. Interestingly, knockdown of c-Myc resulted in down-regulation (∼60%) of Id1, suggesting a positive feedback-loop regulatory mechanism between Id1 and c-Myc. Under anaerobic conditions, both Id1 and c-Myc are down-regulated (50–70%), and overexpression of oxygen-insensitive hypoxia-inducible factor 1α (Hif1α) or its downstream target Mxi1 resulted in a significant reduction

Aberrantly activated AREG-EGFR signaling is required for the growth and survival of CRTC1-MAML2 fusion-positive mucoepidermoid carcinoma cells.

PubMed

Chen, Z; Chen, J; Gu, Y; Hu, C; Li, J-L; Lin, S; Shen, H; Cao, C; Gao, R; Li, J; Ha, P K; Kaye, F J; Griffin, J D; Wu, L

2014-07-17

Salivary gland tumors (SGT) are a group of highly heterogeneous head and neck malignancies with widely varied clinical outcomes and no standard effective treatments. The CRTC1-MAML2 fusion oncogene, encoded by a recurring chromosomal translocation t(11;19)(q14-21;p12-13), is a frequent genetic alteration found in >50% of mucoepidermoid carcinomas (MEC), the most common malignant SGT. In this study, we aimed to define the role of the CRTC1-MAML2 oncogene in the maintenance of MEC tumor growth and to investigate critical downstream target genes and pathways for therapeutic targeting of MEC. By performing gene expression analyses and functional studies via RNA interference and pharmacological modulation, we determined the importance of the CRTC1-MAML2 fusion gene and its downstream AREG-EGFR signaling in human MEC cancer cell growth and survival in vitro and in vivo using human MEC xenograft models. We found that CRTC1-MAML2 fusion oncogene was required for the growth and survival of fusion-positive human MEC cancer cells in vitro and in vivo. The CRTC1-MAML2 oncoprotein induced the upregulation of the epidermal growth factor receptor (EGFR) ligand Amphiregulin (AREG) by co-activating the transcription factor CREB, and AREG subsequently activated EGFR signaling in an autocrine manner that promoted MEC cell growth and survival. Importantly, CRTC1-MAML2-positive MEC cells were highly sensitive to EGFR signaling inhibition. Therefore, our study revealed that aberrantly activated AREG-EGFR signaling is required for CRTC1-MAML2-positive MEC cell growth and survival, suggesting that EGFR-targeted therapies will benefit patients with advanced, unresectable CRTC1-MAML2-positive MEC.

Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting.

PubMed

Jakobsson, Lars; Franco, Claudio A; Bentley, Katie; Collins, Russell T; Ponsioen, Bas; Aspalter, Irene M; Rosewell, Ian; Busse, Marta; Thurston, Gavin; Medvinsky, Alexander; Schulte-Merker, Stefan; Gerhardt, Holger

2010-10-01

Sprouting angiogenesis requires the coordinated behaviour of endothelial cells, regulated by Notch and vascular endothelial growth factor receptor (VEGFR) signalling. Here, we use computational modelling and genetic mosaic sprouting assays in vitro and in vivo to investigate the regulation and dynamics of endothelial cells during tip cell selection. We find that endothelial cells compete for the tip cell position through relative levels of Vegfr1 and Vegfr2, demonstrating a biological role for differential Vegfr regulation in individual endothelial cells. Differential Vegfr levels affect tip selection only in the presence of a functional Notch system by modulating the expression of the ligand Dll4. Time-lapse microscopy imaging of mosaic sprouts identifies dynamic position shuffling of tip and stalk cells in vitro and in vivo, indicating that the VEGFR-Dll4-Notch signalling circuit is constantly re-evaluated as cells meet new neighbours. The regular exchange of the leading tip cell raises novel implications for the concept of guided angiogenic sprouting.

The AP-1 transcription factor Fra1 inhibits follicular B cell differentiation into plasma cells

PubMed Central

Grötsch, Bettina; Brachs, Sebastian; Lang, Christiane; Luther, Julia; Derer, Anja; Schlötzer-Schrehardt, Ursula; Bozec, Aline; Fillatreau, Simon; Berberich, Ingolf; Hobeika, Elias; Reth, Michael; Wagner, Erwin F.; Schett, Georg

2014-01-01

The cornerstone of humoral immunity is the differentiation of B cells into antibody-secreting plasma cells. This process is tightly controlled by a regulatory gene network centered on the transcriptional repressor B lymphocyte–induced maturation protein 1 (Blimp1). Proliferation of activated B cells is required to foster Blimp1 expression but needs to be terminated to avoid overshooting immune reactions. Activator protein 1 (AP-1) transcription factors become quickly up-regulated upon B cell activation. We demonstrate that Fra1, a Fos member of AP-1, enhances activation-induced cell death upon induction in activated B cells. Moreover, mice with B cell–specific deletion of Fra1 show enhanced plasma cell differentiation and exacerbated antibody responses. In contrast, transgenic overexpression of Fra1 blocks plasma cell differentiation and immunoglobulin production, which cannot be rescued by Bcl2. On the molecular level, Fra1 represses Blimp1 expression and interferes with binding of the activating AP-1 member c-Fos to the Blimp1 promoter. Conversely, overexpression of c-Fos in Fra1 transgenic B cells releases Blimp1 repression. As Fra1 lacks transcriptional transactivation domains, we propose that Fra1 inhibits Blimp1 expression and negatively controls plasma cell differentiation through binding to the Blimp1 promoter. In summary, we demonstrate that Fra1 negatively controls plasma cell differentiation by repressing Blimp1 expression. PMID:25288397

A1E reduces stemness and self-renewal in HPV 16-positive cervical cancer stem cells.

PubMed

Kwon, Taeho; Bak, Yesol; Ham, Sun-Young; Yu, Dae-Yeul; Yoon, Do-Young

2016-02-02

Cervical cancer is the second most common cancer in females. Recent reports have revealed the critical role of cervical cancer stem cells (CSCs) in tumorigenicity and metastasis. Previously we demonstrated that A1E exerts an anti-proliferative action, which inhibits the growth of cervical cancer cells. A1E is composed of 11 oriental medicinal herbs. Cervical cancer cell culture, wund healing and invasion assay, flow cytometry, sheroid formation assay, and wstern blot assays were performed in HPV 16-positive SiHa cell and HPV 16-negative C33A cells. A1E targets the E6 and E7 oncogenes; thus, A1E significantly inhibited proliferation of human papilloma virus (HPV) 16-positive SiHa cells, it did not inhibit the proliferation of HPV-negative C33A cells. Accordingly, we investigated whether A1E can regulate epithelial-to-mesenchymal transition (EMT), CSC self-renewal, and stemness-related gene expression in cervical cancer cells. Down rgulation of cell migration, cell invasion, and EMT was observed in A1E-treated SiHa cells. Specifically, A1E-treated SiHa cells showed significant decreases in OCT-3/4 and Sox2 expression levels and in sphere formation. Moreover, CSCs makers ALDH+ and ALDH, CD133 double positive cell were significantly decreased in A1E-treated SiHa cells. However, A1E treatment did not down regulate ALDH+ expression and the number of ALDH/CD133 double positive cells in C33A cells. Taken together, A1E can inhibit CSCs and reduce the expression of stemness markers. Treating CSCs with A1E may be a potential therapy for cervical cancer.

Modification of cytokine-induced killer cells with chimeric antigen receptors (CARs) enhances antitumor immunity to epidermal growth factor receptor (EGFR)-positive malignancies.

PubMed

Ren, Xuequn; Ma, Wanli; Lu, Hong; Yuan, Lei; An, Lei; Wang, Xicai; Cheng, Guanchang; Zuo, Shuguang

2015-12-01

Epidermal growth factor receptor (EGFR, ErbB1, Her-1) is a cell surface molecule overexpressing in a variety of human malignancies and, thus, is an excellent target for immunotherapy. Immunotherapy targeting EGFR-overexpressing malignancies using genetically modified immune effector cells is a novel and promising approach. In the present study, we have developed an adoptive cellular immunotherapy strategy based on the chimeric antigen receptor (CAR)-modified cytokine-induced killer (CAR-CIK) cells specific for the tumor cells expressing EGFR. To generate CAR-CIK cells, a lentiviral vector coding the EGFR-specific CAR was constructed and transduced into the CIK cells. The CAR-CIK cells showed significantly enhanced cytotoxicity and increased production of cytokines IFN-γ and IL-2 when co-cultured with EGFR-positive cancer cells. In tumor xenografts, adoptive immunotherapy of CAR-CIK cells could inhibit tumor growth and prolong the survival of EGFR-overexpressing human tumor xenografts. Moreover, tumor growth inhibition and prolonged survival in mice with EGFR(+) human cancer were associated with the increased persistence of CAR-CIK cells in vivo. Our study indicates that modification with EGFR-specific CAR strongly enhances the antitumor activity of the CIK cells against EGFR-positive malignancies.

Positive MRI contrast enhancement in THP-1 cells with Gd2O3 nanoparticles.

PubMed

Klasson, Anna; Ahrén, Maria; Hellqvist, Eva; Söderlind, Fredrik; Rosén, Anders; Käll, Per-Olov; Uvdal, Kajsa; Engström, Maria

2008-01-01

There is a demand for more efficient and tissue-specific MRI contrast agents and recent developments involve the design of substances useful as molecular markers and magnetic tracers. In this study, nanoparticles of gadolinium oxide (Gd2O3) have been investigated for cell labeling and capacity to generate a positive contrast. THP-1, a monocytic cell line that is phagocytic, was used and results were compared with relaxivity of particles in cell culture medium (RPMI 1640). The results showed that Gd2O3-labeled cells have shorter T1 and T2 relaxation times compared with untreated cells. A prominent difference in signal intensity was observed, indicating that Gd2O3 nanoparticles can be used as a positive contrast agent for cell labeling. The r1 for cell samples was 4.1 and 3.6 s(-1) mm(-1) for cell culture medium. The r2 was 17.4 and 12.9 s(-1) mm(-1), respectively. For r1, there was no significant difference in relaxivity between particles in cells compared to particles in cell culture medium, (p(r1) = 0.36), but r2 was significantly different for the two different series (p(r2) = 0.02). Viability results indicate that THP-1 cells endure treatment with Gd2O3 nanoparticles for an extended period of time and it is therefore concluded that results in this study are based on viable cells. Copyright 2008 John Wiley & Sons, Ltd.

Apical localization of glutamate in GLAST-1, glutamine synthetase positive ciliary body nonpigmented epithelial cells

PubMed Central

Langford, Marlyn P; Gosslee, Jeffrey M; Liang, Chanping; Chen, Dequan; Redens, Thomas B.; Welbourne, Tomas C

2007-01-01

The distribution of glutamate (Glu), the Glu transporter GLAST-1, and glutamine synthetase (GS) in human and monkey anterior uveal tissue, as well as serum (S) to aqueous humor (AH) Glu and glutamine (Gln) gradients were investigated. Cross-linked Glu (xGlu), GLAST-1, and GS were detected using the immunofluorescent antibody technique. S/AH Glu, Gln, and alanine (Ala) concentrations were quantified by high performance liquid chromatography. xGlu immunoreactivity was detected in melanocytes, posterior pigmented epithelial/dilator muscle cells, vascular endothelial cells, and lymphocytes of the iris, as well as the pigmented (PE) and nonpigmented epithelial (NPE) cells and muscle cells of ciliary body. xGlu immunoreactivity was highly concentrated at the apices of GLAST-1, GS positive ciliary body NPE cells, and in GLAST-1 positive iris melanocytes and iris dilator muscle cells. AH Glu concentrations were lower (p < 0.001), while Gln was higher in monkey (p = 0.01) and human cataractous (p = 0.15) AH than serum. The results indicate that Glu is concentrated within GLAST-1, GS positive NPE cells and are consistent with the suggestion that Glu and Gln concentrations in AH may be due in part to GLAST-1 and GS activity in iris and ciliary body epithelial cells. PMID:19668465

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.

Barley disease susceptibility factor RACB acts in epidermal cell polarity and positioning of the nucleus

PubMed Central

Scheler, Björn; Schnepf, Vera; Galgenmüller, Carolina; Ranf, Stefanie; Hückelhoven, Ralph

2016-01-01

RHO GTPases are regulators of cell polarity and immunity in eukaryotes. In plants, RHO-like RAC/ROP GTPases are regulators of cell shaping, hormone responses, and responses to microbial pathogens. The barley (Hordeum vulgare L.) RAC/ROP protein RACB is required for full susceptibility to penetration by Blumeria graminis f.sp. hordei (Bgh), the barley powdery mildew fungus. Disease susceptibility factors often control host immune responses. Here we show that RACB does not interfere with early microbe-associated molecular pattern-triggered immune responses such as the oxidative burst or activation of mitogen-activated protein kinases. RACB also supports rather than restricts expression of defence-related genes in barley. Instead, silencing of RACB expression by RNAi leads to defects in cell polarity. In particular, initiation and maintenance of root hair growth and development of stomatal subsidiary cells by asymmetric cell division is affected by silencing expression of RACB. Nucleus migration is a common factor of developmental cell polarity and cell-autonomous interaction with Bgh. RACB is required for positioning of the nucleus near the site of attack from Bgh. We therefore suggest that Bgh profits from RACB’s function in cell polarity rather than from immunity-regulating functions of RACB. PMID:27056842

Barley disease susceptibility factor RACB acts in epidermal cell polarity and positioning of the nucleus.

PubMed

Scheler, Björn; Schnepf, Vera; Galgenmüller, Carolina; Ranf, Stefanie; Hückelhoven, Ralph

2016-05-01

RHO GTPases are regulators of cell polarity and immunity in eukaryotes. In plants, RHO-like RAC/ROP GTPases are regulators of cell shaping, hormone responses, and responses to microbial pathogens. The barley (Hordeum vulgare L.) RAC/ROP protein RACB is required for full susceptibility to penetration by Blumeria graminis f.sp. hordei (Bgh), the barley powdery mildew fungus. Disease susceptibility factors often control host immune responses. Here we show that RACB does not interfere with early microbe-associated molecular pattern-triggered immune responses such as the oxidative burst or activation of mitogen-activated protein kinases. RACB also supports rather than restricts expression of defence-related genes in barley. Instead, silencing of RACB expression by RNAi leads to defects in cell polarity. In particular, initiation and maintenance of root hair growth and development of stomatal subsidiary cells by asymmetric cell division is affected by silencing expression of RACB. Nucleus migration is a common factor of developmental cell polarity and cell-autonomous interaction with Bgh RACB is required for positioning of the nucleus near the site of attack from Bgh We therefore suggest that Bgh profits from RACB's function in cell polarity rather than from immunity-regulating functions of RACB. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Activation of TGF-β1-CD147 positive feedback loop in hepatic stellate cells promotes liver fibrosis.

PubMed

Li, Hai-Yan; Ju, Di; Zhang, Da-Wei; Li, Hao; Kong, Ling-Min; Guo, Yanhai; Li, Can; Wang, Xi-Long; Chen, Zhi-Nan; Bian, Huijie

2015-11-12

Activation of hepatic stellate cells (HSCs) by transforming growth factor-β1 (TGF-β1) initiates HBV-associated fibrogenesis. The mechanism of TGF-β1 modulating HSC activation is not fully uncovered. We hypothesized a positive feedback signaling loop of TGF-β1-CD147 promoting liver fibrogenesis by activation of HSCs. Human HSC cell line LX-2 and spontaneous liver fibrosis model derived from HBV transgenic mice were used to evaluate the activation of molecules in the signaling loop. Wound healing and cell contraction assay were performed to detect the CD147-overexpressed HSC migration and contraction. The transcriptional regulation of CD147 by TGF-β1/Smad4 was determined using dual-luciferase reporter assay and chromatin immunoprecipitation. We found that a positive reciprocal regulation between TGF-β1 and CD147 mediated HSC activation. CD147 over-expression promoted HSC migration and accelerated TGF-β1-induced cell contraction. Phosphorylation of Smad2 and Smad3 in cooperation with Smad4 mediated the TGF-β1-regulated CD147 expression. Smad4 activated the transcription by direct interaction with CD147 promoter. Meanwhile, CD147 modulated the activated phenotype of HSCs through the ERK1/2 and Sp1 which up-regulated α-SMA, collagen I, and TGF-β1 synthesis. These findings indicate that TGF-β1-CD147 loop plays a key role in regulating the HSC activation and combination of TGF-β receptor inhibitor and anti-CD147 antibody might be promised to reverse fibrogenesis.

Activation of TGF-β1-CD147 positive feedback loop in hepatic stellate cells promotes liver fibrosis

PubMed Central

Li, Hai-Yan; Ju, Di; Zhang, Da-Wei; Li, Hao; Kong, Ling-Min; Guo, Yanhai; Li, Can; Wang, Xi-Long; Chen, Zhi-Nan; Bian, Huijie

2015-01-01

Activation of hepatic stellate cells (HSCs) by transforming growth factor-β1 (TGF-β1) initiates HBV-associated fibrogenesis. The mechanism of TGF-β1 modulating HSC activation is not fully uncovered. We hypothesized a positive feedback signaling loop of TGF-β1-CD147 promoting liver fibrogenesis by activation of HSCs. Human HSC cell line LX-2 and spontaneous liver fibrosis model derived from HBV transgenic mice were used to evaluate the activation of molecules in the signaling loop. Wound healing and cell contraction assay were performed to detect the CD147-overexpressed HSC migration and contraction. The transcriptional regulation of CD147 by TGF-β1/Smad4 was determined using dual-luciferase reporter assay and chromatin immunoprecipitation. We found that a positive reciprocal regulation between TGF-β1 and CD147 mediated HSC activation. CD147 over-expression promoted HSC migration and accelerated TGF-β1-induced cell contraction. Phosphorylation of Smad2 and Smad3 in cooperation with Smad4 mediated the TGF-β1-regulated CD147 expression. Smad4 activated the transcription by direct interaction with CD147 promoter. Meanwhile, CD147 modulated the activated phenotype of HSCs through the ERK1/2 and Sp1 which up-regulated α-SMA, collagen I, and TGF-β1 synthesis. These findings indicate that TGF-β1-CD147 loop plays a key role in regulating the HSC activation and combination of TGF-β receptor inhibitor and anti-CD147 antibody might be promised to reverse fibrogenesis. PMID:26559755

Rice homeobox transcription factor HOX1a positively regulates gibberellin responses by directly suppressing EL1.

PubMed

Wen, Bi-Qing; Xing, Mei-Qing; Zhang, Hua; Dai, Cheng; Xue, Hong-Wei

2011-11-01

Homeobox transcription factors are involved in various aspects of plant development, including maintenance of the biosynthesis and signaling pathways of different hormones. However, few direct targets of homeobox proteins have been identified. We here show that overexpression of rice homeobox gene HOX1a resulted in enhanced gibberellin (GA) response, indicating a positive effect of HOX1a in GA signaling. HOX1a is induced by GA and encodes a homeobox transcription factor with transcription repression activity. In addition, HOX1a suppresses the transcription of early flowering1 (EL1), a negative regulator of GA signaling, and further electrophoretic mobility shift assay and chromatin immunoprecipitation analysis revealed that HOX1a directly bound to the promoter region of EL1 to suppress its expression and stimulate GA signaling. These results demonstrate that HOX1a functions as a positive regulator of GA signaling by suppressing EL1, providing informative hints on the study of GA signaling. © 2011 Institute of Botany, Chinese Academy of Sciences.

CD44-positive cells are candidates for astrocyte precursor cells in developing mouse cerebellum.

PubMed

Cai, Na; Kurachi, Masashi; Shibasaki, Koji; Okano-Uchida, Takayuki; Ishizaki, Yasuki

2012-03-01

Neural stem cells are generally considered to be committed to becoming precursor cells before terminally differentiating into either neurons or glial cells during neural development. Neuronal and oligodendrocyte precursor cells have been identified in several areas in the murine central nervous system. The presence of astrocyte precursor cells (APCs) is not so well understood. The present study provides several lines of evidence that CD44-positive cells are APCs in the early postnatal mouse cerebellum. In developing mouse cerebellum, CD44-positive cells, mostly located in the white matter, were positive for the markers of the astrocyte lineage, but negative for the markers of mature astrocytes. CD44-positive cells were purified from postnatal cerebellum by fluorescence-activated cell sorting and characterized in vitro. In the absence of any signaling molecule, many cells died by apoptosis. The surviving cells gradually expressed glial fibrillary acidic protein, a marker for mature astrocytes, indicating that differentiation into mature astrocytes is the default program for these cells. The cells produced no neurospheres nor neurons nor oligodendrocytes under any condition examined, indicating these cells are not neural stem cells. Leukemia inhibitory factor greatly promoted astrocytic differentiation of CD44-positive cells, whereas bone morphogenetic protein 4 (BMP4) did not. Fibroblast growth factor-2 was a potent mitogen for these cells, but was insufficient for survival. BMP4 inhibited activation of caspase-3 and greatly promoted survival, suggesting a novel role for BMP4 in the control of development of astrocytes in cerebellum. We isolated and characterized only CD44 strongly positive large cells and discarded small and/or CD44 weakly positive cells in this study. Further studies are necessary to characterize these cells to help determine whether CD44 is a selective and specific marker for APCs in the developing mouse cerebellum. In conclusion, we succeeded in

High Glucose Forces a Positive Feedback Loop Connecting Akt Kinase and FoxO1 Transcription Factor to Activate mTORC1 Kinase for Mesangial Cell Hypertrophy and Matrix Protein Expression*

PubMed Central

Das, Falguni; Ghosh-Choudhury, Nandini; Dey, Nirmalya; Bera, Amit; Mariappan, Meenalakshmi M.; Kasinath, Balakuntalam S.; Ghosh Choudhury, Goutam

2014-01-01

High glucose-induced Akt acts as a signaling hub for mesangial cell hypertrophy and matrix expansion, which are recognized as cardinal signatures for the development of diabetic nephropathy. How mesangial cells sustain the activated state of Akt is not clearly understood. Here we show Akt-dependent phosphorylation of the transcription factor FoxO1 by high glucose. Phosphorylation-deficient, constitutively active FoxO1 inhibited the high glucose-induced phosphorylation of Akt to suppress the phosphorylation/inactivation of PRAS40 and mTORC1 activity. In contrast, dominant negative FoxO1 increased the phosphorylation of Akt, resulting in increased mTORC1 activity similar to high glucose treatment. Notably, FoxO1 regulates high glucose-induced protein synthesis, hypertrophy, and expression of fibronectin and PAI-1. High glucose paves the way for complications of diabetic nephropathy through the production of reactive oxygen species (ROS). We considered whether the FoxO1 target antioxidant enzyme catalase contributes to sustained activation of Akt. High glucose-inactivated FoxO1 decreases the expression of catalase to increase the production of ROS. Moreover, we show that catalase blocks high glucose-stimulated Akt phosphorylation to attenuate the inactivation of FoxO1 and PRAS40, resulting in the inhibition of mTORC1 and mesangial cell hypertrophy and fibronectin and PAI-1 expression. Finally, using kidney cortices from type 1 diabetic OVE26 mice, we show that increased FoxO1 phosphorylation is associated with decreased catalase expression and increased fibronectin and PAI-1 expression. Together, our results provide the first evidence for the presence of a positive feedback loop for the sustained activation of Akt involving inactivated FoxO1 and a decrease in catalase expression, leading to increased ROS and mesangial cell hypertrophy and matrix protein expression. PMID:25288788

Up-regulation of CHAF1A, a poor prognostic factor, facilitates cell proliferation of colon cancer

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

Wu, Zehua; Cui, Feifei; Yu, Fudong

2014-06-27

Highlights: • We identified that CHAF1A was up-regulated in colon tumor mucosa in TMA. • The expression pattern of CHAF1A was validated with qPCR and western-blot. • CHAF1A overexpression is an independent indicator for poor colon cancer survival. • CHAF1A facilitates cell proliferation of colon cancer both in vitro and in vivo. - Abstract: Deregulation of chromatin assembly factor 1, p150 subunit A (CHAF1A) has recently been reported to be involved in the development of some cancer types. In this study, we identified that the frequency of positive CHAF1A staining in primary tumor mucosa (45.8%, 93 of 203 samples) wasmore » significantly elevated compared to that in paired normal mucosa (18.7%, 38 of 203 samples). The increased expression was strongly associated with cancer stage, tumor invasion, and histological grade. The five-year survival rate of patients with CHAF1A-positive tumors was remarkably lower than that of patients with CHAF1A-negative tumors. Colon cancer cells with CHAF1A knockdown exhibited decreased cell growth index, reduction in colony formation ability, elevated cell apoptosis rate as well as impaired colon tumorigenicity in nude mice. Hence, CHAF1A upregulation functions as a poor prognostic indicator of colon cancer, potentially contributing to its progression by mediating cancer cell proliferation.« less

Differential requirements for the Ets transcription factor Elf-1 in the development of NKT cells and NK cells

PubMed Central

Choi, Hak-Jong; Geng, Yanbiao; Cho, Hoonsik; Li, Sha; Giri, Pramod Kumar; Felio, Kyrie

2011-01-01

E26 Transformation specific (Ets) family transcription factors control the expression of a large number of genes regulating hematopoietic cell development and function. Two such transcription factors, Ets-1 and myeloid Elf-1–like factor (MEF), have been shown to play critical roles in both natural killer (NK)– and NKT-cell development, but not in the development of conventional T cells. In this study, we address the role of E74-like factor 1 (Elf-1), another Ets family transcription factor that is closely related to MEF but divergent from Ets-1, in NK- and NKT-cell development using Elf-1–deficient (Elf-1−/−) mice. Whereas the proportion of NK cells in Elf-1−/− mice was normal, the proportion of NKT cells was significantly reduced in the thymus and periphery of Elf-1−/− mice compared with wild-type (WT) mice. Although Ets-1–deficient mice lack NKT cells altogether, Elf-1−/− mice exhibited only a partial block in NKT-cell development caused by a cell-intrinsic defect in the selection, survival, and maturation of NKT cells. In addition, residual NKT cells found in Elf-1−/− mice produced less cytokine upon antigen stimulation compared with WT NKT cells. Our data demonstrate that Elf-1 plays an important and nonredundant role in the development and function of NKT cells, but is not involved in NK-cell development. PMID:21148815

FGF7 and cell density are required for final differentiation of pancreatic amylase-positive cells from human ES cells.

PubMed

Takizawa-Shirasawa, Sakiko; Yoshie, Susumu; Yue, Fengming; Mogi, Akimi; Yokoyama, Tadayuki; Tomotsune, Daihachiro; Sasaki, Katsunori

2013-12-01

The major molecular signals of pancreatic exocrine development are largely unknown. We examine the role of fibroblast growth factor 7 (FGF7) in the final induction of pancreatic amylase-containing exocrine cells from induced-pancreatic progenitor cells derived from human embryonic stem (hES) cells. Our protocol consisted in three steps: Step I, differentiation of definitive endoderm (DE) by activin A treatment of hES cell colonies; Step II, differentiation of pancreatic progenitor cells by re-plating of the cells of Step I onto 24-well plates at high density and stimulation with all-trans retinoic acid; Step III, differentiation of pancreatic exocrine cells with a combination of FGF7, glucagon-like peptide 1 and nicotinamide. The expression levels of pancreatic endodermal markers such as Foxa2, Sox17 and gut tube endoderm marker HNF1β were up-regulated in both Step I and II. Moreover, in Step III, the induced cells expressed pancreatic markers such as amylase, carboxypeptidase A and chymotrypsinogen B, which were similar to those in normal human pancreas. From day 8 in Step III, cells immunohistochemically positive for amylase and for carboxypeptidase A, a pancreatic exocrine cell product, were induced by FGF7. Pancreatic progenitor Pdx1-positive cells were localized in proximity to the amylase-positive cells. In the absence of FGF7, few amylase-positive cells were identified. Thus, our three-step culture protocol for human ES cells effectively induces the differentiation of amylase- and carboxypeptidase-A-containing pancreatic exocrine cells.

Induction of Epstein-Barr Virus Oncoprotein LMP1 by Transcription Factors AP-2 and Early B Cell Factor

PubMed Central

Noda, Chieko; Narita, Yohei; Watanabe, Takahiro; Yoshida, Masahiro; Ashio, Keiji; Sato, Yoshitaka; Goshima, Fumi; Kanda, Teru; Yoshiyama, Hironori; Tsurumi, Tatsuya; Kimura, Hiroshi

2016-01-01

ABSTRACT Latent membrane protein 1 (LMP1) is a major oncogene essential for primary B cell transformation by Epstein-Barr virus (EBV). Previous studies suggested that some transcription factors, such as PU.1, RBP-Jκ, NF-κB, and STAT, are involved in this expression, but the underlying mechanism is unclear. Here, we identified binding sites for PAX5, AP-2, and EBF in the proximal LMP1 promoter (ED-L1p). We first confirmed the significance of PU.1 and POU domain transcription factor binding for activation of the promoter in latency III. We then focused on the transcription factors AP-2 and early B cell factor (EBF). Interestingly, among the three AP-2-binding sites in the LMP1 promoter, two motifs were also bound by EBF. Overexpression, knockdown, and mutagenesis in the context of the viral genome indicated that AP-2 plays an important role in LMP1 expression in latency II in epithelial cells. In latency III B cells, on the other hand, the B cell-specific transcription factor EBF binds to the ED-L1p and activates LMP1 transcription from the promoter. IMPORTANCE Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is crucial for B cell transformation and oncogenesis of other EBV-related malignancies, such as nasopharyngeal carcinoma and T/NK lymphoma. Its expression is largely dependent on the cell type or condition, and some transcription factors have been implicated in its regulation. However, these previous reports evaluated the significance of specific factors mostly by reporter assay. In this study, we prepared point-mutated EBV at the binding sites of such transcription factors and confirmed the importance of AP-2, EBF, PU.1, and POU domain factors. Our results will provide insight into the transcriptional regulation of the major oncogene LMP1. PMID:26819314

En1 directs superior olivary complex neuron positioning, survival, and expression of FoxP1.

PubMed

Altieri, Stefanie C; Jalabi, Walid; Zhao, Tianna; Romito-DiGiacomo, Rita R; Maricich, Stephen M

2015-12-01

Little is known about the genetic pathways and transcription factors that control development and maturation of central auditory neurons. En1, a gene expressed by a subset of developing and mature superior olivary complex (SOC) cells, encodes a homeodomain transcription factor important for neuronal development in the midbrain, cerebellum, hindbrain and spinal cord. Using genetic fate-mapping techniques, we show that all En1-lineal cells in the SOC are neurons and that these neurons are glycinergic, cholinergic and GABAergic in neurotransmitter phenotype. En1 deletion does not interfere with specification or neural fate of these cells, but does cause aberrant positioning and subsequent death of all En1-lineal SOC neurons by early postnatal ages. En1-null cells also fail to express the transcription factor FoxP1, suggesting that FoxP1 lies downstream of En1. Our data define important roles for En1 in the development and maturation of a diverse group of brainstem auditory neurons. Copyright © 2015 Elsevier Inc. All rights reserved.

TCPs, WUSs, and WINDs: families of transcription factors that regulate shoot meristem formation, stem cell maintenance, and somatic cell differentiation.

PubMed

Ikeda, Miho; Ohme-Takagi, Masaru

2014-01-01

In contrast to somatic mammalian cells, which cannot alter their fate, plant cells can dedifferentiate to form totipotent callus cells and regenerate a whole plant, following treatment with specific phytohormones. However, the regulatory mechanisms and key factors that control differentiation-dedifferentiation and cell totipotency have not been completely clarified in plants. Recently, several plant transcription factors that regulate meristem formation and dedifferentiation have been identified and include members of the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP), WUSCHEL (WUS), and WOUND INDUCED DEDIFFERENTIATION (WIND1) families. WUS and WIND positively control plant cell totipotency, while TCP negatively controls it. Interestingly, TCP is a transcriptional activator that acts as a negative regulator of shoot meristem formation, and WUS is a transcriptional repressor that positively maintains totipotency of the stem cells of the shoot meristem. We describe here the functions of TCP, WUS, and WIND transcription factors in the regulation of differentiation-dedifferentiation by positive and negative transcriptional regulators.

Cancer cell-associated cytoplasmic B7–H4 is induced by hypoxia through hypoxia-inducible factor-1α and promotes cancer cell proliferation

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

Jeon, You-Kyoung; Advanced Research Center for Multiple Myeloma, Inje University College of Medicine, Busan 614-735; Park, Sae-Gwang

2015-04-03

Aberrant B7–H4 expression in cancer tissues serves as a novel prognostic biomarker for poor survival in patients with cancer. However, the factor(s) that induce cancer cell-associated B7–H4 remain to be fully elucidated. We herein demonstrate that hypoxia upregulates B7–H4 transcription in primary CD138{sup +} multiple myeloma cells and cancer cell lines. In support of this finding, analysis of the Multiple Myeloma Genomics Portal (MMGP) data set revealed a positive correlation between the mRNA expression levels of B7–H4 and the endogenous hypoxia marker carbonic anhydrogenase 9. Hypoxia-induced B7–H4 expression was detected in the cytoplasm, but not in cancer cell membranes. Chromatinmore » immunoprecipitation analysis demonstrated binding of hypoxia-inducible factor-1α (HIF-1α) to proximal hypoxia-response element (HRE) sites within the B7–H4 promoter. Knockdown of HIF-1α and pharmacological inhibition of HIF-1α diminished B7–H4 expression. Furthermore, knockdown of cytoplasmic B7–H4 in MCF-7 decreased the S-phase cell population under hypoxia. Finally, MMGP analysis revealed a positive correlation between the transcript levels of B7–H4 and proliferation-related genes including MKI67, CCNA1, and Myc in several patients with multiple myeloma. Our results provide insight into the mechanisms underlying B7–H4 upregulation and its role in cancer cell proliferation in a hypoxic tumor microenvironment. - Highlights: • Hypoxia upregulates B7–H4 transcription and protein expression. • Hypoxia-induced B7–H4 is detected in the cytoplasm, but not on membrane. • ChIP assay reveals a binding of HIF-1α to B7–H4 promoter at HRE site. • Knockdown and pharmacological inhibition of HIF-1α reduce B7–H4 expression. • B7–H4 knockdown decrease the number of cells in S-phase of cell cycle.« less

Platelet-lymphocyte ratio is an independent prognostic factor in patients with ALK-positive non-small-cell lung cancer.

PubMed

Han, Ying; Wang, Jing; Hong, Liping; Sun, Leina; Zhuang, Hongqing; Sun, Bingsheng; Wang, Hua; Zhang, Xinwei; Ren, Xiubao

2017-01-01

As the prognostic value of the neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR) is unclear in patients with ALK-positive non-small-cell lung cancer (NSCLC), this study assessed the importance of these factors was in this patient subset. In 173 patients with primary ALK-positive NSCLC at pathological stages I-IV, neutrophil, platelet, lymphocyte, D-dimer and eosinophil levels were recorded before starting treatment. The patients' median NLR and PLR values were 2.10 and 127.69, respectively. Univariate analyses showed that NLR and PLR values, the D-dimer level and the eosinophil count were all associated with survival. Although multivariate analysis showed PLR to be an independent prognostic factor for overall survival (p = 0.018), NLR was not. PLR is an independent prognostic factor in ALK-positive NSCLC.

Cell Proliferation and Epidermal Growth Factor Signaling in Non-small Cell Lung Adenocarcinoma Cell Lines Are Dependent on Rin1

PubMed Central

Tomshine, Jin C.; Severson, Sandra R.; Wigle, Dennis A.; Sun, Zhifu; Beleford, Daniah A. T.; Shridhar, Vijayalakshmi; Horazdovsky, Bruce F.

2009-01-01

Rin1 is a Rab5 guanine nucleotide exchange factor that plays an important role in Ras-activated endocytosis and growth factor receptor trafficking in fibroblasts. In this study, we show that Rin1 is expressed at high levels in a large number of non-small cell lung adenocarcinoma cell lines, including Hop62, H650, HCC4006, HCC827, EKVX, HCC2935, and A549. Rin1 depletion from A549 cells resulted in a decrease in cell proliferation that was correlated to a decrease in epidermal growth factor receptor (EGFR) signaling. Expression of wild type Rin1 but not the Rab5 guanine nucleotide exchange factor-deficient Rin1 (Rin1Δ) complemented the Rin1 depletion effects, and overexpression of Rin1Δ had a dominant negative effect on cell proliferation. Rin1 depletion stabilized the cell surface levels of EGFR, suggesting that internalization was necessary for robust signaling in A549 cells. In support of this conclusion, introduction of either dominant negative Rab5 or dominant negative dynamin decreased A549 proliferation and EGFR signaling. These data demonstrate that proper internalization and endocytic trafficking are critical for EGFR-mediated signaling in A549 cells and suggest that up-regulation of Rin1 in A549 cell lines may contribute to their proliferative nature. PMID:19570984

A Positive Feedback Loop between Glial Cells Missing 1 and Human Chorionic Gonadotropin (hCG) Regulates Placental hCGβ Expression and Cell Differentiation

PubMed Central

Cheong, Mei-Leng; Wang, Liang-Jie; Chuang, Pei-Yun; Chang, Ching-Wen; Lee, Yun-Shien; Lo, Hsiao-Fan; Tsai, Ming-Song

2015-01-01

Human chorionic gonadotropin (hCG) is composed of a common α subunit and a placenta-specific β subunit. Importantly, hCG is highly expressed in the differentiated and multinucleated syncytiotrophoblast, which is formed via trophoblast cell fusion and stimulated by cyclic AMP (cAMP). Although the ubiquitous activating protein 2 (AP2) transcription factors TFAP2A and TFAP2C may regulate hCGβ expression, it remains unclear how cAMP stimulates placenta-specific hCGβ gene expression and trophoblastic differentiation. Here we demonstrated that the placental transcription factor glial cells missing 1 (GCM1) binds to a highly conserved promoter region in all six hCGβ paralogues by chromatin immunoprecipitation-on-chip (ChIP-chip) analyses. We further showed that cAMP stimulates GCM1 and the CBP coactivator to activate the hCGβ promoter through a GCM1-binding site (GBS1), which also constitutes a previously identified AP2 site. Given that TFAP2C may compete with GCM1 for GBS1, cAMP enhances the association between the hCGβ promoter and GCM1 but not TFAP2C. Indeed, the hCG-cAMP-protein kinase A (PKA) signaling pathway also stimulates Ser269 and Ser275 phosphorylation of GCM1, which recruits CBP to mediate GCM1 acetylation and stabilization. Consequently, hCG stimulates the expression of GCM1 target genes, including the fusogenic protein syncytin-1, to promote placental cell fusion. Our study reveals a positive feedback loop between GCM1 and hCG regulating placental hCGβ expression and cell differentiation. PMID:26503785

Antitumour effects of PLC-gamma1-(SH2)2-TAT fusion proteins on EGFR/c-erbB-2-positive breast cancer cells.

PubMed

Katterle, Y; Brandt, B H; Dowdy, S F; Niggemann, B; Zänker, K S; Dittmar, T

2004-01-12

Due to its pivotal role in the growth factor-mediated tumour cell migration, the adaptor protein phospholipase C-gamma1 (PLC-gamma1) is an appropriate target to block ultimately the spreading of EGFR/c-erbB-2-positive tumour cells, thereby minimising metastasis formation. Here, we present an approach to block PLC-gamma1 activity by using protein-based PLC-gamma1 inhibitors consisting of PLC-gamma1 SH2 domains, which were fused to the TAT-transduction domain to ensure a high protein transduction efficiency. Two proteins were generated containing one PLC-gamma1-SH2-domain (PS1-TAT) or two PLC-gamma1-SH2 domains (PS2-TAT). PS2-TAT treatment of the EGFR/c-erbB-2-positive cell line MDA-HER2 resulted in a reduction of the EGF-mediated PLC-gamma1 tyrosine phosphorylation of about 30%, concomitant with a complete abrogation of the EGF-driven calcium influx. In addition to this, long-term PS2-TAT treatment both reduces the EGF-mediated migration of about 75% combined with a markedly decreased time locomotion of single MDA-HER2 cells as well as decreases the proliferation of MDA-HER2 cells by about 50%. Due to its antitumoral capacity on EGFR/c-erbB-2-positive breast cancer cells, we conclude from our results that the protein-based PLC-gamma1 inhibitor PS2-TAT may be a means for novel adjuvant antitumour strategies to minimise metastasis formation because of the blockade of cell migration and proliferation.

Plasma soluble factor following two decades prolonged suppressive antiretroviral therapy in HIV-1-positive males: A cross-sectional study.

PubMed

Sperk, Maike; Zhang, Wang; Nowak, Piotr; Neogi, Ujjwal

2018-02-01

Acute human immunodeficiency virus (HIV) infection is associated with a marked induction of several pathways that are linked to inflammation and CD4 T-cell depletion. Many of these processes do not fully resolve on short-term combination antiretroviral therapy (cART) (<5 years), despite complete and durable suppression of viremia. The effects of long-term (>15 years) successful antiretroviral therapy (ART) and the linkage between levels of biomarkers remain unclear. Therefore, the present study aims to assess the host plasma proteome in a well-defined clinical material from HIV-1-positive male patients on successful long-term ART (>15 years) and compared them with age-matched healthy controls and treatment-naïve male patients with viremia in a cross-sectional manner.Plasma samples were obtained from 3 categories of age-matched HIV-1-positive male patients on long-term successfully (ART, n = 10) with a median (Interquartile range, IQR) of 19 (17-20) years, treatment-naïve patients with viremia (VP, n = 14), and HIV-1-negative persons (HC, n = 11). Plasma proteome was analyzed using the proximity extension assay targeting 92 factors. Statistical analyses were performed with GraphPad Prism v7, R-packages, and Qlucore Omics Explorer v3.2. Functional enrichment analysis was performed by Kyoto Encyclopedia of Genes and Genomes (KEGG), and interactions of specific molecules were identified using Path Designer integrated into Ingenuity Pathway Analysis (IPA).Group wise comparison identified 53 soluble factors, which differed between the groups (P < .05). Cluster analysis identified 13 discrete soluble factors (CD8A, CRTAM, CXCL13, EGF, CD5, CD40, CXCL9, Gal-1, IL12RB1, KLRD1, PD-1, CASP-8 and TNFRSF9) between the studied groups (adjusted P < .001). The long-term successfully ART-treated individuals clustered and networked with the HC while VPs clustered separately. All of the proinflammatory cytokines and chemokines were normalized back to levels of

NLR Nod1 signaling promotes survival of BCR-engaged mature B cells through up-regulated Nod1 as a positive outcome

PubMed Central

Asano, Masanao; Li, Yue-Sheng; Núñez, Gabriel

2017-01-01

Although B cell development requires expression of the B cell antigen receptor (BCR), it remains unclear whether engagement of self-antigen provides a positive impact for most B cells. Here, we show that BCR engagement by self-ligand during development in vivo results in up-regulation of the Nod-like receptor member Nod1, which recognizes the products of intestinal commensal bacteria. In anti-thymocyte/Thy-1 autoreactive BCR knock-in mice lacking self–Thy-1 ligand, immunoglobulin light chain editing occurred, generating B cells with up-regulated Nod1, including follicular and marginal zone B cells with natural autoreactivity. This BCR editing with increased Nod1 resulted in preferential survival. In normal adult mice, most mature B cells are enriched for Nod1 up-regulated cells, and signaling through Nod1 promotes competitive survival of mature B cells. These findings demonstrate a role for microbial products in promoting survival of mature B cells through up-regulated Nod1, providing a positive effect of BCR engagement on development of most B cells. PMID:28878001

A Pdx-1-Regulated Soluble Factor Activates Rat and Human Islet Cell Proliferation

PubMed Central

Hayes, Heather L.; Zhang, Lu; Becker, Thomas C.; Haldeman, Jonathan M.; Stephens, Samuel B.; Arlotto, Michelle; Moss, Larry G.; Newgard, Christopher B.

2016-01-01

The homeodomain transcription factor Pdx-1 has important roles in pancreas and islet development as well as in β-cell function and survival. We previously reported that Pdx-1 overexpression stimulates islet cell proliferation, but the mechanism remains unclear. Here, we demonstrate that overexpression of Pdx-1 triggers proliferation largely by a non-cell-autonomous mechanism mediated by soluble factors. Consistent with this idea, overexpression of Pdx-1 under the control of a β-cell-specific promoter (rat insulin promoter [RIP]) stimulates proliferation of both α and β cells, and overexpression of Pdx-1 in islets separated by a Transwell membrane from islets lacking Pdx-1 overexpression activates proliferation in the untreated islets. Microarray and gene ontology (GO) analysis identified inhibin beta-B (Inhbb), an activin subunit and member of the transforming growth factor β (TGF-β) superfamily, as a Pdx-1-responsive gene. Overexpression of Inhbb or addition of activin B stimulates rat islet cell and β-cell proliferation, and the activin receptors RIIA and RIIB are required for the full proliferative effects of Pdx-1 in rat islets. In human islets, Inhbb overexpression stimulates total islet cell proliferation and potentiates Pdx-1-stimulated proliferation of total islet cells and β cells. In sum, this study identifies a mechanism by which Pdx-1 induces a soluble factor that is sufficient to stimulate both rat and human islet cell proliferation. PMID:27620967

Transforming growth factor β-mediated suppression of antitumor T cells requires FoxP1 transcription factor expression.

PubMed

Stephen, Tom L; Rutkowski, Melanie R; Allegrezza, Michael J; Perales-Puchalt, Alfredo; Tesone, Amelia J; Svoronos, Nikolaos; Nguyen, Jenny M; Sarmin, Fahmida; Borowsky, Mark E; Tchou, Julia; Conejo-Garcia, Jose R

2014-09-18

Tumor-reactive T cells become unresponsive in advanced tumors. Here we have characterized a common mechanism of T cell unresponsiveness in cancer driven by the upregulation of the transcription factor Forkhead box protein P1 (Foxp1), which prevents CD8⁺ T cells from proliferating and upregulating Granzyme-B and interferon-γ in response to tumor antigens. Accordingly, Foxp1-deficient lymphocytes induced rejection of incurable tumors and promoted protection against tumor rechallenge. Mechanistically, Foxp1 interacted with the transcription factors Smad2 and Smad3 in preactivated CD8⁺ T cells in response to microenvironmental transforming growth factor-β (TGF-β), and was essential for its suppressive activity. Therefore, Smad2 and Smad3-mediated c-Myc repression requires Foxp1 expression in T cells. Furthermore, Foxp1 directly mediated TGF-β-induced c-Jun transcriptional repression, which abrogated T cell activity. Our results unveil a fundamental mechanism of T cell unresponsiveness different from anergy or exhaustion, driven by TGF-β signaling on tumor-associated lymphocytes undergoing Foxp1-dependent transcriptional regulation. Copyright © 2014 Elsevier Inc. All rights reserved.

Twist1-positive epithelial cells retain adhesive and proliferative capacity throughout dissemination

PubMed Central

Shamir, Eliah R.; Coutinho, Kester; Georgess, Dan; Auer, Manfred

2016-01-01

ABSTRACT Dissemination is the process by which cells detach and migrate away from a multicellular tissue. The epithelial-to-mesenchymal transition (EMT) conceptualizes dissemination in a stepwise fashion, with downregulation of E-cadherin leading to loss of intercellular junctions, induction of motility, and then escape from the epithelium. This gain of migratory activity is proposed to be mutually exclusive with proliferation. We previously developed a dissemination assay based on inducible expression of the transcription factor Twist1 and here utilize it to characterize the timing and dynamics of intercellular adhesion, proliferation and migration during dissemination. Surprisingly, Twist1+ epithelium displayed extensive intercellular junctions, and Twist1– luminal epithelial cells could still adhere to disseminating Twist1+ cells. Although proteolysis and proliferation were both observed throughout dissemination, neither was absolutely required. Finally, Twist1+ cells exhibited a hybrid migration mode; their morphology and nuclear deformation were characteristic of amoeboid cells, whereas their dynamic protrusive activity, pericellular proteolysis and migration speeds were more typical of mesenchymal cells. Our data reveal that epithelial cells can disseminate while retaining competence to adhere and proliferate. PMID:27402962

Histiocyte-like cells expressing factor XIIIa do not belong to the neoplastic cell population in malignant fibrous histiocytoma.

PubMed

Szollosi, Zoltan; Nemeth, Tamas; Egervari, Kristof; Nemes, Zoltan

2005-01-01

The term malignant fibrous histiocytoma (MFH) is widely used for pleomorphic soft tissue sarcomas without a specific line of differentiation. MFH is included in the category of fibrohistiocytic soft tissue tumors. MFH has a broad range of histological appearances, and it has several subtypes. All of these subtypes are composed of spindled fibroblast-like cells, undifferentiated cells, and histiocytic or histiocyte-like cells. A large number of fibroblast-like and pleomorphic cells express factor XIIIa in MFH. The cytological pleomorphism of factor XIIIa cells suggests that these cells may belong to the neoplastic population. It is equally possible that the factor XIIIa-positive cells are only activated stromal cells. The relation of factor XIIIa-positive cells to the neoplastic cell population in MFH is addressed in the present study. A morphometric approach compares the measure of nuclear pleomorphism of the factor XIIIa-positive cells with that of the factor XIIIa-negative tumor cells in high-grade MFH. The immunohistochemical approach compares the factor XIIIa-positive and -negative cell populations with regard to mutations of p53 tumor suppressor gene in p53-positive MFH cases. We selected 58 cases of soft tissue pleomorphic or storiform-pleomorphic MFH on the basis of histopathological examinations. A combination of incident light immunofluorescence for factor XIIIa and transmitted light examination for nuclear staining was used for morphometrical analysis. We found cytoplasmic factor XIIIa positivity in at least 2% of cells in 39 cases; the number of factor XIIIa-positive cells was under 0.5% in two cases, and the number of factor-positive cells ranged between 0.5% and 2% in 13 cases. Eighteen cases were analyzed with nuclear morphometry. We found that mean nuclear area and mean nuclear Ferret diameter in factor XIIIa-positive cells differed significantly from those of the tumor cells in all cases. The mean nuclear roundness factor differed significantly only

Tissue factor expression by endothelial cells in sickle cell anemia.

PubMed

Solovey, A; Gui, L; Key, N S; Hebbel, R P

1998-05-01

The role of the vascular endothelium in activation of the coagulation system, a fundamental homeostatic mechanism of mammalian biology, is uncertain because there is little evidence indicating that endothelial cells in vivo express tissue factor (TF), the system's triggering mechanism. As a surrogate for vessel wall endothelium, we examined circulating endothelial cells (CEC) from normals and patients with sickle cell anemia, a disease associated with activation of coagulation. We find that sickle CEC abnormally express TF antigen (expressed as percent CEC that are TF-positive), with 66+/-13% positive in sickle patients in steady-state, 83+/-19% positive in sickle patients presenting with acute vasoocclusive episodes, and only 10+/-13% positive in normal controls. Repeated samplings confirmed this impression that TF expression is greater when sickle patients develop acute vasoocclusive episodes. Sickle CEC are also positive for TF mRNA, with excellent concurrence between antigen and mRNA expression. The TF expressed on the antigen-positive CEC is functional, as demonstrated by a binding assay for Factor VIIa and a chromogenic assay sensitive to generation of Factor Xa. By establishing that endothelial cells in vivo can express TF, these data imply that the vast endothelial surface area does provide an important pathophysiologic trigger for coagulation activation.

Knockdown of microtubule actin crosslinking factor 1 inhibits cell proliferation in MC3T3-E1 osteoblastic cells

PubMed Central

Hu, Lifang; Su, Peihong; Li, Runzhi; Yan, Kun; Chen, Zhihao; Shang, Peng; Qian, Airong

2015-01-01

Microtubule actin crosslinking factor 1 (MACF1), a widely expressed cytoskeletal linker, plays important roles in various cells by regulating cytoskeleton dynamics. However, its role in osteoblastic cells is not well understood. Based on our previous findings that the association of MACF1 with F-actin and microtubules in osteoblast-like cells was altered under magnetic force conditions, here, by adopting a stable MACF1-knockdown MC3T3-E1 osteoblastic cell line, we found that MACF1 knockdown induced large cells with a binuclear/multinuclear structure. Further, immunofluorescence staining showed disorganization of F-actin and microtubules in MACF1-knockdown cells. Cell counting revealed significant decrease of cell proliferation and cell cycle analysis showed an S phase cell cycle arrest in MACF1-knockdown cells. Moreover and interestingly, MACF1 knockdown showed a potential effect on cellular MTT reduction activity and mitochondrial content, suggesting an impact on cellular metabolic activity. These results together indicate an important role of MACF1 in regulating osteoblastic cell morphology and function. [BMB Reports 2015; 48(10): 583-588] PMID:26277981

Knockdown of microtubule actin crosslinking factor 1 inhibits cell proliferation in MC3T3-E1 osteoblastic cells.

PubMed

Hu, Lifang; Su, Peihong; Li, Runzhi; Yan, Kun; Chen, Zhihao; Shang, Peng; Qian, Airong

2015-10-01

Microtubule actin crosslinking factor 1 (MACF1), a widely expressed cytoskeletal linker, plays important roles in various cells by regulating cytoskeleton dynamics. However, its role in osteoblastic cells is not well understood. Based on our previous findings that the association of MACF1 with F-actin and microtubules in osteoblast-like cells was altered under magnetic force conditions, here, by adopting a stable MACF1-knockdown MC3T3-E1 osteoblastic cell line, we found that MACF1 knockdown induced large cells with a binuclear/multinuclear structure. Further, immunofluorescence staining showed disorganization of F-actin and microtubules in MACF1-knockdown cells. Cell counting revealed significant decrease of cell proliferation and cell cycle analysis showed an S phase cell cycle arrest in MACF1-knockdown cells. Moreover and interestingly, MACF1 knockdown showed a potential effect on cellular MTT reduction activity and mitochondrial content, suggesting an impact on cellular metabolic activity. These results together indicate an important role of MACF1 in regulating osteoblastic cell morphology and function.

The GTPase Rac-1 controls cell fate in the thymus by diverting thymocytes from positive to negative selection.

PubMed

Gomez, M; Kioussis, D; Cantrell, D A

2001-11-01

The positive selection of CD4 or CD8 single-positive mature peripheral T lymphocytes and the deletion of self-reactive cells are crucial for central tolerance in the peripheral immune system. Previously, the guanine nucleotide binding protein Rac-1 has been shown to control pre-T cell development. The present report now describes the actions of Rac-1 in thymocyte selection. The study reveals that this molecule has the striking and unique ability to efficiently divert cells from positive selection into a pathway of negative selection and deletion. The ability of Rac-1 to switch thymocytes from a destiny of positive to negative selection identifies this molecule as a critical regulator of the developmental processes in T cells that are essential for immune homeostasis.

Mouse A6-positive hepatic oval cells derived from embryonic stem cells.

PubMed

Yin, Dong-zhi; Cai, Ji-ye; Zheng, Qi-chang; Chen, Zheng-wei; Zhao, Jing-xian; Yuan, You-neng

2014-02-01

Oval cells have a potential to differentiate into a variety of cell lineages including hepatocytes and biliary epithelia. Several models have been established to activate the oval cells by incorporating a variety of toxins and carcinogens, alone or combined with surgical treatment. Those models are obviously not suitable for the study on human hepatic oval cells. It is necessary to establish a new and efficient model to study the human hepatic oval cells. In this study, the hepatocyte growth factor (HGF) and epidermal growth factor (EGF) were used to induce differentiation of mouse embryonic stem (ES) cells into hepatic oval cells. We first confirmed that hepatic oval cells derived from ES cells, which are bipotential, do exist during the course of mouse ES cells' differentiation into hepatic parenchymal cells. RT-PCR and transmission electron microscopy were applied in this study. The ratio of Sca-1+/CD34+ cells sorted by FACS in the induction group was increased from day 4 and reached the maximum on the day 8, whereas that in the control group remained at a low level. The differentiation ratio of Sca-1+/CD34+ cells in the induction group was significantly higher than that in the control group. About 92.48% of the sorted Sca-1+/CD34+ cells on the day 8 were A6 positive. Highly purified A6+/Sca-1+/CD34+ hepatic oval cells derived from ES cells could be obtained by FACS. The differentiation ratio of hepatic oval cells in the induction group (up to 4.46%) was significantly higher than that in the control group. The number of hepatic oval cells could be increased significantly by HGF and EGF. The study also examined the ultrastructures of ES-derived hepatic oval cells' membrane surface by atomic force microscopy. The ES-derived hepatic oval cells cultured and sorted by our protocols may be available for the future clinical application.

RUNX1 positively regulates the ErbB2/HER2 signaling pathway through modulating SOS1 expression in gastric cancer cells.

PubMed

Mitsuda, Yoshihide; Morita, Ken; Kashiwazaki, Gengo; Taniguchi, Junichi; Bando, Toshikazu; Obara, Moeka; Hirata, Masahiro; Kataoka, Tatsuki R; Muto, Manabu; Kaneda, Yasufumi; Nakahata, Tatsutoshi; Liu, Pu Paul; Adachi, Souichi; Sugiyama, Hiroshi; Kamikubo, Yasuhiko

2018-04-23

The dual function of runt-related transcriptional factor 1 (RUNX1) as an oncogene or oncosuppressor has been extensively studied in various malignancies, yet its role in gastric cancer remains elusive. Up-regulation of the ErbB2/HER2 signaling pathway is frequently-encountered in gastric cancer and contributes to the maintenance of these cancer cells. This signaling cascade is partly mediated by son of sevenless homolog (SOS) family, which function as adaptor proteins in the RTK cascades. Herein we report that RUNX1 regulates the ErbB2/HER2 signaling pathway in gastric cancer cells through transactivating SOS1 expression, rendering itself an ideal target in anti-tumor strategy toward this cancer. Mechanistically, RUNX1 interacts with the RUNX1 binding DNA sequence located in SOS1 promoter and positively regulates it. Knockdown of RUNX1 led to the decreased expression of SOS1 as well as dephosphorylation of ErbB2/HER2, subsequently suppressed the proliferation of gastric cancer cells. We also found that our novel RUNX inhibitor (Chb-M') consistently led to the deactivation of the ErbB2/HER2 signaling pathway and was effective against several gastric cancer cell lines. Taken together, our work identified a novel interaction of RUNX1 and the ErbB2/HER2 signaling pathway in gastric cancer, which can potentially be exploited in the management of this malignancy.

E2F1 transcription factor and its impact on growth factor and cytokine signaling.

PubMed

Ertosun, Mustafa Gokhan; Hapil, Fatma Zehra; Osman Nidai, Ozes

2016-10-01

E2F1 is a transcription factor involved in cell cycle regulation and apoptosis. The transactivation capacity of E2F1 is regulated by pRb. In its hypophosphorylated form, pRb binds and inactivates DNA binding and transactivating functions of E2F1. The growth factor stimulation of cells leads to activation of CDKs (cyclin dependent kinases), which in turn phosphorylate Rb and hyperphosphorylated Rb is released from E2F1 or E2F1/DP complex, and free E2F1 can induce transcription of several genes involved in cell cycle entry, induction or inhibition of apoptosis. Thus, growth factors and cytokines generally utilize E2F1 to direct cells to either fate. Furthermore, E2F1 regulates expressions of various cytokines and growth factor receptors, establishing positive or negative feedback mechanisms. This review focuses on the relationship between E2F1 transcription factor and cytokines (IL-1, IL-2, IL-3, IL-6, TGF-beta, G-CSF, LIF), growth factors (EGF, KGF, VEGF, IGF, FGF, PDGF, HGF, NGF), and interferons (IFN-α, IFN-β and IFN-γ). Copyright © 2016 Elsevier Ltd. All rights reserved.

Behaviour of CD11b-Positive Cells in an Animal Model of Laser-Induced Choroidal Neovascularisation.

PubMed

Li, Lu; Heiduschka, Peter; Alex, Anne F; Niekämper, Daniel; Eter, Nicole

2017-01-01

Immune cells, e.g. microglial cells of the retina, appear to be involved in pathological processes in neovascular age-related macular degeneration. Therefore, the purpose of this study was to immunohistochemically check the expression of various factors and cytokines by CD11b-positive (CD11b+) immune cells in an animal model of choroidal neovascularisation (CNV). We used the animal model of laser-induced CNV in mice. Eyes were isolated at 1, 4, 7, and 14 days after laser treatment. Cryosections were prepared and checked immunohistochemically for the presence of different growth factors and cytokines on microglial cells and other immune cells identified by CD11b immunoreactivity. We found that the number of CD11b+ cells at the laser spots increased dramatically 4 days after laser treatment, the majority of them entering the laser spot most probably by migration. CD11b+ cells in the laser spot were positive for a variety of pro-angiogenic factors, such as PDGF-β, FGF-1, FGF-2, and TGF-β1. They were also positive for some inflammatory cytokines, in particular TNF-α, IL-6, and CXCL1. In non-treated retinas, CD11b+ cells showed almost no immunoreactivity for these proteins. Microglial cells, macrophages, and other CD11b+ cells may promote the neovascularisation in the laser spot and show a moderate inflammatory behaviour. Immunoreactivity for most of these molecules was found to decrease during the time of observation. Modulation of immune cell activity may thus be a tool to reduce the extent of CNV. © 2017 S. Karger AG, Basel.

Treatment with specific soluble factors promotes the functional maturation of transcription factor-mediated, pancreatic transdifferentiated cells.

PubMed

Motoyama, Hiroaki; Kobayashi, Akira; Yokoyama, Takahide; Shimizu, Akira; Sakai, Hiroshi; Notake, Tsuyoshi; Fukushima, Kentaro; Miyagawa, Shin-Ichi

2018-01-01

Pancreatic lineage-specific transcription factors (TFs) display instructive roles in converting adult cells to endocrine pancreatic cells through a process known as transdifferentiation. However, little is known about potential factors capable of accelerating transdifferentiation following transduction to achieve the functional maturation of transdifferentiated cells. In this study, we demonstrated, using adult liver-derived progenitor cells, that soluble factors utilized in pancreatic differentiation protocols of pluripotent stem cells promote functional maturation of TFs-mediated transdifferentiated cells. Treatment with an N2 supplement in combination with three soluble factors (glucagon-like peptide-1 [GLP-1] receptor agonist, notch inhibitor, and transforming growth factor-β [TGF-β] inhibitor) enhanced liver-to-pancreas transdifferentiation based on the following findings: i) the incidence of c-peptide-positive cells increased by approximately 1.2-fold after the aforementioned treatment; ii) the c-peptide expression level in the treated cells increased by approximately 12-fold as compared with the level in the untreated cells; iii) the treated cells secreted insulin in a glucose-dependent manner, whereas the untreated cells did not; and iv) transplantation of treated-transdifferentiated cells into streptozotocin-induced immunodeficient diabetic mice led to the amelioration of hyperglycemia. These results suggest that treatment with specific soluble factors promotes the functional maturation of transdifferentiated cells. Our findings could facilitate the development of new modalities for cell-replacement therapy for patients with diabetes.

Effect of Weight Reduction on Cardiovascular Risk Factors and CD34-positive Cells in Circulation

PubMed Central

Mikirova, Nina A; Casciari, Joseph J; Hunninghake, Ronald E; Beezley, Margaret M

2011-01-01

Being overweight or obese is associated with an increased risk for the development of non-insulin-dependent diabetes mellitus, hypertension, and cardiovascular disease. Dyslipidemia of obesity is characterized by elevated fasting triglycerides and decreased high-density lipoprotein-cholesterol concentrations. Endothelial damage and dysfunction is considered to be a major underlying mechanism for the elevated cardiovascular risk associated with increased adiposity. Alterations in endothelial cells and stem/endothelial progenitor cell function associated with overweight and obesity predispose to atherosclerosis and thrombosis. In our study, we analyzed the effect of a low calorie diet in combination with oral supplementation by vitamins, minerals, probiotics and human chorionic gonadotropin (hCG, 125-180 IUs) on the body composition, lipid profile and CD34-positive cells in circulation. During this dieting program, the following parameters were assessed weekly for all participants: fat free mass, body fat, BMI, extracellular/intracellular water, total body water and basal metabolic rate. For part of participants blood chemistry parameters and circulating CD34-positive cells were determined before and after dieting. The data indicated that the treatments not only reduced body fat mass and total mass but also improved the lipid profile. The changes in body composition correlated with the level of lipoproteins responsible for the increased cardiovascular risk factors. These changes in body composition and lipid profile parameters coincided with the improvement of circulatory progenitor cell numbers. As the result of our study, we concluded that the improvement of body composition affects the number of stem/progenitor cells in circulation. PMID:21850193

Effect of weight reduction on cardiovascular risk factors and CD34-positive cells in circulation.

PubMed

Mikirova, Nina A; Casciari, Joseph J; Hunninghake, Ronald E; Beezley, Margaret M

2011-01-01

Being overweight or obese is associated with an increased risk for the development of non-insulin-dependent diabetes mellitus, hypertension, and cardiovascular disease. Dyslipidemia of obesity is characterized by elevated fasting triglycerides and decreased high-density lipoprotein-cholesterol concentrations. Endothelial damage and dysfunction is considered to be a major underlying mechanism for the elevated cardiovascular risk associated with increased adiposity. Alterations in endothelial cells and stem/endothelial progenitor cell function associated with overweight and obesity predispose to atherosclerosis and thrombosis. In our study, we analyzed the effect of a low calorie diet in combination with oral supplementation by vitamins, minerals, probiotics and human chorionic gonadotropin (hCG, 125-180 IUs) on the body composition, lipid profile and CD34-positive cells in circulation. During this dieting program, the following parameters were assessed weekly for all participants: fat free mass, body fat, BMI, extracellular/intracellular water, total body water and basal metabolic rate. For part of participants blood chemistry parameters and circulating CD34-positive cells were determined before and after dieting. The data indicated that the treatments not only reduced body fat mass and total mass but also improved the lipid profile. The changes in body composition correlated with the level of lipoproteins responsible for the increased cardiovascular risk factors. These changes in body composition and lipid profile parameters coincided with the improvement of circulatory progenitor cell numbers. As the result of our study, we concluded that the improvement of body composition affects the number of stem/progenitor cells in circulation.

Paracrine Engineering of Human Explant-Derived Cardiac Stem Cells to Over-Express Stromal-Cell Derived Factor 1α Enhances Myocardial Repair.

PubMed

Tilokee, Everad L; Latham, Nicholas; Jackson, Robyn; Mayfield, Audrey E; Ye, Bin; Mount, Seth; Lam, Buu-Khanh; Suuronen, Erik J; Ruel, Marc; Stewart, Duncan J; Davis, Darryl R

2016-07-01

First generation cardiac stem cell products provide indirect cardiac repair but variably produce key cardioprotective cytokines, such as stromal-cell derived factor 1α, which opens the prospect of maximizing up-front paracrine-mediated repair. The mesenchymal subpopulation within explant derived human cardiac stem cells underwent lentiviral mediated gene transfer of stromal-cell derived factor 1α. Unlike previous unsuccessful attempts to increase efficacy by boosting the paracrine signature of cardiac stem cells, cytokine profiling revealed that stromal-cell derived factor 1α over-expression prevented lv-mediated "loss of cytokines" through autocrine stimulation of CXCR4+ cardiac stem cells. Stromal-cell derived factor 1α enhanced angiogenesis and stem cell recruitment while priming cardiac stem cells to readily adopt a cardiac identity. As compared to injection with unmodified cardiac stem cells, transplant of stromal-cell derived factor 1α enhanced cells into immunodeficient mice improved myocardial function and angiogenesis while reducing scarring. Increases in myocardial stromal-cell derived factor 1α content paralleled reductions in myocyte apoptosis but did not influence long-term engraftment or the fate of transplanted cells. Transplantation of stromal-cell derived factor 1α transduced cardiac stem cells increased the generation of new myocytes, recruitment of bone marrow cells, new myocyte/vessel formation and the salvage of reversibly damaged myocardium to enhance cardiac repair after experimental infarction. Stem Cells 2016;34:1826-1835. © 2016 AlphaMed Press.

Lack of association of programmed cell death 1 gene (PDCD1) polymorphisms with susceptibility to chronic urticaria in patients with positive autologous serum skin test.

PubMed

Brzoza, Z; Grzeszczak, W; Trautsolt, W; Moczulski, D

2012-01-01

Autoimmune mechanisms play an important role in the pathophysiology of chronic urticaria (CU), and the autologous serum skin test (ASST) helps to identify patients with autoreactive CU. One of the factors involved in autoreactive mechanisms is the cell surface receptor programmed death-1 which is encoded by the programmed cell death 1 gene (PDCD1). To investigate whether PDCD1 polymorphisms influence susceptibility to CU. We enrolled 93 ASST-positive patients with CU and a control group consisting of 105 healthy volunteers. In all individuals, PD1.3 (7146 A/G; rs 11568821) and PD1.5 (7785 C/T; rs 2227981) polymorphisms were analyzed. No statistically significant differences were found between CU patients and controls for allele or genotype distribution. We also did not observe any association between PDCD1 genotypes and severity of urticaria or age of disease onset. PD1.3 and PD1.5 polymorphisms were not proven to be implicated in susceptibility to ASST-positive CU in the Polish population. A more comprehensive analysis of the 2q33-2q37 genomic region might reveal whether variants of 1 or more of the genes in this region are involved in susceptibility to CU.

Stromal cell-derived factor-1 significantly induces proliferation, migration, and collagen type I expression in a human periodontal ligament stem cell subpopulation.

PubMed

Du, Lingqian; Yang, Pishan; Ge, Shaohua

2012-03-01

The pivotal role of chemokine stromal cell-derived factor-1 (SDF-1) in bone marrow mesenchymal stem cells recruitment and tissue regeneration has already been reported. However, its roles in human periodontal ligament stem cells (PDLSCs) remain unknown. PDLSCs are regarded as candidates for periodontal tissue regeneration and are used in stem cell-based periodontal tissue engineering. The expression of chemokine receptors on PDLSCs and the migration of these cells induced by chemokines and their subsequent function in tissue repair may be a crucial procedure for periodontal tissue regeneration. PDL tissues were obtained from clinically healthy premolars extracted for orthodontic reasons and used to isolate single-cell colonies by the limited-dilution method. Immunocytochemical staining was used to detect the expression of the mesenchymal stem cell marker STRO-1. Differentiation potentials were assessed by alizarin-red staining and oil-red O staining. The expression of SDF-1 receptor CXCR4 was evaluated by real-time polymerase chain reaction (PCR) and immunocytochemical staining. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and bromodeoxyuridine incorporation assay were used to determine the viability and proliferation of the PDLSC subpopulation. Expression of collagen type I and alkaline phosphatase was detected by real-time PCR to determine the effect of SDF-1 on cells differentiation. Twenty percent of PDL single-cell colonies expressed STRO-1 positively, and this specific subpopulation was positive for CXCR4 and formed minerals and lipid vacuoles after 4 weeks induction. SDF-1 significantly increased proliferation and stimulated the migration of this PDLSC subpopulation at concentrations between 100 and 400 ng/mL. CXCR4 neutralizing antibody could block cell proliferation and migration, suggesting that SDF-1 exerted its effects on cells through CXCR4. SDF-1 promoted collagen type I level significantly but had little effect on alkaline

Insulin-like growth factor-1 signaling in renal cell carcinoma.

PubMed

Tracz, Adam F; Szczylik, Cezary; Porta, Camillo; Czarnecka, Anna M

2016-07-12

Renal cell carcinoma (RCC) incidence is highest in highly developed countries and it is the seventh most common neoplasm diagnosed. RCC management include nephrectomy and targeted therapies. Type 1 insulin-like growth factor (IGF-1) pathway plays an important role in cell proliferation and apoptosis resistance. IGF-1 and insulin share overlapping downstream signaling pathways in normal and cancer cells. IGF-1 receptor (IGF1R) stimulation may promote malignant transformation promoting cell proliferation, dedifferentiation and inhibiting apoptosis. Clear cell renal cell carcinoma (ccRCC) patients with IGF1R overexpression have 70 % increased risk of death compared to patients who had tumors without IGF1R expression. IGF1R signaling deregulation may results in p53, WT, BRCA1, VHL loss of function. RCC cells with high expression of IGF1R are more resistant to chemotherapy than cells with low expression. Silencing of IGF1R increase the chemosensitivity of ccRCC cells and the effect is greater in VHL mutated cells. Understanding the role of IGF-1 signaling pathway in RCC may result in development of new targeted therapeutic interventions. First preclinical attempts with anti-IGF-1R monoclonal antibodies or fragment antigen-binding (Fab) fragments alone or in combination with an mTOR inhibitor were shown to inhibit in vitro growth and reduced the number of colonies formed by of RCC cells.

The death-inducer obliterator 1 (Dido1) gene regulates embryonic stem cell self-renewal.

PubMed

Liu, Yinyin; Kim, Hyeung; Liang, Jiancong; Lu, Weisi; Ouyang, Bin; Liu, Dan; Songyang, Zhou

2014-02-21

The regulatory network of factors that center on master transcription factors such as Oct4, Nanog, and Sox2 help maintain embryonic stem (ES) cells and ensure their pluripotency. The target genes of these master transcription factors define the ES cell transcriptional landscape. In this study, we report our findings that Dido1, a target of canonical transcription factors such as Oct4, Sox2, and Nanog, plays an important role in regulating ES cell maintenance. We found that depletion of Dido1 in mouse ES cells led to differentiation, and ectopic expression of Dido1 inhibited differentiation induced by leukemia inhibitory factor withdrawal. We further demonstrated that whereas Nanog and Oct4 could occupy the Dido1 locus and promote its transcription, Dido1 could also target to the loci of pluripotency factors such as Nanog and Oct4 and positively regulate their expression. Through this feedback and feedforward loop, Dido1 is able to regulate self-renewal of mouse ES cells.

Recruitment of bone marrow-derived cells to the periodontal ligament via the stromal cell-derived factor-1/C-X-C chemokine receptor type 4 axis.

PubMed

Kaku, M; Kitami, M; Rosales Rocabado, J M; Ida, T; Akiba, Y; Uoshima, K

2017-08-01

The periodontal ligament (PDL) is a non-mineralized connective tissue that exists between the alveolar bone and root surface cementum and plays important roles in tooth function. The PDL harbors a remarkable reserve of multipotent stem cells, which maintain various types of cells. However, the sources of these stem cells, other than their developmental origin, are not well understood. To elucidate the recruitment of bone marrow (BM)-derived stem cells in the PDL, green fluorescent protein (GFP)-expressing BM-derived cells were transplanted into the femoral BM of immunodeficient rats, and the distribution and expression of stem cell markers in the PDL were analyzed in vivo. To evaluate the functional significance of BM-derived cells to the PDL, tooth replantation was performed and the expression of stromal cell-derived factor (SDF)-1, a critical chemotactic signal for mesenchymal stem cell recruitment, was analyzed. To confirm the SDF-1-dependency of BM-derived cell migration to the PDL, PDL-conditioned medium (CM) was prepared, and BM-derived cell migration was analyzed using a transwell culture system. Four weeks after cell transplantation, GFP-positive cells were detected in the PDL, and some of them were also positive for stem cell markers (i.e., CD29, SSEA4, and αSMA). Seven days after tooth replantation, the number of GFP- and SDF-1-positive cells significantly increased in PDL. Concurrently, the concentration of SDF-1 and the number of colony-forming units of fibroblasts in peripheral blood were increased. BM-derived cell migration increased in PDL-CM and was inhibited by an inhibitor of C-X-C chemokine receptor type 4 (CXCR4), an SDF-1 receptor. These results indicate that stem cells and their progeny in PDL are not only derived from their developmental origin but are also supplied from the BM via the blood as the need arises. Moreover, this BM-derived cell recruitment appears to be regulated, at least partially, by the SDF-1/CXCR4 axis. © 2017 John Wiley

Targeted σ factor turnover inserts negative control into a positive feedback loop

PubMed Central

Donohue, Timothy J.

2009-01-01

Summary Since their classification as members of the σ70 super-family, Group IV alternative σ factors have been found to control gene expression in response to diverse environmental or stress signals. Activity of the Streptomyces coelicolor Group IV family member, σR (SigR), is increased by changes in the oxidation-reduction state of cytoplasmic disulphide bonds. Once released by its cognate anti-σ factor RsrA, σR activates expression of gene products that help cells reduce cytoplasmic disulphide bonds. In this issue of Molecular Microbiology, Kim and co-workers provide new insights into positive and negative control of σR activity. The authors show that a transcript derived from the inducible σR-dependent sigRrsrA p2 promoter operon encodes a σR protein of a higher molecular weight (termed σR′) than is found in uninduced cells. One major difference between σR′ and the smaller σR protein found in uninduced cells is the rapid proteolysis of σR′ by the ClpP1/P2 protease system. The genes for the ClpP1/ClpP2 protease subunits are themselves members of the σR regulon. The newly identified positive (σR′ synthesis) and negative control (selective σR′ turnover) aspects of this circuit are either found or predicted to exist in other related Group IV σ factor family members. PMID:19682265

Mushroom acidic glycosphingolipid induction of cytokine secretion from murine T cells and proliferation of NK1.1 {alpha}/{beta} TCR-double positive cells in vitro

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

Nozaki, Hirofumi; Itonori, Saki; Sugita, Mutsumi

2008-08-29

Interferon (IFN)-{gamma} and interleukin (IL)-4 regulate many types of immune responses. Here we report that acidic glycosphingolipids (AGLs) of Hypsizigus marmoreus and Pleurotus eryngii induced secretion of IFN- {gamma} and IL-4 from T cells in a CD11c-positive cell-dependent manner similar to that of {alpha}-galactosylceramide ({alpha}-GalCer) and isoglobotriaosylceramide (iGb3), although activated T cells by AGLs showed less secretion of cytokine than those activated by {alpha}-GalCer. In addition, stimulation of these mushroom AGLs induced proliferation of NK1.1 {alpha}/{beta} TCR-double positive cells in splenocytes. Administration of a mixture of {alpha}-GalCer and AGLs affected the stimulation of {alpha}-GalCer and generally induced a subtle Th1more » bias for splenocytes but induced an extreme Th2 bias for thymocytes. These results suggested that edible mushroom AGLs contribute to immunomodulation.« less

EWS-FLI1 reprograms the metabolism of Ewing sarcoma cells via positive regulation of glutamine import and serine-glycine biosynthesis.

PubMed

Sen, Nirmalya; Cross, Allison M; Lorenzi, Philip L; Khan, Javed; Gryder, Berkley E; Kim, Suntae; Caplen, Natasha J

2018-06-06

Ewing sarcoma (EWS) is a soft tissue and bone tumor that occurs primarily in adolescents and young adults. In most cases of EWS, the chimeric transcription factor, EWS-FLI1 is the primary oncogenic driver. The epigenome of EWS cells reflects EWS-FLI1 binding and activation or repression of transcription. Here, we demonstrate that EWS-FLI1 positively regulates the expression of proteins required for serine-glycine biosynthesis and uptake of the alternative nutrient source glutamine. Specifically, we show that EWS-FLI1 activates expression of PHGDH, PSAT1, PSPH, and SHMT2. Using cell-based studies, we also establish that EWS cells are dependent on glutamine for cell survival and that EWS-FLI1 positively regulates expression of the glutamine transporter, SLC1A5 and two enzymes involved in the one-carbon cycle, MTHFD2 and MTHFD1L. Inhibition of serine-glycine biosynthesis in EWS cells impacts their redox state leading to an accumulation of reactive oxygen species, DNA damage, and apoptosis. Importantly, analysis of EWS primary tumor transcriptome data confirmed that the aforementioned genes we identified as regulated by EWS-FLI1 exhibit increased expression compared with normal tissues. Furthermore, retrospective analysis of an independent data set generated a significant stratification of the overall survival of EWS patients into low- and high-risk groups based on the expression of PHGDH, PSAT1, PSPH, SHMT2, SLC1A5, MTHFD2, and MTHFD1L. In summary, our study demonstrates that EWS-FLI1 reprograms the metabolism of EWS cells and that serine-glycine metabolism or glutamine uptake are potential targetable vulnerabilities in this tumor type. © 2018 The Authors. Molecular Carcinogenesis Published by WileyPeriodicals, Inc.

CDK4/6 Inhibitors Sensitize Rb-positive Sarcoma Cells to Wee1 Kinase Inhibition through Reversible Cell-Cycle Arrest.

PubMed

Francis, Ashleigh M; Alexander, Angela; Liu, Yanna; Vijayaraghavan, Smruthi; Low, Kwang Hui; Yang, Dong; Bui, Tuyen; Somaiah, Neeta; Ravi, Vinod; Keyomarsi, Khandan; Hunt, Kelly K

2017-09-01

Research into the biology of soft tissue sarcomas has uncovered very few effective treatment strategies that improve upon the current standard of care which usually involves surgery, radiation, and chemotherapy. Many patients with large (>5 cm), high-grade sarcomas develop recurrence, and at that point have limited treatment options available. One challenge is the heterogeneity of genetic drivers of sarcomas, and many of these are not validated targets. Even when such genes are tractable targets, the rarity of each subtype of sarcoma makes advances in research slow. Here we describe the development of a synergistic combination treatment strategy that may be applicable in both soft tissue sarcomas as well as sarcomas of bone that takes advantage of targeting the cell cycle. We show that Rb-positive cell lines treated with the CDK4/6 inhibitor palbociclib reversibly arrest in the G 1 phase of the cell cycle, and upon drug removal cells progress through the cell cycle as expected within 6-24 hours. Using a long-term high-throughput assay that allows us to examine drugs in different sequences or concurrently, we found that palbociclib-induced cell-cycle arrest poises Rb-positive sarcoma cells (SK-LMS1 and HT-1080) to be more sensitive to agents that work preferentially in S-G 2 phase such as doxorubicin and Wee1 kinase inhibitors (AZD1775). The synergy between palbociclib and AZD1775 was also validated in vivo using SK-LMS1 xenografts as well as Rb-positive patient-derived xenografts (PDX) developed from leiomyosarcoma patients. This work provides the necessary preclinical data in support of a clinical trial utilizing this treatment strategy. Mol Cancer Ther; 16(9); 1751-64. ©2017 AACR . ©2017 American Association for Cancer Research.

High Myeloperoxidase Positive Cell Infiltration in Colorectal Cancer Is an Independent Favorable Prognostic Factor

PubMed Central

Eppenberger-Castori, Serenella; Zlobec, Inti; Viehl, Carsten T.; Frey, Daniel M.; Nebiker, Christian A.; Rosso, Raffaele; Zuber, Markus; Amicarella, Francesca; Iezzi, Giandomenica; Sconocchia, Giuseppe; Heberer, Michael; Lugli, Alessandro; Tornillo, Luigi; Oertli, Daniel

2013-01-01

Background Colorectal cancer (CRC) infiltration by adaptive immune system cells correlates with favorable prognosis. The role of the innate immune system is still debated. Here we addressed the prognostic impact of CRC infiltration by neutrophil granulocytes (NG). Methods A TMA including healthy mucosa and clinically annotated CRC specimens (n = 1491) was stained with MPO and CD15 specific antibodies. MPO+ and CD15+ positive immune cells were counted by three independent observers. Phenotypic profiles of CRC infiltrating MPO+ and CD15+ cells were validated by flow cytometry on cell suspensions derived from enzymatically digested surgical specimens. Survival analysis was performed by splitting randomized data in training and validation subsets. Results MPO+ and CD15+ cell infiltration were significantly correlated (p<0.0001; r = 0.76). However, only high density of MPO+ cell infiltration was associated with significantly improved survival in training (P = 0.038) and validation (P = 0.002) sets. In multivariate analysis including T and N stage, vascular invasion, tumor border configuration and microsatellite instability status, MPO+ cell infiltration proved an independent prognostic marker overall (P = 0.004; HR = 0.65; CI:±0.15) and in both training (P = 0.048) and validation (P = 0.036) sets. Flow-cytometry analysis of CRC cell suspensions derived from clinical specimens showed that while MPO+ cells were largely CD15+/CD66b+, sizeable percentages of CD15+ and CD66b+ cells were MPO−. Conclusions High density MPO+ cell infiltration is a novel independent favorable prognostic factor in CRC. PMID:23734221

Antitumorigenic effect of atmospheric-pressure dielectric barrier discharge on human colorectal cancer cells via regulation of Sp1 transcription factor

NASA Astrophysics Data System (ADS)

Han, Duksun; Cho, Jin Hyoung; Lee, Ra Ham; Bang, Woong; Park, Kyungho; Kim, Minseok S.; Shim, Jung-Hyun; Chae, Jung-Il; Moon, Se Youn

2017-02-01

Human colorectal cancer cell lines (HT29 and HCT116) were exposed to dielectric barrier discharge (DBD) plasma at atmospheric pressure to investigate the anticancer capacity of the plasma. The dose- and time-dependent effects of DBDP on cell viability, regulation of transcription factor Sp1, cell-cycle analysis, and colony formation were investigated by means of MTS assay, DAPI staining, propidium iodide staining, annexin V-FITC staining, Western blot analysis, RT-PCR analysis, fluorescence microscopy, and anchorage-independent cell transformation assay. By increasing the duration of plasma dose times, significant reductions in the levels of both Sp1 protein and Sp1 mRNA were observed in both cell lines. Also, expression of negative regulators related to the cell cycle (such as p53, p21, and p27) was increased and of the positive regulator cyclin D1 was decreased, indicating that the plasma treatment led to apoptosis and cell-cycle arrest. In addition, the sizes and quantities of colony formation were significantly suppressed even though two cancer promoters, such as TPA and epidermal growth factor, accompanied the plasma treatment. Thus, plasma treatment inhibited cell viability and colony formation by suppressing Sp1, which induced apoptosis and cell-cycle arrest in these two human colorectal cancer cell lines.

Human umbilical vein endothelial cells protect against hypoxic-ischemic damage in neonatal brain via stromal cell-derived factor 1/C-X-C chemokine receptor type 4.

PubMed

Wu, Chia-Ching; Chen, Yi-Chi; Chang, Ying-Chao; Wang, Lan-Wan; Lin, Yung-Chieh; Chiang, Yi-Lun; Ho, Chien-Jung; Huang, Chao-Ching

2013-05-01

Agents that protect against neurovascular damage provide a powerful neuroprotective strategy. Human umbilical vein endothelial cells (HUVECs) may be used to treat neonates with hypoxic-ischemia (HI) because of its autologous capability. We hypothesized that peripherally injected HUVECs entered the brain after HI, protected against neurovascular damage, and provided protection via stromal cell-derived factor 1/C-X-C chemokine receptor type 4 pathway in neonatal brain. Postpartum day 7 rat pups received intraperitoneal injections of low-passage HUVEC-P4, high-passage HUVEC-P8, or conditioned medium before and immediately after HI. HUVECs were transfected with adenovirus-green fluorescent protein for cell tracing. Oxygen-glucose deprivation was established by coculturing HUVEC-P4 with mouse neuroblastoma neuronal cells (Neuro-2a) and with mouse immortalized cerebral vascular endothelial cells (b.End3). HUVEC-P4-treated group had more blood levels of green fluorescent protein-positive cells than HUVEC-P8-treated group 3 hours postinjection. Intraperitoneally injected HUVEC-P4, but not HUVEC-P8, entered the cortex after HI and positioned closed to the neurons and microvessels. Compared with the condition medium-treated group, the HUVEC-P4-treated but not the HUVEC-P8-treated group showed significantly less neuronal apoptosis and blood-brain barrier damage and more preservation of microvessels in the cortex 24 hours after HI. On postpartum day 14, the HUVEC-P4-treated group showed significant neuroprotection compared with the condition medium-treated group. Stromal cell-derived factor 1 was upregulated in the ipsilateral cortex 3 hours after HI, and inhibiting the stromal cell-derived factor 1/C-X-C chemokine receptor type 4 reduced the protective effect of HUVEC-P4. In vitro transwell coculturing of HUVEC-P4 also significantly protected against oxygen-glucose deprivation cell death in neurons and endothelial cells. Cell therapy using HUVECs may provide a powerful

Valproic acid exhibits different cell growth arrest effect in three HPV-positive/negative cervical cancer cells and possibly via inducing Notch1 cleavage and E6 downregulation.

PubMed

Feng, Shuyu; Yang, Yue; Lv, Jingyi; Sun, Lichun; Liu, Mingqiu

2016-07-01

We investigated the effect of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, and the mechanism of VPA-induced growth inhibition on three cervical cancer cell lines with different molecular and genetic background. We found that VPA induced proliferation suppression, cell apoptosis and cell cycle arrest in all tested cell lines, with an increase of Notch1 active form ICN1 as a tumor suppressor and its target gene HES1. Noteworthy, blocking of Notch signaling with DAPT resulted in growth inhibition in ICN1-overexpressing CaSki and HT-3 cells. Thus, endogenous Notch signaling may be necessary for survival of ICN1-overexpressing cervical cancer cell lines. Furthermore, G1 phase arrest was induced in HeLa and CaSki cells by VPA while G2 phase arrest was induced in HT-3 cells, suggesting different mechanism in this cycle arrest. We also found VPA suppressed oncogene E6 in a Notch-independent manner, and induced significant apoptosis in E6-overexpressing HPV positive CaSki cells. Cell morphological change was also observed in HeLa and HT-3 cell lines after VPA treatment with an upregulation of EMT transcription factor Snail1. Notch signaling inhibitor DAPT partly reversed VPA-induced Snail1 upregulation in HeLa cells. This discovery supports that VPA may induce EMT at least partly via Notch activation.

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

Nuclear factor I-A represses expression of the cell adhesion molecule L1

PubMed Central

2009-01-01

Background The neural cell adhesion molecule L1 plays a crucial role in development and plasticity of the nervous system. Neural cells thus require precise control of L1 expression. Results We identified a full binding site for nuclear factor I (NFI) transcription factors in the regulatory region of the mouse L1 gene. Electrophoretic mobility shift assay (EMSA) showed binding of nuclear factor I-A (NFI-A) to this site. Moreover, for a brain-specific isoform of NFI-A (NFI-A bs), we confirmed the interaction in vivo using chromatin immunoprecipitation (ChIP). Reporter gene assays showed that in neuroblastoma cells, overexpression of NFI-A bs repressed L1 expression threefold. Conclusion Our findings suggest that NFI-A, in particular its brain-specific isoform, represses L1 gene expression, and might act as a second silencer of L1 in addition to the neural restrictive silencer factor (NRSF). PMID:20003413

Nuclear respiratory factor-1 and bioenergetics in tamoxifen-resistant breast cancer cells

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

Radde, Brandie N.; Ivanova, Margarita M.; Mai, Huy Xuan

Acquired tamoxifen (TAM) resistance is a significant clinical problem in treating patients with estrogen receptor α (ERα)+ breast cancer. We reported that ERα increases nuclear respiratory factor-1 (NRF-1), which regulates nuclear-encoded mitochondrial gene transcription, in MCF-7 breast cancer cells and NRF-1 knockdown stimulates apoptosis. Whether NRF-1 and target gene expression is altered in endocrine resistant breast cancer cells is unknown. We measured NRF-1and metabolic features in a cell model of progressive TAM-resistance. NRF-1 and its target mitochondrial transcription factor A (TFAM) were higher in TAM-resistant LCC2 and LCC9 cells than TAM-sensitive MCF-7 cells. Using extracellular flux assays we observed thatmore » LCC1, LCC2, and LCC9 cells showed similar oxygen consumption rate (OCR), but lower mitochondrial reserve capacity which was correlated with lower Succinate Dehydrogenase Complex, Subunit B in LCC1 and LCC2 cells. Complex III activity was lower in LCC9 than MCF-7 cells. LCC1, LCC2, and LCC9 cells had higher basal extracellular acidification (ECAR), indicating higher aerobic glycolysis, relative to MCF-7 cells. Mitochondrial bioenergetic responses to estradiol and 4-hydroxytamoxifen were reduced in the endocrine-resistant cells compared to MCF-7 cells. These results suggest the acquisition of altered metabolic phenotypes in response to long term antiestrogen treatment may increase vulnerability to metabolic stress. - Highlights: • NRF-1 and TFAM expression are higher in endocrine-resistant breast cancer cells. • Oxygen consumption rate is similar in endocrine-sensitive and resistant cells. • Mitochondrial reserve capacity is lower in endocrine-resistant cells. • Endocrine-resistant breast cancer cells have increased glycolysis. • Bioenergetic responses to E2 and tamoxifen are lower in endocrine-resistant cells.« less

Frequent downregulation of BTB and CNC homology 2 expression in Epstein-Barr virus-positive diffuse large B-cell lymphoma.

PubMed

Noujima-Harada, Mai; Takata, Katsuyoshi; Miyata-Takata, Tomoko; Sakurai, Hiroaki; Igarashi, Kazuhiko; Ito, Etsuro; Nagakita, Keina; Taniguchi, Kohei; Ohnishi, Nobuhiko; Omote, Shizuma; Tabata, Tetsuya; Sato, Yasuharu; Yoshino, Tadashi

2017-05-01

Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell lymphoma subtype, and the Epstein-Barr virus (EBV)-positive subtype of DLBCL is known to show a more aggressive clinical behavior than the EBV-negative one. BTB and CNC homology 2 (BACH2) has been highlighted as a tumor suppressor in hematopoietic malignancies; however, the role of BACH2 in EBV-positive DLBCL is unclear. In the present study, BACH2 expression and its significance were studied in 23 EBV-positive and 43 EBV-negative patient samples. Immunohistochemistry revealed BACH2 downregulation in EBV-positive cases (P < 0.0001), although biallelic deletion of BACH2 was not detected by FISH. Next, we analyzed the contribution of BACH2 negativity to aggressiveness in EBV-positive B-cell lymphomas using FL-18 (EBV-negative) and FL-18-EB cells (FL-18 sister cell line, EBV-positive). In BACH2-transfected FL-18-EB cells, downregulation of phosphorylated transforming growth factor-β-activated kinase 1 (pTAK1) and suppression in p65 nuclear fractions were observed by Western blot analysis contrary to non-transfected FL-18-EB cells. In patient samples, pTAK1 expression and significant nuclear p65, p50, and p52 localization were detected immunohistochemically in BACH2-negative DLBCL (P < 0.0001, P = 0.006, and P = 0.001, respectively), suggesting that BACH2 downregulation contributes to constitutive activation of the nuclear factor-κB pathway through TAK1 phosphorylation in BACH2-negative DLBCL (most EBV-positive cases). Although further molecular and pathological studies are warranted to clarify the detailed mechanisms, downregulation of BACH2 may contribute to constitutive activation of the nuclear factor-κB pathway through TAK1 activation. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Ctip2-, Satb2-, Prox1-, and GAD65-Expressing Neurons in Rat Cultures: Preponderance of Single- and Double-Positive Cells, and Cell Type-Specific Expression of Neuron-Specific Gene Family Members, Nsg-1 (NEEP21) and Nsg-2 (P19).

PubMed

Digilio, Laura; Yap, Chan Choo; Winckler, Bettina

2015-01-01

The brain consists of many distinct neuronal cell types, but which cell types are present in widely used primary cultures of embryonic rodent brain is often not known. We characterized how abundantly four cell type markers (Ctip2, Satb2, Prox1, GAD65) were represented in cultured rat neurons, how easily neurons expressing different markers can be transfected with commonly used plasmids, and whether neuronal-enriched endosomal proteins Nsg-1 (NEEP21) and Nsg-2 (P19) are ubiquitously expressed in all types of cultured neurons. We found that cultured neurons stably maintain cell type identities that are reflective of cell types in vivo. This includes neurons maintaining simultaneous expression of two transcription factors, such as Ctip2+/Satb2+ or Prox1+/Ctip2+ double-positive cells, which have also been described in vivo. Secondly, we established the superior efficiency of CAG promoters for both Lipofectamine-mediated transfection as well as for electroporation. Thirdly, we discovered that Nsg-1 and Nsg-2 were not expressed equally in all neurons: whereas high levels of both Nsg-1 and Nsg-2 were found in Satb2-, Ctip2-, and GAD65-positive neurons, Prox1-positive neurons in hippocampal cultures expressed low levels of both. Our findings thus highlight the importance of identifying neuronal cell types for doing cell biology in cultured neurons: Keeping track of neuronal cell type might uncover effects in assays that might otherwise be masked by the mixture of responsive and non-responsive neurons in the dish.

Bone marrow mesenchymal stem cells ameliorate inflammatory factor-induced dysfunction of INS-1 cells on chip.

PubMed

Sun, Yu; Yao, Zhina; Lin, Peng; Hou, Xinguo; Chen, Li

2014-05-01

Using a microfluidic chip, we have investigated whether bone marrow mesenchymal stem cells (BM-MSCs) could ameliorate IL-1β/IFN-γ-induced dysfunction of INS-1 cells. BM-MSCs were obtained from diabetes mellitus patients and their cell surface antigen expression profiles were analyzed by flow cytometric. INS-1 cells were cocultured with BM-MSCs on a microfluidic chip with persistent perfusion of medium containing 1 ng/mL IL-1β and 2.5 U/mL IFN-γ for 72 h. BM-MSCs could partially rescue INS-1 cells from cytokine-induced dysfunction and ameliorate the expression of insulin and PDX-1 gene in INS-1 cells. Thus BM-MSCs can be viewed as a promising stem cell source to depress inflammatory factor-induced dysfunction of pancreatic β cells in diabetic patients. © 2014 International Federation for Cell Biology.

A positive immunoselection method to isolate villous cytotrophoblast cells from first trimester and term placenta to high purity.

PubMed

Pötgens, A J G; Kataoka, H; Ferstl, S; Frank, H-G; Kaufmann, P

2003-04-01

We developed a method for isolating highly pure villous cytotrophoblast cells from first trimester and term placenta that excludes extravillous trophoblast and syncytiotrophoblast fragments. The method is based on positive immunoselection using an antibody (mAb C76/18) reacting with hepatocyte growth factor activator inhibitor 1, HAI-1, a membrane antigen on villous cytotrophoblast. As a comparison, we also immunopurified cells using an antibody against CD105, present on syncytiotrophoblast and some extravillous trophoblast cells. The isolates were characterized by flow cytometry. HAI-1-positive cells from first trimester and term placentae were highly pure (>98 per cent cytokeratin 7-positive) mononuclear trophoblast cells. These isolations were contaminated with only very small percentages of vimentin and CD45-positive cells. HAI-1-positive trophoblast cells lacked CD105 and also HLA class I, a marker for extravillous trophoblast. In culture HAI-1-positive cells adhered, displayed an epithelial morphology, and survived for more than three days. In contrast, CD105-positive cell fractions from first trimester placenta were a heterogeneous mixture of mononuclear and multinuclear elements consisting of syncytiotrophoblast fragments, extravillous trophoblast cells, as well as around 5 per cent non-trophoblastic contaminants. In conclusion, the positive immunoselection method using antibody C76/18 yielded highly pure villous cytotrophoblast cells devoid of elements derived from syncytiotrophoblast or extravillous trophoblast.

Predomination of IL-17-producing tryptase-positive/chymase-positive mast cells in azoospermic chronic testicular inflammation.

PubMed

Chen, S-J; Duan, Y-G; Haidl, G; Allam, J-P

2016-08-01

Chronic testicular inflammation and infection have been regarded as important factors in the pathogenesis of azoospermia. As key effector cells in innate and adaptive immune system, mast cells (MCs) were observed in inflammation and autoimmune disease. Furthermore, increased expression of tryptase-positive MCs has been reported in testicular disorders associated with male infertility/subfertility. However, little is known about the potential relationship between MCs and chronic testicular inflammation in azoospermic patients. Moreover, the preferential expression of MCs' subtypes in testis of these patients is still far from being understood. Thus, this study aimed to investigate characteristics of testicular MCs as well as their subtypes in azoospermic men with chronic testicular inflammation (AZI, n = 5) by immunohistochemical techniques. Our results showed significant increase of MCs in AZI, and more importantly, considerable numbers of tryptase-positive/chymase-positive MCs could also be demonstrated in AZI, when compared to control groups representing azoospermia without chronic testicular inflammation (AZW, n = 5) and normal spermatogenesis (NT, n = 5) respectively. Most interestingly, immunofluorescence staining revealed autoimmune-associated interleukin (IL)-17-producing MCs in AZI, whereas co-expression of MC markers with tumour necrosis factor (TNF)-α, IL-10 and IL-1β could not be detected. In conclusion, AZI is associated with significant increase of tryptase-positive/chymase-positive MCs expressing IL-17, and these MCs might contribute to the pathogenesis of AZI. © 2015 Blackwell Verlag GmbH.

Transgenic Overexpression of the Transcription Factor Nkx6.1 in β-Cells of Mice Does Not Increase β-Cell Proliferation, β-Cell Mass, or Improve Glucose Clearance

PubMed Central

Schaffer, Ashleigh E.; Yang, Almira J.; Thorel, Fabrizio; Herrera, Pedro L.

2011-01-01

The loss or dysfunction of the pancreatic endocrine β-cell results in diabetes. Recent innovative therapeutic approaches for diabetes aim to induce β-cell proliferation in vivo by pharmacological intervention. Based on the finding that overexpression of the transcription factor Nkx6.1 in islets in vitro increases β-cell proliferation while maintaining β-cell function, Nkx6.1 has been proposed as a potential target for diabetes therapy. However, it is unknown whether elevated Nkx6.1 levels in β-cells in vivo have similar effects as observed in isolated islets. To this end, we sought to investigate whether overexpression of Nkx6.1 in β-cells in vivo could increase β-cell mass and/or improve β-cell function in normal or β-cell-depleted mice. Using a bigenic inducible Cre-recombinase-based transgenic model, we analyzed the effects of Nkx6.1 overexpression on β-cell proliferation, β-cell mass, and glucose metabolism. We found that mice overexpressing Nkx6.1 in β-cells displayed similar β-cell proliferation rates and β-cell mass as control mice. Furthermore, after partial β-cell ablation, Nkx6.1 overexpression was not sufficient to induce β-cell regeneration under either nondiabetic or diabetic conditions. Together these results demonstrate that sustained Nkx6.1 overexpression in vivo does not stimulate β-cell proliferation, expand β-cell mass, or improve glucose metabolism in either normal or β-cell-depleted pancreata. Thus, raising cellular Nkx6.1 levels in β-cells in vivo is unlikely to have a positive impact on type 2 diabetes. PMID:21964593

The hematopoietic cell-specific transcription factor PU.1 is critical for expression of CD11c.

PubMed

Yashiro, Takuya; Kasakura, Kazumi; Oda, Yoshihito; Kitamura, Nao; Inoue, Akihito; Nakamura, Shusuke; Yokoyama, Hokuto; Fukuyama, Kanako; Hara, Mutsuko; Ogawa, Hideoki; Okumura, Ko; Nishiyama, Makoto; Nishiyama, Chiharu

2017-02-01

PU.1 is a hematopoietic cell-specific transcription factor belonging to the Ets family, which plays an important role in the development of dendritic cells (DCs). CD11c (encoded by Itgax) is well established as a characteristic marker of hematopoietic lineages including DCs. In the present study, we analyzed the role of PU.1 (encoded by Spi-1) in the expression of CD11c. When small interfering RNA (siRNA) for Spi-1 was introduced into bone marrow-derived DCs (BMDCs), the mRNA level and cell surface expression of CD11c were dramatically reduced. Using reporter assays, the TTCC sequence at -56/-53 was identified to be critical for PU.1-mediated activation of the promoter. An EMSA showed that PU.1 directly bound to this region. ChIP assays demonstrated that a significant amount of PU.1 bound to this region on chromosomal DNA in BMDCs, which was decreased in LPS-stimulated BMDCs in accordance with the reduced levels of mRNAs of Itgax and Spi-1, and the histone acetylation degree. Enforced expression of exogenous PU.1 induced the expression of the CD11c protein on the cell surface of mast cells, whereas control transfectants rarely expressed CD11c. Quantitative RT-PCR also showed that the expression of a transcription factor Irf4, which is a partner molecule of PU.1, was reduced in PU.1-knocked down BMDCs. IRF4 transactivated the Itgax gene in a synergistic manner with PU.1. Taken together, these results indicate that PU.1 functions as a positive regulator of CD11c gene expression by directly binding to the Itgax promoter and through transactivation of the Irf4 gene. © The Japanese Society for Immunology. 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Id-1 promotes TGF-{beta}1-induced cell motility through HSP27 activation and disassembly of adherens junction in prostate epithelial cells

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

Di Kaijun; Wong, Y.C.; Wang Xianghong

Id-1 (inhibitor of differentiation or DNA binding-1) has been positively associated with cell proliferation, cell cycle progression, and invasiveness during tumorigenesis. In addition, Id-1 has been shown to modulate cellular sensitivity to TGF-{beta}1 (transforming growth factor {beta}1). Here we demonstrate a novel role of Id-1 in promoting TGF-{beta}1-induced cell motility in a non-malignant prostate epithelial cell line, NPTX. We found that Id-1 promoted F-actin stress fiber formation in response to TGF-{beta}1, which was associated with increased cell-substrate adhesion and cell migration in NPTX cells. In addition, this positive effect of Id-1 on TGF-{beta}1-induced cell motility was mediated through activation ofmore » MEK-ERK signaling pathway and subsequent phosphorylation of HSP27 (heat shock protein 27). Furthermore, Id-1 disrupted the adherens junction complex in TGF-{beta}1-treated cells through down-regulation of E-cadherin, redistribution of {beta}-catenin, along with up-regulation of N-cadherin. These lines of evidence reveal a novel tumorigenic role of Id-1 through reorganization of actin cytoskeleton and disassembly of cell-cell adhesion in response to TGF-{beta}1 in human prostate epithelial cells, and suggest that intracellular Id-1 levels might be a determining factor for switching TGF-{beta}1 from a growth inhibitor to a tumor promoter during prostate carcinogenesis.« less

Can transforming growth factor-beta1 and retinoids modify the activity of estradiol and antiestrogens in MCF-7 breast cancer cells?.

PubMed

Czeczuga-Semeniuk, Ewa; Anchim, Tomasz; Dziecioł, Janusz; Dabrowska, Milena; Wołczyński, Sławomir

2004-01-01

Retinoic acid and transforming growth factor-beta (TGF-beta) affect differentiation, proliferation and carcinogenesis of epithelial cells. The effect of both compounds on the proliferation of cells of the hormone sensitive human breast cancer cell line (ER+) MCF-7 was assessed in the presence of estradiol and tamoxifen. The assay was based on [3H]thymidine incorporation and the proliferative activity of PCNA- and Ki 67-positive cells. The apoptotic index and expression of the Bcl-2 and p53 antigens in MCF-7 cells were also determined. Exogenous TGF-beta1 added to the cell culture showed antiproliferative activity within the concentration range of 0.003-30 ng/ml. Irrespective of TGF-beta1 concentrations, a marked reduction in the stimulatory action of estradiol (10(-9) and 10(-8) M) was observed whereas in combination with tamoxifen (10(-7) and 10(-6) M) only 30 ng/ml TGF-beta1 caused a statistically significant reduction to approximately 30% of the proliferative cells. In further experiments we examined the effect of exposure of breast cancer cells to retinoids in combination with TGF-beta1. The incorporation of [3H]thymidine into MCF-7 cells was inhibited to 52 +/- 19% (control =100%) by 3 ng/ml TGF-beta1, and this dose was used throughout. It was found that addition of TGF-beta1 and isotretinoin to the culture did not decrease proliferation, while TGF-beta1 and tretinoin at low concentrations (3 x 10(-8) and 3 x 10(-7) M) reduced the percentage of proliferating cells by approximately 30% (67+/-8% and 67+/-5%, P<0.05 compared to values in the tretinoin group). Both retinoids also led to a statistically significant decrease in the stimulatory effect of 10(-9) M estradiol, attenuated by TGF-beta1. In addition, the retinoids in combination with TGF-beta1 and tamoxifen (10(-6) M) caused a further reduction in the percentage of proliferating cells. Immunocytochemical analysis showed that all the examined compounds gave a statistically significant reduction in the

Human Epidermal Growth Factor Receptor 2 (HER2) -Specific Chimeric Antigen Receptor-Modified T Cells for the Immunotherapy of HER2-Positive Sarcoma.

PubMed

Ahmed, Nabil; Brawley, Vita S; Hegde, Meenakshi; Robertson, Catherine; Ghazi, Alexia; Gerken, Claudia; Liu, Enli; Dakhova, Olga; Ashoori, Aidin; Corder, Amanda; Gray, Tara; Wu, Meng-Fen; Liu, Hao; Hicks, John; Rainusso, Nino; Dotti, Gianpietro; Mei, Zhuyong; Grilley, Bambi; Gee, Adrian; Rooney, Cliona M; Brenner, Malcolm K; Heslop, Helen E; Wels, Winfried S; Wang, Lisa L; Anderson, Peter; Gottschalk, Stephen

2015-05-20

The outcome for patients with metastatic or recurrent sarcoma remains poor. Adoptive therapy with tumor-directed T cells is an attractive therapeutic option but has never been evaluated in sarcoma. We conducted a phase I/II clinical study in which patients with recurrent/refractory human epidermal growth factor receptor 2 (HER2) -positive sarcoma received escalating doses (1 × 10(4)/m(2) to 1 × 10(8)/m(2)) of T cells expressing an HER2-specific chimeric antigen receptor with a CD28.ζ signaling domain (HER2-CAR T cells). We enrolled 19 patients with HER2-positive tumors (16 osteosarcomas, one Ewing sarcoma, one primitive neuroectodermal tumor, and one desmoplastic small round cell tumor). HER2-CAR T-cell infusions were well tolerated with no dose-limiting toxicity. At dose level 3 (1 × 10(5)/m(2)) and above, we detected HER2-CAR T cells 3 hours after infusion by quantitative polymerase chain reaction in 14 of 16 patients. HER2-CAR T cells persisted for at least 6 weeks in seven of the nine evaluable patients who received greater than 1 × 10(6)/m(2) HER2-CAR T cells (P = .005). HER2-CAR T cells were detected at tumor sites of two of two patients examined. Of 17 evaluable patients, four had stable disease for 12 weeks to 14 months. Three of these patients had their tumor removed, with one showing ≥ 90% necrosis. The median overall survival of all 19 infused patients was 10.3 months (range, 5.1 to 29.1 months). This first evaluation of the safety and efficacy of HER2-CAR T cells in patients with cancer shows the cells can persist for 6 weeks without evident toxicities, setting the stage for studies that combine HER2-CAR T cells with other immunomodulatory approaches to enhance their expansion and persistence. © 2015 by American Society of Clinical Oncology.

Fibroblast growth factor rescues brain endothelial cells lacking presenilin 1 from apoptotic cell death following serum starvation.

PubMed

Gama Sosa, Miguel A; De Gasperi, Rita; Hof, Patrick R; Elder, Gregory A

2016-07-22

Presenilin 1 (Psen1) is important for vascular brain development and is known to influence cellular stress responses. To understand the role of Psen1 in endothelial stress responses, we investigated the effects of serum withdrawal on wild type (wt) and Psen1-/- embryonic brain endothelial cells. Serum starvation induced apoptosis in Psen1-/- cells but did not affect wt cells. PI3K/AKT signaling was reduced in serum-starved Psen1-/- cells, and this was associated with elevated levels of phospho-p38 consistent with decreased pro-survival AKT signaling in the absence of Psen1. Fibroblast growth factor (FGF1 and FGF2), but not vascular endothelial growth factor (VEGF) rescued Psen1-/- cells from serum starvation induced apoptosis. Inhibition of FGF signaling induced apoptosis in wt cells under serum withdrawal, while blocking γ-secretase activity had no effect. In the absence of serum, FGF2 immunoreactivity was distributed diffusely in cytoplasmic and nuclear vesicles of wt and Psen1-/- cells, as levels of FGF2 in nuclear and cytosolic fractions were not significantly different. Thus, sensitivity of Psen1-/- cells to serum starvation is not due to lack of FGF synthesis but likely to effects of Psen1 on FGF release onto the cell surface and impaired activation of the PI3K/AKT survival pathway.

Canopy1, a positive feedback regulator of FGF signaling, controls progenitor cell clustering during Kupffer's vesicle organogenesis

PubMed Central

Matsui, Takaaki; Thitamadee, Siripong; Murata, Tomoko; Kakinuma, Hisaya; Nabetani, Takuji; Hirabayashi, Yoshio; Hirate, Yoshikazu; Okamoto, Hitoshi; Bessho, Yasumasa

2011-01-01

The assembly of progenitor cells is a crucial step for organ formation during vertebrate development. Kupffer's vesicle (KV), a key organ required for the left–right asymmetric body plan in zebrafish, is generated from a cluster of ∼20 dorsal forerunner cells (DFCs). Although several genes are known to be involved in KV formation, how DFC clustering is regulated and how cluster formation then contributes to KV formation remain unclear. Here we show that positive feedback regulation of FGF signaling by Canopy1 (Cnpy1) controls DFC clustering. Cnpy1 positively regulates FGF signals within DFCs, which in turn promote Cadherin1-mediated cell adhesion between adjacent DFCs to sustain cell cluster formation. When this FGF positive feedback loop is disrupted, the DFC cluster fails to form, eventually leading to KV malformation and defects in the establishment of laterality. Our results therefore uncover both a previously unidentified role of FGF signaling during vertebrate organogenesis and a regulatory mechanism underlying cell cluster formation, which is an indispensable step for formation of a functional KV and establishment of the left–right asymmetric body plan. PMID:21628557

Overexpression of ANXA1 in penile carcinomas positive for high-risk HPVs.

PubMed

Calmon, Marilia Freitas; Mota, Mânlio Tasso de Oliveira; Babeto, Érica; Candido, Natália Maria; Girol, Ana Paula; Mendiburu, Carlos Fabian; Bonilha, Jane Lopes; Silvestre, Rodrigo Vellasco Duarte; Rosa, Bruno Miziara; Thomé, Jorge Alberto; Medeiros, Gustavo Hernandez Américo; Soares, Fernando Augusto; Guimarães, Gustavo Cardoso; de Arruda, José Germano Ferraz; Oliani, Sonia Maria; Villa, Luisa Lina; Vassallo, José; Rahal, Paula

2013-01-01

The incidence of penile cancer varies between populations but is rare in developed nations. Penile cancer is associated with a number of established risk factors and associated diseases including phimosis with chronic inflammation, human papillomavirus (HPV) infection, poor hygiene and smoking. The objective of this study was to identify genes related to this type of cancer. The detection of HPV was analyzed in 47 penile squamous cell carcinoma samples. HPV DNA was detected in 48.9% of penile squamous cell carcinoma cases. High-risk HPV were present in 42.5% of cases and low-risk HPV were detected in 10.6% of penile squamous cell carcinomas. The RaSH approach identified differential expression of Annexin A1 (ANXA1), p16, RPL6, PBEF1 and KIAA1033 in high-risk HPV positive penile carcinoma; ANXA1 and p16 were overexpressed in penile squamous cells positive for high-risk HPVs compared to normal penile samples by qPCR. ANXA1 and p16 proteins were significantly more expressed in the cells from high-risk HPV-positive penile carcinoma as compared to HPV-negative tumors (p<0.0001) independently of the subtype of the carcinoma. Overexpression of ANXA1 might be mediated by HPV E6 in penile squamous cell carcinoma of patients with high-risk HPVs, suggesting that this gene plays an important role in penile cancer.

Overexpression of ANXA1 in Penile Carcinomas Positive for High-Risk HPVs

PubMed Central

Calmon, Marilia Freitas; Mota, Mânlio Tasso de Oliveira; Babeto, Érica; Candido, Natália Maria; Girol, Ana Paula; Mendiburu, Carlos Fabian; Bonilha, Jane Lopes; Silvestre, Rodrigo Vellasco Duarte; Rosa, Bruno Miziara; Thomé, Jorge Alberto; Medeiros, Gustavo Hernandez Américo; Soares, Fernando Augusto; Guimarães, Gustavo Cardoso; de Arruda, José Germano Ferraz; Oliani, Sonia Maria; Villa, Luisa Lina; Vassallo, José; Rahal, Paula

2013-01-01

The incidence of penile cancer varies between populations but is rare in developed nations. Penile cancer is associated with a number of established risk factors and associated diseases including phimosis with chronic inflammation, human papillomavirus (HPV) infection, poor hygiene and smoking. The objective of this study was to identify genes related to this type of cancer. The detection of HPV was analyzed in 47 penile squamous cell carcinoma samples. HPV DNA was detected in 48.9% of penile squamous cell carcinoma cases. High-risk HPV were present in 42.5% of cases and low-risk HPV were detected in 10.6% of penile squamous cell carcinomas. The RaSH approach identified differential expression of Annexin A1 (ANXA1), p16, RPL6, PBEF1 and KIAA1033 in high-risk HPV positive penile carcinoma; ANXA1 and p16 were overexpressed in penile squamous cells positive for high-risk HPVs compared to normal penile samples by qPCR. ANXA1 and p16 proteins were significantly more expressed in the cells from high-risk HPV-positive penile carcinoma as compared to HPV-negative tumors (p<0.0001) independently of the subtype of the carcinoma. Overexpression of ANXA1 might be mediated by HPV E6 in penile squamous cell carcinoma of patients with high-risk HPVs, suggesting that this gene plays an important role in penile cancer. PMID:23341933

DETERMINANT FACTORS OF VISUAL INSPECTION WITH ACETIC ACID (VIA) POSITIVE LESIONS AMONG HIV POSITIVE WOMEN IN MEKELLE HOSPITAL, NORTHERN ETHIOPIA: A CASE CONTROL STUDY.

PubMed

Gessesse, Zekariase; Tadesse, Zemen; Alemayehu, Mussie; Hiruye, Abiy; Getachew, Yeneneh; Derbew, Miliard; Mariam, Damen Haile; Mammo, Dereje; Eva, Kantelhardt; Yebyo, Henock; Michael, Hailay Gebre

2015-07-01

Cervical cancer is the second commonest type and third cause of cancer death among women in low-income countries. Women living with HIV/AIDS are at greater risk of developing cervical cancer. The study aimed to identify the determinant factors forsuspected precancerous cervical lesions among HIV- positive women in Mekelle hospital, Ethiopia. Anunmatched case-control study was conducted among randomly selected HIV positive women in Mekelle hospital in 2014. In Mekelle Hospital, routine screening for lesions of the cervix uteri by visual inspection with acetic acid (VIA) is done in HIV positive women by trained nurses. Suspicious findings are treated by cryotherapy or referred to the Gynaecologist. A number of 116 cases, who had suspicious findings on VIA, and 232 HIV-positive controls without suspicious findings on VIA were randomly selected and enrolled into the study The determinant factors for precancerous cervical lesion were analyzed using multiple logistic regression and described as adjusted odds ratio (AOR). HIV positive women who had CD4 cells less than 350/mm3 were two times more likely to have precancerous cervical lesion compared to those with CD4 cells above 350/mm3. Women with two (AOR = 3.6; 95% CI: 1.7, 7.7) and three (AOR = 2.5; 95% CI: 1.2, 5.4) sexual partners were four and three times more likely to have precancerous cervical lesion, respectively, as compared to those who had one sexual partner. Age, History of STI and duration of ART had no influence on presence of VIA positive lesions in HIV positive women. CD4 count cells and number of sexual partners were predictors of VIA positive cervical lesion among HIV positive women.

Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation

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

Nagata, Yosuke, E-mail: cynagata@mail.ecc.u-tokyo.ac.jp; Ohashi, Kazuya; Wada, Eiji

2014-08-01

Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermalmore » growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor. - Highlights: • EGF in combination with insulin induces proliferation of quiescent C2C12 cells. • Sphingosine kinase activity increases when reserve cells are stimulated with EGF. • EGF-induced activation of reserve cells is dependent on sphingosine kinase and ERK. • The S1P receptor S1P2 is involved in EGF-induced reserve cell activation. • EGF-induced reserve cell activation is mediated by S1P and

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.

Insulin-like growth factor 1 enhances the migratory capacity of mesenchymal stem cells

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

Li, Yangxin; Yu, XiYong; Lin, ShuGuang

2007-05-11

Mesenchymal stem cells (MSCs) are attractive candidates for cell based therapies. However, the mechanisms responsible for stem cell migration and homing after transplantation remain unknown. It has been shown that insulin-like growth factor-1 (IGF-1) induces proliferation and migration of some cell types, but its effects on stem cells have not been investigated. We isolated and cultured MSC from rat bone marrow, and found that IGF-1 increased the expression levels of the chemokine receptor CXCR4 (receptor for stromal cell-derived factor-1, SDF-1). Moreover, IGF-1 markedly increased the migratory response of MSC to SDF-1. The IGF-1-induced increase in MSC migration in response tomore » SDF-1 was attenuated by PI3 kinase inhibitor (LY294002 and wortmannin) but not by mitogen-activated protein/ERK kinase inhibitor PD98059. Our data indicate that IGF-1 increases MSC migratory responses via CXCR4 chemokine receptor signaling which is PI3/Akt dependent. These findings provide a new paradigm for biological effects of IGF-1 on MSC and have implications for the development of novel stem cell therapeutic strategies.« less

Tumor necrosis factor-{alpha} enhances IL-15-induced natural killer cell differentiation

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

Lee, Jiwon; Lee, Suk Hyung; Korea University of Science and Technology, Yusong, Daejeon 305-333

2009-09-04

The differentiation of natural killer (NK) cells is regulated by various factors including soluble growth factors and transcription factors. Here, we have demonstrated that tumor necrosis factor-{alpha} (TNF-{alpha}) is a positive regulator of NK cell differentiation. TNF-{alpha} augmented the IL-15-induced expression of NK1.1 and CD122 in mature NK cells, and TNF-{alpha} alone also induced NK cell maturation as well as IL-15. TNF-{alpha} also increased IFN-{gamma} production in NK cells in the presence of IL-15. Meanwhile, mRNA expression of several transcription factors, including T-bet and GATA-3, was increased by the addition of TNF-{alpha} and IL-15. In addition, TNF-{alpha} increased nuclear factor-kappamore » B (NF-{kappa}B) activity in NK cells and inhibition of NF-{kappa}B impeded TNF-{alpha}-enhanced NK cell maturation. Overall, these data suggest that TNF-{alpha} significantly increased IL-15-driven NK cell differentiation by increasing the expression of transcription factors that play crucial roles in NK cell maturation and inducing the NF-{kappa}B activity.« less

B-cell transcription factors Pax-5, Oct-2, BOB.1, Bcl-6, and MUM1 are useful markers for the diagnosis of nodular lymphocyte predominant Hodgkin lymphoma.

PubMed

Herbeck, Rosemarie; Teodorescu Brînzeu, D; Giubelan, Marioara; Lazăr, Elena; Dema, Alis; Ioniţă, Hortensia

2011-01-01

In some instances, the overlap in morphologic features and antigen expression between nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) and classical Hodgkin lymphoma (cHL) can cause confusion in the diagnosis. In these cases, the transcription factors (TFs) B-cell specific activator protein (BSAP)/Pax-5, octamer binding protein-2 (Oct-2), B-lymphocyte-specific co-activator BOB.1/OBF.1, Bcl-6 protein and multiple myeloma-1/interferon regulatory factor-4 (MUM1/IRF-4) may aid in clarifying the diagnosis. Twenty-two cases of NLPHL were studied for the immunohistochemical expression of Pax-5, Oct-2, BOB.1, Bcl-6 protein and MUM1/IRF-4. Our results sustain the usefulness of the selected set of TFs to diagnose and distinguish NLPHL from cHL since Pax-5, Oct-2, BOB.1 and Bcl-6 are consistently expressed by lymphocyte predominant (LP) cells and reported by others to be often unexpressed in Hodgkin and Reed-Sternberg cells. By contrast, MUM1/IRF-4 protein scored negative in the majority of LP cells, but is reported to be expressed in almost all cases of cHL. Thus, although the expression of transcription factors is very heterogeneous, their simultaneous implementation for positive and differential diagnosis may be useful.

Functional Impairment of Myeloid Dendritic Cells during Advanced Stage of HIV-1 Infection: Role of Factors Regulating Cytokine Signaling.

PubMed

Sachdeva, Meenakshi; Sharma, Aman; Arora, Sunil K

2015-01-01

Severely immunocompromised state during advanced stage of HIV-1 infection has been linked to functionally defective antigen presentation by dendritic cells (DCs). The molecular mechanisms behind DC impairment are still obscure. We investigated changes in DC function and association of key regulators of cytokine signaling during different stages of HIV-1 infection and following antiretroviral therapy (ART). Phenotypic and functional characteristics of circulating myeloid DCs (mDCs) in 56 ART-naive patients (23 in early and 33 in advanced stage of disease), 36 on ART and 24 healthy controls were evaluated. Sixteen patients were studied longitudinally prior-to and 6 months after the start of ART. For functional studies, monocyte-derived DCs (Mo-DCs) were evaluated for endocytosis, allo-stimulation and cytokine secretion. The expression of suppressor of cytokine signaling (SOCS)-1 and other regulators of cytokine signaling was evaluated by real-time RT-PCR. The ability to respond to an antigenic stimulation was severely impaired in patients in advanced HIV-1 disease which showed partial recovery in the treated group. Mo-DCs from patients with advanced HIV-disease remained immature with low allo-stimulation and reduced cytokine secretion even after TLR-4 mediated stimulation ex-vivo. The cells had an increased expression of negative regulatory factors like SOCS-1, SOCS-3, SH2-containing phosphatase (SHP)-1 and a reduced expression of positive regulators like Janus kinase (JAK)2 and Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)1. A functional recovery after siRNA mediated silencing of SOCS-1 in these mo-DCs confirms the role of negative regulatory factors in functional impairment of these cells. Functionally defective DCs in advanced stage of HIV-1 infection seems to be due to imbalanced state of negative and positive regulatory gene expression. Whether this is a cause or effect of increased viral replication at this stage of disease, needs

PDGFBB promotes PDGFR{alpha}-positive cell migration into artificial bone in vivo

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

Yoshida, Shigeyuki; Center for Human Metabolomic Systems Biology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582; Iwasaki, Ryotaro

2012-05-18

Highlights: Black-Right-Pointing-Pointer We examined effects of PDGFBB in PDGFR{alpha} positive cell migration in artificial bones. Black-Right-Pointing-Pointer PDGFBB was not expressed in osteoblastic cells but was expressed in peripheral blood cells. Black-Right-Pointing-Pointer PDGFBB promoted PDGFR{alpha} positive cell migration into artificial bones but not osteoblast proliferation. Black-Right-Pointing-Pointer PDGFBB did not inhibit osteoblastogenesis. -- Abstract: Bone defects caused by traumatic bone loss or tumor dissection are now treated with auto- or allo-bone graft, and also occasionally by artificial bone transplantation, particularly in the case of large bone defects. However, artificial bones often exhibit poor affinity to host bones followed by bony union failure.more » Thus therapies combining artificial bones with growth factors have been sought. Here we report that platelet derived growth factor bb (PDGFBB) promotes a significant increase in migration of PDGF receptor {alpha} (PDGFR{alpha})-positive mesenchymal stem cells/pre-osteoblastic cells into artificial bone in vivo. Growth factors such as transforming growth factor beta (TGF{beta}) and hepatocyte growth factor (HGF) reportedly inhibit osteoblast differentiation; however, PDGFBB did not exhibit such inhibitory effects and in fact stimulated osteoblast differentiation in vitro, suggesting that combining artificial bones with PDGFBB treatment could promote host cell migration into artificial bones without inhibiting osteoblastogenesis.« less

Circulating CD34-positive cells, glomerular filtration rate and triglycerides in relation to hypertension.

PubMed

Shimizu, Yuji; Sato, Shimpei; Koyamatsu, Jun; Yamanashi, Hirotomo; Nagayoshi, Mako; Kadota, Koichiro; Maeda, Takahiro

2015-11-01

Serum triglycerides have been reported to be independently associated with the development of chronic kidney disease (CKD), which is known to play a role in vascular disturbance. On the other hand, circulating CD34-positve cells, including endothelial progenitor cells, are reported to contribute to vascular repair. However, no studies have reported on the correlation between triglycerides and the number of CD34-positive cells. Since hypertension is well known factor for vascular impairment, the degree of correlation between serum triglycerides and circulating CD34-positve cells should account for hypertension status. We conducted a cross-sectional study of 274 elderly Japanese men aged ≥ 60 years (range 60-79 years) undergoing general health checkups. Multiple linear regression analysis of non-hypertensive subjects adjusting for classical cardiovascular risk factors showed that although triglyceride levels (1SD increments; 64 mg/dL) did not significantly correlate with glomerular filtration rate (GFR) (β = -2.06, p = 0.163), a significant positive correlation was seen between triglycerides and the number of circulating CD34-positive cells (β = 0.50, p = 0.004). In hypertensive subjects, a significant inverse correlation between triglycerides and GFR was observed (β = -2.66, p = 0.035), whereas no significant correlation between triglycerides and the number of circulating CD34-positive cells was noted (β = -0.004, p = 0.974). Since endothelial progenitor cells (CD34-positive cells) have been reported to contribute to vascular repair, our results indicate that in non-hypertensive subjects, triglycerides may stimulate an increase in circulating CD34-positive cells (vascular repair) by inducing vascular disturbance. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Steroid receptor coactivator-3 regulates glucose metabolism in bladder cancer cells through coactivation of hypoxia inducible factor 1α.

PubMed

Zhao, Wei; Chang, Cunjie; Cui, Yangyan; Zhao, Xiaozhi; Yang, Jun; Shen, Lan; Zhou, Ji; Hou, Zhibo; Zhang, Zhen; Ye, Changxiao; Hasenmayer, Donald; Perkins, Robert; Huang, Xiaojing; Yao, Xin; Yu, Like; Huang, Ruimin; Zhang, Dianzheng; Guo, Hongqian; Yan, Jun

2014-04-18

Cancer cell proliferation is a metabolically demanding process, requiring high glycolysis, which is known as "Warburg effect," to support anabolic growth. Steroid receptor coactivator-3 (SRC-3), a steroid receptor coactivator, is overexpressed and/or amplified in multiple cancer types, including non-steroid targeted cancers, such as urinary bladder cancer (UBC). However, whether SRC-3 regulates the metabolic reprogramming for cancer cell growth is unknown. Here, we reported that overexpression of SRC-3 accelerated UBC cell growth, accompanied by the increased expression of genes involved in glycolysis. Knockdown of SRC-3 reduced the UBC cell glycolytic rate under hypoxia, decreased tumor growth in nude mice, with reduction of proliferating cell nuclear antigen and lactate dehydrogenase expression levels. We further revealed that SRC-3 could interact with hypoxia inducible factor 1α (HIF1α), which is a key transcription factor required for glycolysis, and coactivate its transcriptional activity. SRC-3 was recruited to the promoters of HIF1α-target genes, such as glut1 and pgk1. The positive correlation of expression levels between SRC-3 and Glut1 proteins was demonstrated in human UBC patient samples. Inhibition of glycolysis through targeting HK2 or LDHA decelerated SRC-3 overexpression-induced cell growth. In summary, overexpression of SRC-3 promoted glycolysis in bladder cancer cells through HIF1α to facilitate tumorigenesis, which may be an intriguing drug target for bladder cancer therapy.

[Promotion effect of stromal cell-derived factor 1 on the migration of epidermal stem cells in the healing process of frostbite-wound model ex vivo].

PubMed

Gan, Lu; Cao, Chuan; Li, Shi-rong; Chai, Lin-lin; Guo, Rui; Xiang, Guang-jin; Zhao, Shu-wen

2010-06-01

To study the promotion effect of stromal cell-derived factor 1 (SDF-1) on the migration of epidermal stem cells (ESC) in the healing process of frostbite-wound model ex vivo. A three-dimensional model of full-thickness frostbite of skin was constructed (with slot-like wound) out of skin equivalent. The expression of SDF-1 in wound stroma was observed with immunohistochemistry staining on post injury days (PID) 3 and 7. The model frostbite wounds were divided into control group (treated with PBS 50 microL per wound), SDF-1 group (treated with 100 ng/mL SDF-1, 50 microL per wound), and AMD3100 group [treated with 100 ng/mL AMD3100 (50 microL per wound) for 30 minutes, and then SDF-1 50 microL was added per wound]. The redistribution of ESC around wound was observed. The expression of SDF-1 in wound stroma increased gradually on PID 3 and 7. Compared with those in control and AMD3100 groups, there were more ESC and epithelial cell layers, and more integrin beta(1)-positive cells appeared at the basal layer of wound in SDF-1 group, and some of the positive cells migrated upward to epidermis. SDF-1 contributes to wound repair through promoting ESC to migrate toward and gather around wound edge. This may be one of the mechanisms of ESC participating in wound repair.

Glial cell line-derived neurotrophic factor in combination with insulin-like growth factor 1 and basic fibroblast growth factor promote in vitro culture of goat spermatogonial stem cells.

PubMed

Bahadorani, M; Hosseini, S M; Abedi, P; Abbasi, H; Nasr-Esfahani, M H

2015-01-01

Growth factors are increasingly considered as important regulators of spermatogonial stem cells (SSCs). This study investigated the effects of various growth factors (GDNF, IGF1, bFGF, EGF and GFRalpha-1) on purification and colonization of undifferentiated goat SSCs under in vitro and in vivo conditions. Irrespective of the culture condition used, the first signs of developing colonies were observed from day 4 of culture onwards. The number of colonies developed in GDNF + IGF1 + bFGF culture condition was significantly higher than the other groups (p < 0.05). In contrast, the size of colonies developed in GDNF + EGF + LIF culture condition was significantly higher than the other groups (p < 0.05). Immunocytochemical stationing for specific biomarkers of somatic cells (vimentin, alpha-inhibin and α-SMA) and spermatogonial cells (PLZF, THY 1, VASA, alpha-1 integrin, bet-1 integrin and DBA) revealed that both cell types existed in developing colonies, irrespective of the culture condition used. Even though, the relative abundance of VASA, FGFR3, OCT4, PLZF, BCL6B and THY1 transcription factors in GDNF + IGF1 + bFGF treatment group was significantly higher than the other groups (p < 0.05). Additionally, goat SSCs developed in the latter culture condition could colonize within the seminiferous tubules of the germ-cell depleted recipient mice following xenotransplantation. Obtained results demonstrated that combination of GDNF with IGF1 and bFGF promote in vitro culture of goat SSCs while precludes uncontrolled proliferation of somatic cells.

Lysophosphatidylcholine acyltransferase1 overexpression promotes oral squamous cell carcinoma progression via enhanced biosynthesis of platelet-activating factor.

PubMed

Shida-Sakazume, Tomomi; Endo-Sakamoto, Yosuke; Unozawa, Motoharu; Fukumoto, Chonji; Shimada, Ken; Kasamatsu, Atsushi; Ogawara, Katsunori; Yokoe, Hidetaka; Shiiba, Masashi; Tanzawa, Hideki; Uzawa, Katsuhiro

2015-01-01

The relevance of lysophosphatidylcholine acyltransferase1 (LPCAT1), a cytosolic enzyme in the remodeling pathway of phosphatidylcholine metabolism, in oral squamous cell carcinoma (OSCC) is unknown. We investigated LPCAT1 expression and its functional mechanism in OSCCs. We analyzed LPCAT1 mRNA and protein expression levels in OSCC-derived cell lines. Immunohistochemistry was performed to identify correlations between LPCAT1 expression levels and primary OSCCs clinicopathological status. We established LPCAT1 knockdown models of the OSCC-derived cell lines (SAS, Ca9-22) for functional analysis and examined the association between LPCAT1 expression and the platelet-activating factor (PAF) concentration and PAF-receptor (PAFR) expression. LPCAT1 mRNA and protein were up-regulated significantly (p<0.05) in OSCC-derived cell lines compared with human normal oral keratinocytes. Immunohistochemistry showed significantly (p<0.05) elevated LPCAT1 expression in primary OSCCs compared with normal counterparts and a strong correlation between LPCAT1-positive OSCCs and tumoral size and regional lymph node metastasis. In LPCAT1 knockdown cells, cellular proliferation and invasiveness decreased significantly (p<0.05); cellular migration was inhibited compared with control cells. Down-regulation of LPCAT1 resulted in a decreased intercellular PAF concentration and PAFR expression. LPCAT1 was overexpressed in OSCCs and correlated with cellular invasiveness and migration. LPCAT1 may contribute to tumoral growth and metastasis in oral cancer.

Clinicopathologic Risk Factor Distributions for MLH1 Promoter Region Methylation in CIMP-Positive Tumors.

PubMed

Levine, A Joan; Phipps, Amanda I; Baron, John A; Buchanan, Daniel D; Ahnen, Dennis J; Cohen, Stacey A; Lindor, Noralane M; Newcomb, Polly A; Rosty, Christophe; Haile, Robert W; Laird, Peter W; Weisenberger, Daniel J

2016-01-01

The CpG island methylator phenotype (CIMP) is a major molecular pathway in colorectal cancer. Approximately 25% to 60% of CIMP tumors are microsatellite unstable (MSI-H) due to DNA hypermethylation of the MLH1 gene promoter. Our aim was to determine if the distributions of clinicopathologic factors in CIMP-positive tumors with MLH1 DNA methylation differed from those in CIMP-positive tumors without DNA methylation of MLH1. We assessed the associations between age, sex, tumor-site, MSI status BRAF and KRAS mutations, and family colorectal cancer history with MLH1 methylation status in a large population-based sample of CIMP-positive colorectal cancers defined by a 5-marker panel using unconditional logistic regression to assess the odds of MLH1 methylation by study variables. Subjects with CIMP-positive tumors without MLH1 methylation were significantly younger, more likely to be male, and more likely to have distal colon or rectal primaries and the MSI-L phenotype. CIMP-positive MLH1-unmethylated tumors were significantly less likely than CIMP-positive MLH1-methylated tumors to harbor a BRAF V600E mutation and significantly more likely to harbor a KRAS mutation. MLH1 methylation was associated with significantly better overall survival (HR, 0.50; 95% confidence interval, 0.31-0.82). These data suggest that MLH1 methylation in CIMP-positive tumors is not a completely random event and implies that there are environmental or genetic determinants that modify the probability that MLH1 will become methylated during CIMP pathogenesis. MLH1 DNA methylation status should be taken into account in etiologic studies. ©2015 American Association for Cancer Research.

Clinicopathological risk factor distributions for MLH1 promoter region methylation in CIMP positive tumors

PubMed Central

Levine, A. Joan; Phipps, Amanda I.; Baron, John A.; Buchanan, Daniel D.; Ahnen, Dennis J.; Cohen, Stacey A.; Lindor, Noralane M.; Newcomb, Polly A.; Rosty, Christophe; Haile, Robert W.; Laird, Peter W.; Weisenberger, Daniel J.

2015-01-01

Background The CpG Island Methylator Phenotype (CIMP) is a major molecular pathway in colorectal cancer (CRC). Approximately 25% to 60% of CIMP tumors are microsatellite unstable (MSI-H) due to DNA hypermethylation of the MLH1 gene promoter. Our aim was to determine if the distributions of clinicopathologic factors in CIMP-positive tumors with MLH1 DNA methylation differed from those in CIMP-positive tumors without DNA methylation of MLH1. Methods We assessed the associations between age, sex, tumor-site, MSI status BRAF and KRAS mutations and family CRC history with MLH1 methylation status in a large population-based sample of CIMP-positive CRCs defined by a 5-marker panel using unconditional logistic regression to assess the odds of MLH1 methylation by study variables. Results Subjects with CIMP-positive tumors without MLH1 methylation were significantly younger, more likely to be male, more likely to have distal colon or rectal primaries and the MSI-L phenotype. CIMP-positive MLH1-unmethylated tumors were significantly less likely than CIMP-positive MLH1-methylated tumors to harbor a BRAF V600E mutation and significantly more likely to harbor a KRAS mutation. MLH1 methylation was associated with significantly better overall survival (HR=0.50; 95% Confidence Interval (0.31, 0.82)). Conclusions These data suggest that MLH1 methylation in CIMP-positive tumors is not a completely random event and implies that there are environmental or genetic determinants that modify the probability that MLH1 will become methylated during CIMP pathogenesis. Impact MLH1 DNA methylation status should be taken into account in etiologic studies. PMID:26512054

Differences in expression of the cancer stem cell marker aldehyde dehydrogenase 1 among estrogen receptor-positive/human epidermal growth factor receptor type 2-negative breast cancer cases with early, late, and no recurrence.

PubMed

Miyoshi, Yuichiro; Shien, Tadahiko; Ogiya, Akiko; Ishida, Naoko; Yamazaki, Kieko; Horii, Rie; Horimoto, Yoshiya; Masuda, Norikazu; Yasojima, Hiroyuki; Inao, Touko; Osako, Tomofumi; Takahashi, Masato; Tomioka, Nobumoto; Endo, Yumi; Hosoda, Mitsuchika; Doihara, Hiroyoshi; Miyoshi, Shinichiro; Yamashita, Hiroko

2016-07-02

The significance of the expression of aldehyde dehydrogenase 1 (ALDH1), a cancer stem cell marker, for predicting the recurrence of estrogen receptor (ER)-positive/human epidermal growth factor receptor type 2 (HER2)-negative breast cancer is still poorly understood. The value of ALDH1 in predicting the time of recurrence remains unknown. In total, 184 patients with early distant recurrence, 134 patients with late distant recurrence, and 321 control patients without recurrence for more than 10 years after starting initial treatment for ER-positive/HER2-negative breast cancer, registered in 9 institutions, were analyzed. We assessed relationships between ALDH1 and other clinicopathological features, and ALDH1 expression was compared among the three groups. The relationship between ALDH1 expression and overall survival after recurrence was also evaluated in each group. The rates of ALDH1 expression positivity (more than 1 %) in the early, late, and no recurrence groups were 18.4 %, 13.4 %, and 8.4 %, respectively. ALDH1 expression correlated significantly with lymph node metastases (p = 0.048) and the Ki-67 labeling index (p < 0.001) in the early recurrence group. Multivariate analysis revealed ALDH1 expression to be significantly higher in the early recurrence group than in the no recurrence group (adjusted OR 2.140, 95 % CI 1.144-4.003, p = 0.016). Moreover, there was a significant difference in ALDH1 expression between the early and no recurrence groups receiving adjuvant endocrine therapy and chemotherapy (adjusted OR 4.625, 95 % CI 1.881-12.474, p < 0.001). However, there was no difference in ALDH1 expression between the late and no recurrence groups in univariate analysis (OR 1.507, 95 % CI 0.738-2.998, p = 0.253). In multivariate analysis, ALDH1 was not a factor independently predicting overall survival after the detection of recurrence (adjusted OR 1.451, 95 % CI 0.985-2.085, p = 0.059). Among patients with ER-positive/HER2

Ferrous glycinate regulates cell energy metabolism by restrictinghypoxia-induced factor-1α expression in human A549 cells.

PubMed

Kuo, Yung-Ting; Jheng, Jhong-Huei; Lo, Mei-Chen; Chen, Wei-Lu; Wang, Shyang-Guang; Lee, Horng-Mo

2018-06-04

Iron or oxygen regulates the stability of hypoxia inducible factor-1α (HIF-1α). We investigated whether ferrous glycinate would affect HIF-1α accumulation, aerobic glycolysis and mitochondrial energy metabolism in human A549 lung cancer cells. Incubation of A549 cells with ferrous glycinate decreased the protein levels of HIF-1α, which was abrogated by proteosome inhibitor, or prolyl hydroxylase inhibitor. The addition of ferrous glycinate decreased protein levels of glucose transporter-1, hexokinase-2, and lactate dehydrogenase A, and decreased pyruvate dehydrogenase kinase-1 (PDK-1) and pyruvate dehydrogenase (PDH) phosphorylation in A549 cells. Ferrous glycinate also increased the expression of the mitochondrial transcription factor A (TFAM), and the mitochondrial protein, cytochrome c oxidase (COX-IV). Silencing of HIF-1α expression mimicked the effects of ferrous glycinate on PDK-1, PDH, TFAM and COX-IV in A549 cells. Ferrous glycinate increased mitochondrial membrane potential and ATP production in A549 cells. These results suggest that ferrous glycinate may reverse Warburg effect through down regulating HIF-1α in A549 cells.

Positive Results Bias and Impact Factor in Ophthalmology.

PubMed

Mimouni, Michael; Krauthammer, Mark; Gershoni, Assaf; Mimouni, Francis; Nesher, Ronit

2015-01-01

Previous studies in several fields of medicine have reported an association between the result of a trial (positive versus negative) and the impact factor of the journal in which it is published. The purpose of this study was to test the hypotheses that in the field of ophthalmology: (1) studies with positive results have a greater chance of being published in journals with a higher impact factor; (2) likewise, studies with a larger number of participants are more likely to be published in journals with a higher impact factor. In this retrospective study, consecutive randomized, controlled trials conducted in the field of ophthalmology between 1 January 2010 and 1 January 2013 were retrieved from PubMed. Each study was classified as having either a positive or negative result. A positive result was defined as a study in which there was a statistically significant difference between groups (p < 0.05). The impact factor of the journal in which the study was published was retrieved. The number of patients enrolled and whether or not the trial was placebo controlled was documented as well. Out of 2524 studies identified, 892 met the inclusion criteria. Studies with positive results were published in journals with a significantly higher impact factor than that of the journals in which negative result studies were published (p < 0.001). Studies with positive results had a slightly larger number of participants than studies with negative results (p = 0.028). In multiple regression analysis, the ranked impact factor was significantly predicted by the primary outcome (positive versus negative results) and the number of participants in a study (total R(2 )= 2.95, p < 0.001). In the field of ophthalmology, articles with positive results are currently published in journals with a higher impact factor. This finding supports the ongoing occurrence of positive results bias in the field of ophthalmology.

Helicobacter pylori induces vascular endothelial growth factor production in gastric epithelial cells through hypoxia-inducible factor-1α-dependent pathway.

PubMed

Kang, Min-Jung; Song, Eun-Jung; Kim, Bo-Yeon; Kim, Dong-Jae; Park, Jong-Hwan

2014-12-01

Although Helicobacter pylori have been known to induce vascular endothelial growth factor (VEGF) production in gastric epithelial cells, the precise mechanism for cellular signaling is incompletely understood. In this study, we investigated the role of bacterial virulence factor and host cellular signaling in VEGF production of H. pylori-infected gastric epithelial cells. We evaluated production of VEGF, activation of nuclear factor nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinases (MAPKs) and hypoxia-inducible factor-1α (HIF-1α) stabilization in gastric epithelial cells infected with H. pylori WT or isogenic mutants deficient in type IV secretion system (T4SS). H. pylori induced VEGF production in gastric epithelial cells via both T4SS-dependent and T4SS-independent pathways, although T4SS-independent pathway seems to be the dominant signaling. The inhibitor assay implicated that activation of NF-κB and MAPKs is dispensable for H. pylori-induced VEGF production in gastric epithelial cells. H. pylori led to HIF-1α stabilization in gastric epithelial cells independently of T4SS, NF-κB, and MAPKs, which was essential for VEGF production in these cells. N-acetyl-cysteine (NAC), a reactive oxygen species (ROS) inhibitor, treatment impaired H. pylori-induced HIF-1α stabilization and VEGF production in gastric epithelial cells. We defined the important role of ROS-HIF-1α axis in VEGF production of H. pylori-infected gastric epithelial cells, and bacterial T4SS has a minor role in H. pylori-induced VEGF production of gastric epithelial cells. © 2014 John Wiley & Sons Ltd.

SDF-1 overexpression by mesenchymal stem cells enhances GAP-43-positive axonal growth following spinal cord injury.

PubMed

Stewart, Andrew Nathaniel; Matyas, Jessica Jane; Welchko, Ryan Matthew; Goldsmith, Alison Delanie; Zeiler, Sarah Elizabeth; Hochgeschwender, Ute; Lu, Ming; Nan, Zhenhong; Rossignol, Julien; Dunbar, Gary Leo

2017-01-01

Utilizing genetic overexpression of trophic molecules in cell populations has been a promising strategy to develop cell replacement therapies for spinal cord injury (SCI). Over-expressing the chemokine, stromal derived factor-1 (SDF-1α), which has chemotactic effects on many cells of the nervous system, offers a promising strategy to promote axonal regrowth following SCI. The purpose of this study was to explore the effects of human SDF-1α, when overexpressed by mesenchymal stem cells (MSCs), on axonal growth and motor behavior in a contusive rat model of SCI. Using a transwell migration assay, the paracrine effects of MSCs, which were engineered to secrete human SDF-1α (SDF-1-MSCs), were assessed on cultured neural stem cells (NSCs). For in vivo analyses, the SDF-1-MSCs, unaltered MSCs, or Hanks Buffered Saline Solution (vehicle) were injected into the lesion epicenter of rats at 9-days post-SCI. Behavior was analyzed for 7-weeks post-injury, using the Basso, Beattie, and Bresnahan (BBB) scale of locomotor functions. Immunohistochemistry was performed to evaluate major histopathological outcomes, including gliosis, inflammation, white matter sparing, and cavitation. New axonal outgrowth was characterized using immunohistochemistry against the neuron specific growth-associated protein-43 (GAP-43). The results of these experiments demonstrate that the overexpression of SDF-1α by MSCs can enhance the migration of NSCs in vitro. Although only modest functional improvements were observed following transplantation of SDF-1-MSCs, a significant reduction in cavitation surrounding the lesion, and an increased density of GAP-43-positive axons inside the SCI lesion/graft site were found. The results from these experiments support the potential role for utilizing SDF-1α as a treatment for enhancing growth and regeneration of axons after traumatic SCI.

Posttransplantation primary cutaneous CD30 (Ki-1)-positive large-cell lymphoma.

PubMed

Seçkin, D; Demirhan, B; Oğuz Güleç, T; Arikan, U; Haberal, M

2001-12-01

We describe the case of a 51-year-old female renal transplant recipient with primary cutaneous CD30-positive large-cell lymphoma of T-cell origin. Cutaneous T-cell lymphomas are rarely reported in organ transplant recipients, and we believe they should be considered in the differential diagnosis of cutaneous neoplastic and infectious diseases affecting this patient group.

MicroRNA-214 Reduces Insulin-like Growth Factor-1 (IGF-1) Receptor Expression and Downstream mTORC1 Signaling in Renal Carcinoma Cells*

PubMed Central

Das, Falguni; Dey, Nirmalya; Bera, Amit; Kasinath, Balakuntalam S.; Ghosh-Choudhury, Nandini; Choudhury, Goutam Ghosh

2016-01-01

Elevated IGF-1/insulin-like growth factor-1 receptor (IGF-1R) autocrine/paracrine signaling in patients with renal cell carcinoma is associated with poor prognosis of the disease independent of their von Hippel-Lindau (VHL) status. Increased expression of IGF-1R in renal cancer cells correlates with their potency of tumor development and progression. The mechanism by which expression of IGF-1R is increased in renal carcinoma is not known. We report that VHL-deficient and VHL-positive renal cancer cells possess significantly decreased levels of mature, pre-, and pri-miR-214 than normal proximal tubular epithelial cells. We identified an miR-214 recognition element in the 3′UTR of IGF-1R mRNA and confirmed its responsiveness to miR-214. Overexpression of miR-214 decreased the IGF-1R protein levels, resulting in the inhibition of Akt kinase activity in both types of renal cancer cells. IGF-1 provoked phosphorylation and inactivation of PRAS40 in an Akt-dependent manner, leading to the activation of mTORC1 signal transduction to increase phosphorylation of S6 kinase and 4EBP-1. Phosphorylation-deficient mutants of PRAS40 and 4EBP-1 significantly inhibited IGF-1R-driven proliferation of renal cancer cells. Expression of miR-214 suppressed IGF-1R-induced phosphorylation of PRAS40, S6 kinase, and 4EBP-1, indicating inhibition of mTORC1 activity. Finally, miR-214 significantly blocked IGF-1R-forced renal cancer cell proliferation, which was reversed by expression of 3′UTR-less IGF-1R and constitutively active mTORC1. Together, our results identify a reciprocal regulation of IGF-1R levels and miR-214 expression in renal cancer cells independent of VHL status. Our data provide evidence for a novel mechanism for IGF-1R-driven renal cancer cell proliferation involving miR-214 and mTORC1. PMID:27226530

Fas Promotes T Helper 17 Cell Differentiation and Inhibits T Helper 1 Cell Development by Binding and Sequestering Transcription Factor STAT1.

PubMed

Meyer Zu Horste, Gerd; Przybylski, Dariusz; Schramm, Markus A; Wang, Chao; Schnell, Alexandra; Lee, Youjin; Sobel, Raymond; Regev, Aviv; Kuchroo, Vijay K

2018-03-20

The death receptor Fas removes activated lymphocytes through apoptosis. Previous transcriptional profiling predicted that Fas positively regulates interleukin-17 (IL-17)-producing T helper 17 (Th17) cells. Here, we demonstrate that Fas promoted the generation and stability of Th17 cells and prevented their differentiation into Th1 cells. Mice with T-cell- and Th17-cell-specific deletion of Fas were protected from induced autoimmunity, and Th17 cell differentiation and stability were impaired. Fas-deficient Th17 cells instead developed a Th1-cell-like transcriptional profile, which a new algorithm predicted to depend on STAT1. Experimentally, Fas indeed bound and sequestered STAT1, and Fas deficiency enhanced IL-6-induced STAT1 activation and nuclear translocation, whereas deficiency of STAT1 reversed the transcriptional changes induced by Fas deficiency. Thus, our computational and experimental approach identified Fas as a regulator of the Th17-to-Th1 cell balance by controlling the availability of opposing STAT1 and STAT3 to have a direct impact on autoimmunity. Copyright © 2018. Published by Elsevier Inc.

Involvement of Mst1 in tumor necrosis factor-{alpha}-induced apoptosis of endothelial cells

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

Ohtsubo, Hideki; Ichiki, Toshihiro; Imayama, Ikuyo

2008-03-07

Mammalian sterile 20-kinase 1 (Mst1), a member of the sterile-20 family protein kinase, plays an important role in the induction of apoptosis. However, little is know about the physiological activator of Mst1 and the role of Mst1 in endothelial cells (ECs). We examined whether Mst1 is involved in the tumor necrosis factor (TNF)-{alpha}-induced apoptosis of ECs. Western blot analysis revealed that TNF-{alpha} induced activation of caspase 3 and Mst1 in a time- and dose-dependent manner. TNF-{alpha}-induced Mst1 activation is almost completely prevented by pretreatment with Z-DEVD-FMK, a caspase 3 inhibitor. Nuclear staining with Hoechst 33258 and fluorescence-activated cell sorting ofmore » propidium iodide-stained cells showed that TNF-{alpha} induced apoptosis of EC. Diphenyleneiodonium, an inhibitor of NADPH oxidase, and N-acetylcysteine, a potent antioxidant, also inhibited TNF-{alpha}-induced activation of Mst1 and caspase 3, as well as apoptosis. Knockdown of Mst1 expression by short interfering RNA attenuated TNF-{alpha}-induced apoptosis but not cleavage of caspase 3. These results suggest that Mst1 plays an important role in the induction of TNF-{alpha}-induced apoptosis of EC. However, positive feedback mechanism between Mst1 and caspase 3, which was shown in the previous studies, was not observed. Inhibition of Mst1 function may be beneficial for maintaining the endothelial integrity and inhibition of atherogenesis.« less

Human Epidermal Growth Factor Receptor 2 (HER2) –Specific Chimeric Antigen Receptor–Modified T Cells for the Immunotherapy of HER2-Positive Sarcoma

PubMed Central

Ahmed, Nabil; Brawley, Vita S.; Hegde, Meenakshi; Robertson, Catherine; Ghazi, Alexia; Gerken, Claudia; Liu, Enli; Dakhova, Olga; Ashoori, Aidin; Corder, Amanda; Gray, Tara; Wu, Meng-Fen; Liu, Hao; Hicks, John; Rainusso, Nino; Dotti, Gianpietro; Mei, Zhuyong; Grilley, Bambi; Gee, Adrian; Rooney, Cliona M.; Brenner, Malcolm K.; Heslop, Helen E.; Wels, Winfried S.; Wang, Lisa L.; Anderson, Peter; Gottschalk, Stephen

2015-01-01

Purpose The outcome for patients with metastatic or recurrent sarcoma remains poor. Adoptive therapy with tumor-directed T cells is an attractive therapeutic option but has never been evaluated in sarcoma. Patients and Methods We conducted a phase I/II clinical study in which patients with recurrent/refractory human epidermal growth factor receptor 2 (HER2) –positive sarcoma received escalating doses (1 × 104/m2 to 1 × 108/m2) of T cells expressing an HER2-specific chimeric antigen receptor with a CD28.ζ signaling domain (HER2-CAR T cells). Results We enrolled 19 patients with HER2-positive tumors (16 osteosarcomas, one Ewing sarcoma, one primitive neuroectodermal tumor, and one desmoplastic small round cell tumor). HER2-CAR T-cell infusions were well tolerated with no dose-limiting toxicity. At dose level 3 (1 × 105/m2) and above, we detected HER2-CAR T cells 3 hours after infusion by quantitative polymerase chain reaction in 14 of 16 patients. HER2-CAR T cells persisted for at least 6 weeks in seven of the nine evaluable patients who received greater than 1 × 106/m2 HER2-CAR T cells (P = .005). HER2-CAR T cells were detected at tumor sites of two of two patients examined. Of 17 evaluable patients, four had stable disease for 12 weeks to 14 months. Three of these patients had their tumor removed, with one showing ≥ 90% necrosis. The median overall survival of all 19 infused patients was 10.3 months (range, 5.1 to 29.1 months). Conclusion This first evaluation of the safety and efficacy of HER2-CAR T cells in patients with cancer shows the cells can persist for 6 weeks without evident toxicities, setting the stage for studies that combine HER2-CAR T cells with other immunomodulatory approaches to enhance their expansion and persistence. PMID:25800760

Risk factors associated with low CD4+ lymphocyte count among HIV-positive pregnant women in Nigeria.

PubMed

Abimiku, Alash'le; Villalba-Diebold, Pacha; Dadik, Jelpe; Okolo, Felicia; Mang, Edwina; Charurat, Man

2009-09-01

To determine the risk factors for CD4+ lymphocyte counts of 200 cells/mm(3) or lower in HIV-positive pregnant women in Nigeria. A cross-sectional data analysis from a prospective cohort of 515 HIV-positive women attending a prenatal clinic. Risk of a low CD4+ count was estimated using logistic regression analysis. CD4+ lymphocyte counts of 200 cells/mm(3) or lower (280+/-182 cells/mm(3)) were recorded in 187 (36.3%) out of 515 HIV-positive pregnant women included in the study. Low CD4+ count was associated with older age (adjusted odds ratio [aOR] 10.71; 95% confidence interval [CI], 1.20-95.53), lack of condom use (aOR, 5.16; 95% CI, 1.12-23.8), history of genital ulcers (aOR, 1.78; 95% CI, 1.12-2.82), and history of vaginal discharge (aOR; 1.62; 1.06-2.48). Over 35% of the HIV-positive pregnant women had low CD4+ counts, indicating the need for treatment. The findings underscore the need to integrate prevention of mother-to-child transmission with HIV treatment and care, particularly services for sexually transmitted infections.

Apoptosis-Resistant Cardiac Progenitor Cells Modified With Apurinic/Apyrimidinic Endonuclease/Redox Factor 1 Gene Overexpression Regulate Cardiac Repair After Myocardial Infarction.

PubMed

Aonuma, Tatsuya; Takehara, Naofumi; Maruyama, Keisuke; Kabara, Maki; Matsuki, Motoki; Yamauchi, Atsushi; Kawabe, Jun-Ichi; Hasebe, Naoyuki

2016-08-01

: Overcoming the insufficient survival of cell grafts is an essential objective in cell-based therapy. Apurinic/apyrimidinic endonuclease/redox factor 1 (APE1) promotes cell survival and may enhance the therapeutic effect of engrafted cells. The aim of this study is to determine whether APE1 overexpression in cardiac progenitor cells (CPCs) could ameliorate the efficiency of cell-based therapy. CPCs isolated from 8- to 10-week-old C57BL/6 mouse hearts were infected with retrovirus harboring APE1-DsRed (APE1-CPC) or a DsRed control (control-CPC). Oxidative stress-induced apoptosis was then assessed in APE1-CPCs, control-CPCs, and neonatal rat ventricular myocytes (NRVMs) cocultured with these CPCs. This analysis revealed that APE1 overexpression inhibited CPC apoptosis with activation of transforming growth factor β-activated kinase 1 (TAK1) and nuclear factor (NF)-κB. In the coculture model, NRVM apoptosis was inhibited to a greater extent in the presence of APE1-CPCs compared with control-CPCs. Moreover, the number of surviving DsRed-positive CPC grafts was significantly higher 7 days after the transplant of APE1-CPCs into a mouse myocardial infarction model, and the left ventricular ejection fraction showed greater improvement with attenuation of fibrosis 28 days after the transplant of APE1-CPCs compared with control-CPCs. Additionally, fewer inflammatory macrophages and a higher percentage of cardiac α-sarcomeric actinin-positive CPC-grafts were observed in mice injected with APE1-CPCs compared with control-CPCs after 7 days. In conclusion, antiapoptotic APE1-CPC graft, which increased TAK1-NF-κB pathway activation, survived effectively in the ischemic heart, restored cardiac function, and reduced cardiac inflammation and fibrosis. APE1 overexpression in CPCs may serve as a novel strategy to improve cardiac cell therapy. Improving the survival of cell grafts is essential to maximize the efficacy of cell therapy. The authors investigated the role of APE1 in

B-cell subset alterations and correlated factors in HIV-1 infection.

PubMed

Pensieroso, Simone; Galli, Laura; Nozza, Silvia; Ruffin, Nicolas; Castagna, Antonella; Tambussi, Giuseppe; Hejdeman, Bo; Misciagna, Donatella; Riva, Agostino; Malnati, Mauro; Chiodi, Francesca; Scarlatti, Gabriella

2013-05-15

During HIV-1 infection, the development, phenotype, and functionality of B cells are impaired. Transitional B cells and aberrant B-cell populations arise in blood, whereas a declined percentage of resting memory B cells is detected. Our study aimed at pinpointing the demographic, immunological, and viral factors driving these pathological findings, and the role of antiretroviral therapy in reverting these alterations. B-cell phenotype and correlating factors were evaluated. Variations in B-cell subsets were evaluated by flow cytometry in HIV-1-infected individuals naive to therapy, elite controllers, and patients treated with antiretroviral drugs (virological control or failure). Multivariable analysis was performed to identify variables independently associated with the B-cell alterations. Significant differences were observed among patients' groups in relation to all B-cell subsets. Resting memory B cells were preserved in patients naive to therapy and elite controllers, but reduced in treated patients. Individuals naive to therapy and experiencing multidrug failure, as well as elite controllers, had significantly higher levels of activated memory B cells compared to healthy controls. In the multivariate analysis, plasma viral load and nadir CD4 T cells independently correlated with major B-cell alterations. Coinfection with hepatitis C but not hepatitis B virus also showed an impact on specific B-cell subsets. Successful protracted antiretroviral treatment led to normalization of all B-cell subsets with exception of resting memory B cells. Our results indicate that viremia and nadir CD4 T cells are important prognostic markers of B-cell perturbations and provide evidence that resting memory B-cell depletion during chronic infection is not reverted upon successful antiretroviral therapy.

A Role for MEK-Interacting Protein 1 in Hormone Responsiveness of ER Positive Breast Cancer Cells

DTIC Science & Technology

2010-07-01

positive, but not ER-negative, breast cancer cell lines. 2) The cell death observed in ER- positiv e cell lin es was associated with an a pproximate...and stained after 24 h, then counted. Top panel: photographs of stained cells. Bottom panel: Quantitation of migrated cells, normalized to control...function and breast cancer biology. W e therefore hypothesized that MP1 m ight play an im portant role in ER positiv e breast cancer cells. To test this

Side population cells and Bcrp1 expression in lung.

PubMed

Summer, Ross; Kotton, Darrell N; Sun, Xi; Ma, Bei; Fitzsimmons, Kathleen; Fine, Alan

2003-07-01

Side population (SP) cells are a rare subset of cells found in various tissues that are highly enriched for stem cell activity. SP cells can be isolated by dual-wavelength flow cytometry because of their capacity to efflux Hoechst dye, a process mediated by the ATP-binding cassette transporter breast cancer resistance protein (Bcrp) 1. By performing flow cytometry of enzymedigested mouse lung stained with Hoechst dye, we found that SP cells comprise 0.03-0.07% of total lung cells and are evenly distributed in proximal and distal lung regions. By RT-PCR, we found that lung SP cells express hepatocyte nuclear factor-3beta, but not thyroid transcription factor-1. Surface marker analysis revealed lung SP cells to be stem cell antigen 1 positive, Bcrp1 positive, lineage marker negative, and heterogeneous at the CD45 locus. As expected, we did not detect lung SP cells in Bcrp1-deficient animals. We, therefore, employed nonisotopic in situ hybridization and immunostaining for Bcrp1 as a strategy to localize these cells in vivo. Expression was observed in distinct lung cell types: bronchial and vascular smooth muscle cells and round cells within the distal air space. We confirmed the expression of Bcrp1 in primary bronchial smooth muscle cell cultures (BSMC) and in lavaged distal airway cells, but neither possessed the capacity to efflux Hoechst dye. In BSMC, Bcrp1 was localized to an intracellular compartment, suggesting that the molecular site of Bcrp1 expression regulates SP phenotype.

Induced trefoil factor family 1 expression by trans-differentiating Clara cells in a murine asthma model.

PubMed

Kouznetsova, Irina; Chwieralski, Caroline E; Bälder, Ralf; Hinz, Margitta; Braun, Armin; Krug, Norbert; Hoffmann, Werner

2007-03-01

Asthma is a chronic inflammatory disease of the airways that is accompanied by goblet cell metaplasia and mucus hypersecretion. Trefoil factor family (TFF) peptides represent major secretory products of the respiratory tract and are synthesized together with mucins. In the murine lung, TFF2 is mainly expressed, whereas TFF1 transcripts represent only a minor species. TFF peptides are well known for their motogenic and anti-apoptotic effects, and they modulate the inflammatory response of bronchial epithelial cells. Here, an established mouse model of asthma was investigated (i.e., exposure to Aspergillus fumigatus [AF] antigens). RT-PCR analysis of lung tissue showed elevated levels particularly of TFF1 transcripts in AF-sensitized/challenged animals. In contrast, transcripts encoding Clara cell secretory protein (CCSP/CC10) were strongly diminished in these animals. For comparison, the expression of the goblet cell secretory granule marker mCLCA3/Gob-5, the mucins Muc1-Muc6 and Muc19, and the secretoglobins ScgB3A1 and ScgB3A2, as well as the mammalian ependymin-related gene MERP2, were monitored. Immunohistochemistry localized TFF1 mainly in cells with a mixed phenotype (e.g., TFF1-positive cells stain with the lectin wheat germ agglutinin (WGA), which recognizes mucins characteristic of goblet cells). In addition, these cells express CCSP/CC10, a Clara cell marker. When compared with mucins or CCSP/CC10, TFF1 was stored in a different population of secretory granules localized at the more basolateral portion of these cells. Thus, the results presented indicate for the first time that allergen exposure leads to the trans-differentiation of Clara cells toward a TFF1-expressing mucous phenotype.

Saxagliptin Induces β-Cell Proliferation through Increasing Stromal Cell-Derived Factor-1α In Vivo and In Vitro.

PubMed

Li, Chun-Jun; Sun, Bei; Fang, Qian-Hua; Ding, Min; Xing, Yun-Zhi; Chen, Li-Ming; Yu, De-Min

2017-01-01

Dipeptidyl peptidase-4 inhibitors, such as saxagliptin, have been reported to have beneficial effects on β-cell function, but the specific underlying mechanism remains unclear. Stromal cell-derived factor-1α (SDF-1α), a chemokine produced in multiple organs, has been considered as a crucial regulator in promoting β-cell survival. Here, we speculate that SDF-1α might mediate the effect of saxagliptin on improving β-cell function. After 12-week saxagliptin treatment in high-fat diet/streptozotocin-induced diabetic rats, significant improvement in pancreas insulin secretion capacity evaluated by hyperglycemia clamp and increased β-cell to α-cell areas ratio were observed. Saxagliptin significantly induced β-cell proliferation and upregulated the expression of proliferation-related factors including c-myc and cyclind D1 determined with western blotting from the isolated islets. The expression/activity of DPP-4 was significantly reduced and paralleled with the restoration of SDF-1α levels in the saxagliptin-treated diabetic rats, subsequently the key WNT-signaling regulators, β-catenin, and AKT were activated. However, the effect of saxagliptin inducing β-cell proliferation was attenuated when we silenced the SDF-1α receptor (CXCR4) with RNAi in INS cell lines. Collectively, our data indicate that SDF-1α mediates the protective effect of saxagliptin on β-cell proliferation, suggesting that DPP-4 inhibitors have the potential role on delaying β-cell failure and SDF-1α could be a therapeutic target of β-cell regeneration.

Saxagliptin Induces β-Cell Proliferation through Increasing Stromal Cell-Derived Factor-1α In Vivo and In Vitro

PubMed Central

Li, Chun-Jun; Sun, Bei; Fang, Qian-Hua; Ding, Min; Xing, Yun-Zhi; Chen, Li-Ming; Yu, De-Min

2017-01-01

Dipeptidyl peptidase-4 inhibitors, such as saxagliptin, have been reported to have beneficial effects on β-cell function, but the specific underlying mechanism remains unclear. Stromal cell-derived factor-1α (SDF-1α), a chemokine produced in multiple organs, has been considered as a crucial regulator in promoting β-cell survival. Here, we speculate that SDF-1α might mediate the effect of saxagliptin on improving β-cell function. After 12-week saxagliptin treatment in high-fat diet/streptozotocin-induced diabetic rats, significant improvement in pancreas insulin secretion capacity evaluated by hyperglycemia clamp and increased β-cell to α-cell areas ratio were observed. Saxagliptin significantly induced β-cell proliferation and upregulated the expression of proliferation-related factors including c-myc and cyclind D1 determined with western blotting from the isolated islets. The expression/activity of DPP-4 was significantly reduced and paralleled with the restoration of SDF-1α levels in the saxagliptin-treated diabetic rats, subsequently the key WNT-signaling regulators, β-catenin, and AKT were activated. However, the effect of saxagliptin inducing β-cell proliferation was attenuated when we silenced the SDF-1α receptor (CXCR4) with RNAi in INS cell lines. Collectively, our data indicate that SDF-1α mediates the protective effect of saxagliptin on β-cell proliferation, suggesting that DPP-4 inhibitors have the potential role on delaying β-cell failure and SDF-1α could be a therapeutic target of β-cell regeneration. PMID:29230196

Hypoxia-inducible factor-1α promotes cell survival during ammonia stress response in ovarian cancer stem-like cells

PubMed Central

Kitajima, Shojiro; Lee, Kian Leong; Hikasa, Hiroki; Sun, Wendi; Huang, Ruby Yun-Ju; Yang, Henry; Matsunaga, Shinji; Yamaguchi, Takehiro; Araki, Marito; Kato, Hiroyuki

2017-01-01

Ammonia is a toxic by-product of metabolism that causes cellular stresses. Although a number of proteins are involved in adaptive stress response, specific factors that counteract ammonia-induced cellular stress and regulate cell metabolism to survive against its toxicity have yet to be identified. We demonstrated that the hypoxia-inducible factor-1α (HIF-1α) is stabilized and activated by ammonia stress. HIF-1α activated by ammonium chloride compromises ammonia-induced apoptosis. Furthermore, we identified glutamine synthetase (GS) as a key driver of cancer cell proliferation under ammonia stress and glutamine-dependent metabolism in ovarian cancer stem-like cells expressing CD90. Interestingly, activated HIF-1α counteracts glutamine synthetase function in glutamine metabolism by facilitating glycolysis and elevating glucose dependency. Our studies reveal the hitherto unknown functions of HIF-1α in a biphasic ammonia stress management in the cancer stem-like cells where GS facilitates cell proliferation and HIF-1α contributes to the metabolic remodeling in energy fuel usage resulting in attenuated proliferation but conversely promoting cell survival. PMID:29383096

Thrombopoietin/MPL signaling confers growth and survival capacity to CD41-positive cells in a mouse model of Evi1 leukemia.

PubMed

Nishikawa, Satoshi; Arai, Shunya; Masamoto, Yosuke; Kagoya, Yuki; Toya, Takashi; Watanabe-Okochi, Naoko; Kurokawa, Mineo

2014-12-04

Ecotropic viral integration site 1 (Evi1) is a transcription factor that is highly expressed in hematopoietic stem cells and is crucial for their self-renewal capacity. Aberrant expression of Evi1 is observed in 5% to 10% of de novo acute myeloid leukemia (AML) patients and predicts poor prognosis, reflecting multiple leukemogenic properties of Evi1. Here, we show that thrombopoietin (THPO) signaling is implicated in growth and survival of Evi1-expressing cells using a mouse model of Evi1 leukemia. We first identified that the expression of megakaryocytic surface molecules such as ITGA2B (CD41) and the THPO receptor, MPL, positively correlates with EVI1 expression in AML patients. In agreement with this finding, a subpopulation of bone marrow and spleen cells derived from Evi1 leukemia mice expressed both CD41 and Mpl. CD41(+) Evi1 leukemia cells induced secondary leukemia more efficiently than CD41(-) cells in a serial bone marrow transplantation assay. Importantly, the CD41(+) cells predominantly expressing Mpl effectively proliferated and survived on OP9 stromal cells in the presence of THPO via upregulating BCL-xL expression, suggesting an essential role of the THPO/MPL/BCL-xL cascade in enhancing the progression of Evi1 leukemia. These observations provide a novel aspect of the diverse functions of Evi1 in leukemogenesis. © 2014 by The American Society of Hematology.

Deep Proteome Profiling Reveals Common Prevalence of MZB1-Positive Plasma B Cells in Human Lung and Skin Fibrosis.

PubMed

Schiller, Herbert B; Mayr, Christoph H; Leuschner, Gabriela; Strunz, Maximilian; Staab-Weijnitz, Claudia; Preisendörfer, Stefan; Eckes, Beate; Moinzadeh, Pia; Krieg, Thomas; Schwartz, David A; Hatz, Rudolf A; Behr, Jürgen; Mann, Matthias; Eickelberg, Oliver

2017-11-15

Analyzing the molecular heterogeneity of different forms of organ fibrosis may reveal common and specific factors and thus identify potential future therapeutic targets. We sought to use proteome-wide profiling of human tissue fibrosis to (1) identify common and specific signatures across end-stage interstitial lung disease (ILD) cases, (2) characterize ILD subgroups in an unbiased fashion, and (3) identify common and specific features of lung and skin fibrosis. We collected samples of ILD tissue (n = 45) and healthy donor control samples (n = 10), as well as fibrotic skin lesions from localized scleroderma and uninvolved skin (n = 6). Samples were profiled by quantitative label-free mass spectrometry, Western blotting, or confocal imaging. We determined the abundance of more than 7,900 proteins and stratified these proteins according to their detergent solubility profiles. Common protein regulations across all ILD cases, as well as distinct ILD subsets, were observed. Proteomic comparison of lung and skin fibrosis identified a common upregulation of marginal zone B- and B1-cell-specific protein (MZB1), the expression of which identified MZB1 + /CD38 + /CD138 + /CD27 + /CD45 - /CD20 - plasma B cells in fibrotic lung and skin tissue. MZB1 levels correlated positively with tissue IgG and negatively with diffusing capacity of the lung for carbon monoxide. Despite the presumably high molecular and cellular heterogeneity of ILD, common protein regulations are observed, even across organ boundaries. The surprisingly high prevalence of MZB1-positive plasma B cells in tissue fibrosis warrants future investigations regarding the causative role of antibody-mediated autoimmunity in idiopathic cases of organ fibrosis, such as idiopathic pulmonary fibrosis.

[Clinical significance of HPV L1 capsid protein detection in cervical exfoliated cells in high-risk HPV positive women].

PubMed

Wang, Jiajian; Tian, Qifang; Zhang, Su; Lyu, Liping; Dong, Jie; Lyu, Weiguo

2015-04-01

To explore the clinical significance of human papillomavirus L1 capsid protein detection in cervical exfoliated cells in high-risk HPV positive women. From November 2012 to June 2013, 386 high-risk HPV positive (detected by hybrid capture II) cases were enrolled as eligible women from Huzhou Maternity & Child Care Hospital and Women's Hospital, School of Medicine, Zhejiang University. All eligible women underwent liquid-based cytology (ThinPrep) followed by colposcopy. Biopsies were taken if indicated. Cervical exfoliated cells were collected for HPV L1 capsid protein detection by immunocytochemistry. Expression of HPV L1 capsid protein in groups with different histological diagnosis were compared, and the role of HPV L1 capsid protein detection in cervical exfoliated cells in cervical lesions screening was accessed. Total 386 enrolled eligible women were finally diagnosed histologically as follwed: 162 normal cervix, 94 low-grade squamous intraepithelial lesion (LSIL), 128 high-grade squamous intraepithelial lesion (HSIL) and 2 squamous cervical cancer (SCC). The positive expression rate of HPV L1 in HSIL+ (HSIL or worse) group was significantly lower than that in LSIL- (LSIL or better) group (19.2% vs 66.4%, P=0.000). While identifying HSIL+ in HPV positive cases and compared with cytology, HPV L1 detection resulted in significant higher sensitivity (80.77% vs 50.77%, P=0.000) and negative predictive value (NPV; 87.18% vs 76.47%, P=0.004), significant lower specificity (66.41% vs 81.25%, P=0.000), and comparable positive predictive value (PPV; 54.97% vs 57.89%, P=0.619). To identify HSIL+ in HPV-positive/cytology-negative women, the sensitivity, specificity, PPV, and NPV of HPV L1 detection were 87.50%, 61.54%, 41.18%, and 94.12% respectively, while 80.00%, 86.36%, 80.00% and 86.36% respectively in HPV-positive/atypical squamous cell of undetermined significance (ASCUS) women. HPV L1 capsid detection in cervical exfoliated cells have a role in cervical lesions

Human RNA polymerase II associated factor 1 complex promotes tumorigenesis by activating c-MYC transcription in non-small cell lung cancer

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

Zhi, Xiuyi; Giroux-Leprieur, Etienne; Respiratory Diseases and Thoracic Oncology Department, Ambroise Pare Hospital – APHP, Versailles Saint Quentin en Yvelines University, 9 Avenue Charles de Gaulle, 92100, Boulogne-Billancourt

2015-10-02

Human RNA polymerase II (RNAPII)-associated factor 1 complex (hPAF1C) plays a crucial role in protein-coding gene transcription. Overexpression of hPAF1C has been implicated in the initiation and progression of various human cancers. However, the molecular pathways involved in tumorigenesis through hPAF1C remain to be elucidated. The current study suggested hPAF1C expression as a prognostic biomarker for early stage non-small cell lung cancer (NSCLC) and patients with low hPAF1C expression levels had significantly better overall survival. Furthermore, the expression of hPAF1C was found to be positively correlated with c-MYC expression in patient tumor samples and in cancer cell lines. Mechanistic studiesmore » indicated that hPAF1C could promote lung cancer cell proliferation through regulating c-MYC transcription. These results demonstrated the prognostic value of hPAF1C in early-stage NSCLC and the role of hPAF1C in the transcriptional regulation of c-MYC oncogene during NSCLC tumorigenesis. - Highlights: • hPAF1C expression is a prognostic biomarker for early stage non-small cell lung cancer. • The expression of hPAF1C was positively correlated with c-MYC in tumor samples of patients and in several NSCLC cell lines. • hPAF1C could promote lung cancer cell proliferation through regulating c-MYC transcription.« less

Role of insulin-like growth factor-1 (IGF-1) in regulating cell cycle progression

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

Ma, Qi-lin; Yang, Tian-lun; Yin, Ji-ye

2009-11-06

Aims: Insulin-like growth factor-1 (IGF-1) is a polypeptide protein hormone, similar in molecular structure to insulin, which plays an important role in cell migration, cell cycle progression, cell survival and proliferation. In this study, we investigated the possible mechanisms of IGF-1 mediated cell cycle redistribution and apoptosis of vascular endothelial cells. Method: Human umbilical vein endothelial cells (HUVECs) were pretreated with 0.1, 0.5, or 2.5 {mu}g/mL of IGF-1 for 30 min before the addition of Ang II. Cell cycle redistribution and apoptosis were examined by flow cytometry. Expression of Ang II type 1 (AT{sub 1}) mRNA and cyclin E proteinmore » were determined by RT-PCR and Western blot, respectively. Results: Ang II (1 {mu}mol/L) induced HUVECs arrested at G{sub 0}/G{sub 1}, enhanced the expression level of AT{sub 1} mRNA in a time-dependent manner, reduced the enzymatic activity of nitric oxide synthase (NOS) and nitric oxide (NO) content as well as the expression level of cyclin E protein. However, IGF-1 enhanced NOS activity, NO content, and the expression level of cyclin E protein, and reduced the expression level of AT{sub 1} mRNA. L-NAME significantly counteracted these effects of IGF-1. Conclusions: Our data suggests that IGF-1 can reverse vascular endothelial cells arrested at G{sub 0}/G{sub 1} and apoptosis induced by Ang II, which might be mediated via a NOS-NO signaling pathway and is likely associated with the expression levels of AT1 mRNA and cyclin E proteins.« less

Significant expression of thyroid transcription factor-1 in pulmonary squamous cell carcinoma detected by SPT24 monoclonal antibody and CSA-II system.

PubMed

Kashima, Kenji; Hashimoto, Hisashi; Nishida, Haruto; Arakane, Motoki; Yada, Naomi; Daa, Tsutomu; Yokoyama, Shigeo

2014-01-01

In contrast to the usefulness of thyroid transcription factor-1 (TTF-1) in distinguishing primary adenocarcinoma of the lung from metastatic lesions, TTF-1 expression in pulmonary squamous cell carcinoma is reported to be at low level and not a suitable immunohistochemical marker. We hypothesized that the highly sensitive detection system, CSA-II, can visualize even faint expression of TTF-1 in pulmonary squamous cell carcinoma. In this study, 2 commercially available clones of TTF-1 monoclonal antibody, 8G7G3/1 and SPT24, were used for staining 38 cases of pulmonary squamous cell carcinoma, in combination with the CSA-II and the conventional detection system, EnVision. The combined use of the 8G7G3/1 clone with EnVision and CSA-II showed a positive reaction in only 1 and 4 cases, respectively. The use of SPT24 clone showed positive staining in 5 cases with EnVision and in 20 of 38 cases (52.6%) with the CSA-II. Interestingly, positive staining by the SPT24-CSA-II technique of samples from tissue blocks preserved for <2 years was 73.6% compared with only 31.5% in those preserved for >2 years. In addition, a 6-month preservation of the cut sections resulted in stain fading and decreased positivity (50%), compared with freshly cut sections. We conclude that the use of the SPT24 monoclonal antibody with the CSA-II system can detect even weak expression of TTF-1 in pulmonary squamous cell carcinoma. This staining technique can potentially allow the discrimination of primary squamous cell carcinoma of the lung from metastatic lesions, especially in freshly prepared paraffin sections.

Effects of hypoxia-inducible factor-1α silencing on the proliferation of CBRH-7919 hepatoma cells

PubMed Central

Xu, Lin-Feng; Ni, Jia-Yan; Sun, Hong-Liang; Chen, Yao-Ting; Wu, Yu-Dan

2013-01-01

AIM: To study the effects of hypoxia-inducible factor-1α (HIF-1α) silencing on the proliferation of hypoxic CBRH-7919 rat hepatoma cells. METHODS: The CBRH-7919 rat hepatoma cell line was used in this study and the hypoxic model was constructed using CoCl2. The HIF-1α-specific RNAi sequences were designed according to the gene coding sequence of rat HIF-1α obtained from GeneBank. The secondary structure of the HIF-1α gene sequence was analyzed using RNA draw software. The small interfering RNA (siRNA) transfection mixture was produced by mixing the siRNA and Lipofectamine2000TM, and transfected into the hypoxic hepatoma cells. Real time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting assay were used to detect the expression levels of mRNA and protein. HIF-1α and vascular endothelial growth factor (VEGF) mRNA was determined using real time RT-PCR; the protein expression levels of AKT, p-AKT, p21 and cyclinD1 were determined using Western blotting. The proliferation of hepatoma cells was observed using the methyl thiazolyl tetrazolium (MTT) assay and the bromodeoxyuridine (BrdU) incorporation cell proliferation assay. RESULTS: Under induced hypoxia, the viability of the hepatoma cells reached a minimum at 800 μmol/L CoCl2; the viability of the cells was relatively high at CoCl2 concentrations between 100 μmol/L and 200 μmol/L. Under hypoxia, the mRNA and protein expression levels of HIF-1α and VEGF were significantly higher than that of hepatoma cells that were cultured in normaxia. HIF-1α-specific RNAi sequences were successfully transfected into hepatoma cells. The transfection of specific siRNAs significantly inhibited the mRNA and protein expression levels of HIF-1α and VEGF, along with the protein expression levels of p-AKT and cyclinD1; the protein expression of p21 was significantly increased, and there was no significant difference in the expression of AKT. The MTT assay showed that the amount of hepatoma cells in S

Stromal cell-derived factor 1 (SDF-1) accelerated skin wound healing by promoting the migration and proliferation of epidermal stem cells.

PubMed

Guo, Rui; Chai, Linlin; Chen, Liang; Chen, Wenguang; Ge, Liangpeng; Li, Xiaoge; Li, Hongli; Li, Shirong; Cao, Chuan

2015-06-01

Epidermal stem cells could contribute to skin repair through the migration of cells from the neighboring uninjured epidermis, infundibulum, hair follicle, or sebaceous gland. However, little is known about the factors responsible for the complex biological processes in wound healing. Herein, we will show that the attracting chemokine, SDF-1/CXCR4, is a major regulator involved in the migration of epidermal stem cells during wound repair. We found that the SDF-1 levels were markedly increased at the wound margins following injury and CXCR4 expressed in epidermal stem cells and proliferating epithelial cells. Blocking the SDF-1/CXCR4 axis resulted in a significant reduction in epidermal stem cell migration toward SDF-1 in vitro and delayed wound healing in vivo, while an SDF-1 treatment enhanced epidermal stem cell migration and proliferation and accelerated wound healing. These results provide direct evidence that SDF-1 promotes epidermal stem cell migration, accelerates skin regeneration, and makes the development of new regenerative therapeutic strategies for wound healing possible.

Gab1 Mediates Hepatocyte Growth Factor-Stimulated Mitogenicity and Morphogenesis in Multipotent Myeloid Cells

PubMed Central

Felici, Angelina; Giubellino, Alessio; Bottaro, Donald P.

2012-01-01

Hepatocyte growth factor (HGF)-stimulated mitogenesis, motogenesis and morphogenesis in various cell types begins with activation of the Met receptor tyrosine kinase and the recruitment of intracellular adaptors and kinase substrates. The adapter protein Gab1 is a critical effector and substrate of activated Met, mediating morphogenesis, among other activities, in epithelial cells. To define its role downstream of Met in hematopoietic cells, Gab1 was expressed in the HGF-responsive, Gab1-negative murine myeloid cell line 32D. Interestingly, the adhesion and motility of Gab1-expressing cells were significantly greater than parental cells, independent of growth factor treatment. Downstream of activated Met, Gab1 expression was specifically associated with rapid Shp-2 recruitment and activation, increased mitogenic potency, suppression of GATA-1 expression and concomitant upregulation of GATA-2 transcription. In addition to enhanced proliferation, continuous culture of Gab1-expressing 32D cells in HGF resulted in cell attachment, filopodia extension and phenotypic changes suggestive of monocytic differentiation. Our results suggest that in myeloid cells, Gab1 is likely to enhance HGF mitogenicity by coupling Met to Shp-2 and GATA-2 expression, thereby potentially contributing to normal myeloid differentiation as well as oncogenic transformation. PMID:20506405

Heregulin/ErbB3 Signaling Enhances CXCR4-Driven Rac1 Activation and Breast Cancer Cell Motility via Hypoxia-Inducible Factor 1α

PubMed Central

Lopez-Haber, Cynthia; Barrio-Real, Laura; Casado-Medrano, Victoria

2016-01-01

The growth factor heregulin (HRG), a ligand of ErbB3 and ErbB4 receptors, contributes to breast cancer development and the promotion of metastatic disease, and its expression in breast tumors has been associated with poor clinical outcome and resistance to therapy. In this study, we found that breast cancer cells exposed to sustained HRG treatment show markedly enhanced Rac1 activation and migratory activity in response to the CXCR4 ligand SDF-1/CXCL12, effects mediated by P-Rex1, a Rac-guanine nucleotide exchange factor (GEF) aberrantly expressed in breast cancer. Notably, HRG treatment upregulates surface expression levels of CXCR4, a G protein-coupled receptor (GPCR) implicated in breast cancer metastasis and an indicator of poor prognosis in breast cancer patients. A detailed mechanistic analysis revealed that CXCR4 upregulation and sensitization of the Rac response/motility by HRG are mediated by the transcription factor hypoxia-inducible factor 1α (HIF-1α) via ErbB3 and independently of ErbB4. HRG caused prominent induction in the nuclear expression of HIF-1α, which transcriptionally activates the CXCR4 gene via binding to a responsive element located in positions −1376 to −1372 in the CXCR4 promoter, as revealed by mutagenesis analysis and chromatin immunoprecipitation (ChIP). Our results uncovered a novel function for ErbB3 in enhancing breast cancer cell motility and sensitization of the P-Rex1/Rac1 pathway through HIF-1α-mediated transcriptional induction of CXCR4. PMID:27185877

A subset of high Gleason grade prostate carcinomas contain a large burden of prostate cancer syndecan-1 positive stromal cells.

PubMed

Sharpe, Benjamin; Alghezi, Dhafer A; Cattermole, Claire; Beresford, Mark; Bowen, Rebecca; Mitchard, John; Chalmers, Andrew D

2017-05-01

There is a pressing need to identify prognostic and predictive biomarkers for prostate cancer to aid treatment decisions in both early and advanced disease settings. Syndecan-1, a heparan sulfate proteoglycan, has been previously identified as a potential prognostic biomarker by multiple studies at the tissue and serum level. However, other studies have questioned its utility. Anti-Syndecan-1 immunohistochemistry was carried out on 157 prostate tissue samples (including cancerous, adjacent normal tissue, and non-diseased prostate) from three independent cohorts of patients. A population of Syndecan-1 positive stromal cells was identified and the number and morphological parameters of these cells quantified. The identity of the Syndecan-1-positive stromal cells was assessed by multiplex immunofluorescence using a range of common cell lineage markers. Finally, the burden of Syndecan-1 positive stromal cells was tested for association with clinical parameters. We identified a previously unreported cell type which is marked by Syndecan-1 expression and is found in the stroma of prostate tumors and adjacent normal tissue but not in non-diseased prostate. We call these cells Prostate Cancer Syndecan-1 Positive (PCSP) cells. Immunofluorescence analysis revealed that the PCSP cell population did not co-stain with markers of common prostate epithelial, stromal, or immune cell populations. However, morphological analysis revealed that PCSP cells are often elongated and displayed prominent lamellipodia, suggesting they are an unidentified migratory cell population. Analysis of clinical parameters showed that PCSP cells were found with a frequency of 20-35% of all tumors evaluated, but were not present in non-diseased normal tissue. Interestingly, a subset of primary Gleason 5 prostate tumors had a high burden of PCSP cells. The current study identifies PCSP cells as a novel, potentially migratory cell type, which is marked by Syndecan-1 expression and is found in the stroma

Monocarboxylate transporter 1 contributes to growth factor-induced tumor cell migration independent of transporter activity

PubMed Central

Gray, Alana L.; Coleman, David T.; Shi, Runhua; Cardelli, James A.

2016-01-01

Tumor progression to metastatic disease contributes to the vast majority of incurable cancer. Understanding the processes leading to advanced stage cancer is important for the development of future therapeutic strategies. Here, we establish a connection between tumor cell migration, a prerequisite to metastasis, and monocarboxylate transporter 1 (MCT1). MCT1 transporter activity is known to regulate aspects of tumor progression and, as such, is a clinically relevant target for treating cancer. Knockdown of MCT1 expression caused decreased hepatocyte growth factor (HGF)-induced as well as epidermal growth factor (EGF)-induced tumor cell scattering and wound healing. Western blot analysis suggested that MCT1 knockdown (KD) hinders signaling through the HGF receptor (c-Met) but not the EGF receptor. Exogenous, membrane-permeable MCT1 substrates were not able to rescue motility in MCT1 KD cells, nor was pharmacologic inhibition of MCT1 able to recapitulate decreased cell motility as seen with MCT1 KD cells, indicating transporter activity of MCT1 was dispensable for EGF- and HGF-induced motility. These results indicate MCT1 expression, independent of transporter activity, is required for growth factor-induced tumor cell motility. The findings presented herein suggest a novel function for MCT1 in tumor progression independent of its role as a monocarboxylate transporter. PMID:27127175

Membrane-To-Nucleus Signaling Links Insulin-Like Growth Factor-1- and Stem Cell Factor-Activated Pathways

PubMed Central

Hayashi, Yujiro; Asuzu, David T.; Gibbons, Simon J.; Aarsvold, Kirsten H.; Bardsley, Michael R.; Lomberk, Gwen A.; Mathison, Angela J.; Kendrick, Michael L.; Shen, K. Robert; Taguchi, Takahiro; Gupta, Anu; Rubin, Brian P.; Fletcher, Jonathan A.; Farrugia, Gianrico; Urrutia, Raul A.; Ordog, Tamas

2013-01-01

Stem cell factor (mouse: Kitl, human: KITLG) and insulin-like growth factor-1 (IGF1), acting via KIT and IGF1 receptor (IGF1R), respectively, are critical for the development and integrity of several tissues. Autocrine/paracrine KITLG-KIT and IGF1-IGF1R signaling are also activated in several cancers including gastrointestinal stromal tumors (GIST), the most common sarcoma. In murine gastric muscles, IGF1 promotes Kitl-dependent development of interstitial cells of Cajal (ICC), the non-neoplastic counterpart of GIST, suggesting cooperation between these pathways. Here, we report a novel mechanism linking IGF1-IGF1R and KITLG-KIT signaling in both normal and neoplastic cells. In murine gastric muscles, the microenvironment for ICC and GIST, human hepatic stellate cells (LX-2), a model for cancer niches, and GIST cells, IGF1 stimulated Kitl/KITLG protein and mRNA expression and promoter activity by activating several signaling pathways including AKT-mediated glycogen synthase kinase-3β inhibition (GSK3i). GSK3i alone also stimulated Kitl/KITLG expression without activating mitogenic pathways. Both IGF1 and GSK3i induced chromatin-level changes favoring transcriptional activation at the Kitl promoter including increased histone H3/H4 acetylation and H3 lysine (K) 4 methylation, reduced H3K9 and H3K27 methylation and reduced occupancy by the H3K27 methyltransferase EZH2. By pharmacological or RNA interference-mediated inhibition of chromatin modifiers we demonstrated that these changes have the predicted impact on KITLG expression. KITLG knock-down and immunoneutralization inhibited the proliferation of GIST cells expressing wild-type KIT, signifying oncogenic autocrine/paracrine KITLG-KIT signaling. We conclude that membrane-to-nucleus signaling involving GSK3i establishes a previously unrecognized link between the IGF1-IGF1R and KITLG-KIT pathways, which is active in both physiologic and oncogenic contexts and can be exploited for therapeutic purposes. PMID:24116170

Higher B-cell activating factor levels at birth are positively associated with maternal dairy farm exposure and negatively related to allergy development.

PubMed

Lundell, Anna-Carin; Hesselmar, Bill; Nordström, Inger; Adlerberth, Ingegerd; Wold, Agnes E; Rudin, Anna

2015-10-01

A high proportion of circulating immature/naive CD5(+) B cells during early infancy is a risk factor for allergy development. B-cell activating factor (BAFF) is an important cytokine for B-cell maturation. We sought to investigate whether BAFF levels are related to environmental exposures during pregnancy and early childhood and whether BAFF levels are associated with postnatal B-cell maturation and allergic disease. In the FARMFLORA study, including both farming and nonfarming families, we measured BAFF levels in plasma from mothers and their children at birth and at 1, 4, 18, and 36 months of age. Infants' blood samples were also analyzed for B-cell numbers and proportions of CD5(+) and CD27(+) B cells. Allergic disease was clinically evaluated at 18 and 36 months of age. Circulating BAFF levels were maximal at birth, and farmers' children had higher BAFF levels than nonfarmers' children. Higher BAFF levels at birth were positively associated with proportions of CD27(+) memory B cells among farmers' children and inversely related to proportions of CD5(+) immature/naive B cells among nonfarmers' children. Children with allergic disease at 18 months of age had lower cord blood BAFF levels than nonallergic children. At birth, girls had higher BAFF levels and lower proportions of CD5(+) B cells than boys. Farm exposure during pregnancy appears to induce BAFF production in the newborn child, and high neonatal BAFF levels were associated with more accelerated postnatal B-cell maturation, which lend further strength to the role of B cells in the hygiene hypothesis. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

The Role of Activator Protein-1 (AP-1) Family Members in CD30-Positive Lymphomas

PubMed Central

Garces de los Fayos Alonso, Ines; Lagger, Sabine; Merkel, Olaf; Kenner, Lukas

2018-01-01

The Activator Protein-1 (AP-1) transcription factor (TF) family, composed of a variety of members including c-JUN, c-FOS and ATF, is involved in mediating many biological processes such as proliferation, differentiation and cell death. Since their discovery, the role of AP-1 TFs in cancer development has been extensively analysed. Multiple in vitro and in vivo studies have highlighted the complexity of these TFs, mainly due to their cell-type specific homo- or hetero-dimerization resulting in diverse transcriptional response profiles. However, as a result of the increasing knowledge of the role of AP-1 TFs in disease, these TFs are being recognized as promising therapeutic targets for various malignancies. In this review, we focus on the impact of deregulated expression of AP-1 TFs in CD30-positive lymphomas including Classical Hodgkin Lymphoma and Anaplastic Large Cell Lymphoma. PMID:29597249

SUMO-modified insulin-like growth factor 1 receptor (IGF-1R) increases cell cycle progression and cell proliferation.

PubMed

Lin, Yingbo; Liu, Hongyu; Waraky, Ahmed; Haglund, Felix; Agarwal, Prasoon; Jernberg-Wiklund, Helena; Warsito, Dudi; Larsson, Olle

2017-10-01

Increasing number of studies have shown nuclear localization of the insulin-like growth factor 1 receptor (nIGF-1R) in tumor cells and its links to adverse clinical outcome in various cancers. Any obvious cell physiological roles of nIGF-1R have, however, still not been disclosed. Previously, we reported that IGF-1R translocates to cell nucleus and modulates gene expression by binding to enhancers, provided that the receptor is SUMOylated. In this study, we constructed stable transfectants of wild type IGF1R (WT) and triple-SUMO-site-mutated IGF1R (TSM) using igf1r knockout mouse fibroblasts (R-). Cell clones (R-WT and R-TSM) expressing equal amounts of IGF-1R were selected for experiments. Phosphorylation of IGF-1R, Akt, and Erk upon IGF-1 stimulation was equal in R-WT and R-TSM. WT was confirmed to enter nuclei. TSM did also undergo nuclear translocation, although to a lesser extent. This may be explained by that TSM heterodimerizes with insulin receptor, which is known to translocate to cell nuclei. R-WT proliferated substantially faster than R-TSM, which did not differ significantly from the empty vector control. Upon IGF-1 stimulation G1-S-phase progression of R-WT increased from 12 to 38%, compared to 13 to 20% of R-TSM. The G1-S progression of R-WT correlated with increased expression of cyclin D1, A, and CDK2, as well as downregulation of p27. This suggests that SUMO-IGF-1R affects upstream mechanisms that control and coordinate expression of cell cycle regulators. Further studies to identify such SUMO-IGF-1R dependent mechanisms seem important. © 2017 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc.

The Correlation Between PARP1 and BRCA1 in AR Positive Triple-negative Breast Cancer.

PubMed

Luo, Jiayan; Jin, Juan; Yang, Fang; Sun, Zijia; Zhang, Wenwen; Shi, Yaqin; Xu, Jing; Guan, Xiaoxiang

2016-01-01

Triple-negative breast cancer (TNBC) lacks estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2) expression and thus cannot benefit from conventional hormonal or anti-HER2 targeted therapies. Anti-androgen therapy has shown a certain effect on androgen receptor (AR) positive TNBC. The emerging researches have proved that poly (ADP-ribose) polymerase (PARP) inhibitor is effective in BRCA1-deficient breast cancers. We demonstrated that combination of AR antagonist (bicalutamide) and PARP inhibitor (ABT-888) could inhibit cell viability and induce cell apoptosis significantly whatever in vitro or in vivo setting in AR-positive TNBC. Previous studies have proved that both BRCA1 and PARP1 have close connections with AR in prostate cancer. We explored the correlation among AR, PARP1 and BRCA1 in TNBC for the first time. After BRCA1 overexpression, the expression of AR and PARP1 were decreased in mRNA and protein levels. Additionally, AR positively regulated PARP1 while PARP1 also up-regulated AR expression in vitro. We also confirmed BRCA1 expression was negatively correlated with AR and PARP1 in TNBC patients using a tissue microarray with TNBC patient samples. These results suggest that the combination of bicalutamide and PARP inhibitor may be a potential strategy for TNBC patients and merits further evaluation.

ATP1B3 Protein Modulates the Restriction of HIV-1 Production and Nuclear Factor κ Light Chain Enhancer of Activated B Cells (NF-κB) Activation by BST-2*

PubMed Central

Nishitsuji, Hironori; Sugiyama, Ryuichi; Abe, Makoto; Takaku, Hiroshi

2016-01-01

Here, we identify ATP1B3 and fibrillin-1 as novel BST-2-binding proteins. ATP1B3 depletion in HeLa cells (BST-2-positive cells), but not 293T cells (BST-2-negative cells), induced the restriction of HIV-1 production in a BST-2-dependent manner. In contrast, fibrillin-1 knockdown reduced HIV-1 production in 293T and HeLa cells in a BST-2-independent manner. Moreover, NF-κB activation was enhanced by siATP1B3 treatment in HIV-1- and HIV-1ΔVpu-infected HeLa cells. In addition, ATP1B3 silencing induced high level BST-2 expression on the surface of HeLa cells. These results indicate that ATP1B3 is a co-factor that accelerates BST-2 degradation and reduces BST-2-mediated restriction of HIV-1 production and NF-κB activation. PMID:26694617

Role of monocyte-lineage cells in prostate cancer cell invasion and tissue factor expression.

PubMed

Lindholm, Paul F; Lu, Yi; Adley, Brian P; Vladislav, Tudor; Jovanovic, Borko; Sivapurapu, Neela; Yang, Ximing J; Kajdacsy-Balla, André

2010-11-01

Tissue factor (TF) is a cell surface glycoprotein intricately related to blood coagulation and inflammation. This study was performed to investigate the role of monocyte-lineage cells in prostate cancer cell TF expression and cell invasion. Prostate cancer cell invasion was tested with and without added peripheral blood monocytes or human monocyte-lineage cell lines. TF neutralizing antibodies were used to determine the TF requirement for prostate cancer cell invasion activity. Immunohistochemistry was performed to identify prostate tissue CD68 positive monocyte-derived cells and prostate epithelial TF expression. Co-culture of PC-3, DU145, and LNCaP cells with isolated human monocytes significantly stimulated prostate cancer cell invasion activity. TF expression was greater in highly invasive prostate cancer cells and was induced in PC-3, DU145, and LNCaP cells by co-culture with U-937 cells, but not with THP-1 cells. TF neutralizing antibodies inhibited PC-3 cell invasion in co-cultures with monocyte-lineage U-937 or THP-1 cells. Prostate cancer tissues contained more CD68 positive cells in the stroma and epithelium (145 ± 53/mm(2)) than benign prostate (108 ± 31/mm(2)). Samples from advanced stage prostate cancer tended to contain more CD68 positive cells when compared with lower stage lesions. Prostatic adenocarcinoma demonstrated significantly increased TF expression compared with benign prostatic epithelium. This study shows that co-culture with monocyte-lineage cells induced prostate cancer cell invasion activity. PC-3 invasion and TF expression was induced in co-culture with U-937 cells and partially inhibited with TF neutralizing antibodies.

FOXN1 Transcription Factor in Epithelial Growth and Wound Healing.

PubMed

Grabowska, Anna I; Wilanowski, Tomasz

2017-09-01

FOXN1 is a prodifferentiation transcription factor in the skin epithelium. Recently, it has also emerged as an important player in controlling the skin wound healing process, as it actively participates in reepithelialization and is thought to be responsible for scar formation. FOXN1 positivity is also a feature of pigmented keratinocytes, including nevi, and FOXN1 is an attribute of benign epithelial tumors. The lack of FOXN1 favors the skin regeneration process displayed by nude mice, pointing to FOXN1 as a switch between regeneration and reparative processes. The stem cell niche provides a functional source of cells after the loss of tissue following wounding. The involvement of prodifferentiation factors in the regulation of this pool of stem cells is suggested. However, the exact mechanism is still under question, and we speculate that the FOXN1 transcription factor is involved in this process. This review analyzes the pleiotropic effects of FOXN1 in the skin, its function in the tumorigenesis process, and its potential role in depletion of the stem cell niche after injury, as well as its suggested mechanistic role, acting in a cell-autonomous and a non-cell-autonomous manner during skin self-renewal. Copyright © 2017 American Society for Microbiology.

Influence of periostin-positive cell-specific Klf5 deletion on aortic thickening in DOCA-salt hypertensive mice.

PubMed

Zempo, Hirofumi; Suzuki, Jun-Ichi; Ogawa, Masahito; Watanabe, Ryo; Fujiu, Katsuhito; Manabe, Ichiro; Conway, Simon J; Taniyama, Yoshiaki; Morishita, Ryuichi; Hirata, Yasunobu; Isobe, Mitsuaki; Nagai, Ryozo

2016-11-01

Chronic hypertension causes vascular remodeling that is associated with an increase in periostin- (postn) positive cells, including fibroblasts and smooth muscle cells. Krüppel-like factor (KLF) 5, a transcription factor, is also observed in vascular remodeling; however, it is unknown what role KLF5 plays in postn-positive cells during vascular remodeling induced by deoxycorticosterone-acetate (DOCA) salt. We used postn-positive cell-specific Klf5-deficient mice (Klf5 Postn KO: Klf5 flox/flox ; Postn Cre/- ) and wild-type mice (WT: Klf5 flox/flox ; Postn -/- ). We implanted a DOCA pellet and provided drinking water containing 0.9% NaCl for 8 weeks. The DOCA-salt treatment induced hypertension in both genotypes, as observed by increases in systolic blood pressure. In WT animals, DOCA-salt treatment increased the aortic medial area compared with the non-treated controls. Similarly, Tgfb1 was overexpressed in the aortas of the DOCA-salt treated WT mice compared with the controls. Immunofluorescence staining revealed that fibroblast-specific protein 1 (FSP1) + -α smooth muscle actin (αSMA) + myofibroblasts exist in the medial area of the WT aortas after DOCA-salt intervention. Importantly, these changes were not observed in the Klf5 Postn KO animals. In conclusion, the results of this study suggest that the presence of KLF5 in postn-positive cells contributes to the pathogenesis of aortic thickening induced by DOCA-salt hypertension.

Myeloid Leukemia Factor 1 inhibits erythropoietin-induced differentiation, cell cycle exit and p27Kip1 accumulation.

PubMed

Winteringham, Louise Natalie; Kobelke, Simon; Williams, James Howard; Ingley, Evan; Klinken, Svend Peter

2004-06-24

Myeloid leukemia factor 1 (MLF1) is a novel oncoprotein involved in translocations associated with acute myeloid leukemia (AML), especially erythroleukemias. In this study, we demonstrate that ectopic expression of Mlf1 prevented J2E erythroleukemic cells from undergoing biological and morphological maturation in response to erythropoietin (Epo). We show that Mlf1 inhibited Epo-induced cell cycle exit and suppressed a rise in the cell cycle inhibitor p27(Kip1). Unlike differentiating J2E cells, Mlf1-expressing cells did not downregulate Cul1 and Skp2, components of the ubiquitin E3 ligase complex SCF(Skp2) involved in the proteasomal degradation of p27(Kip1). In contrast, Mlf1 did not interfere with increases in p27(Kip1) and terminal differentiation initiated by thyroid hormone withdrawal from erythroid cells, or cytokine-stimulated maturation of myeloid cells. These data demonstrate that Mlf1 interferes with an Epo-responsive pathway involving p27(Kip1) accumulation, which inhibits cell cycle arrest essential for erythroid terminal differentiation.

Genistein promotes DNA demethylation of the steroidogenic factor 1 (SF-1) promoter in endometrial stromal cells

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

Matsukura, Hiroshi, E-mail: hmatsukura.epi@mri.tmd.ac.jp; Aisaki, Ken-ichi; Igarashi, Katsuhide

2011-08-26

Highlights: {yields} Genistein (GEN) is a phytoestrogen found in soy products. {yields} GEN demethylated/unsilenced the steroidogenic factor 1 gene in endometrial tissue. {yields} GEN thus altered mRNA expression in uteri of ovariectomized (OVX) mice. {yields} A high-resolution melting assay was used to screen for epigenetic change. {yields} We isolated an endometrial cell clone that was epigenetically modulated by GEN. -- Abstract: It has recently been demonstrated that genistein (GEN), a phytoestrogen in soy products, is an epigenetic modulator in various types of cells; but its effect on endometrium has not yet been determined. We investigated the effects of GEN onmore » mouse uterine cells, in vivo and in vitro. Oral administration of GEN for 1 week induced mild proliferation of the endometrium in ovariectomized (OVX) mice, which was accompanied by the induction of steroidogenic factor 1 (SF-1) gene expression. GEN administration induced demethylation of multiple CpG sites in the SF-1 promoter; these sites are extensively methylated and thus silenced in normal endometrium. The GEN-mediated promoter demethylation occurred predominantly on the luminal side, as opposed to myometrium side, indicating that the epigenetic change was mainly shown in regenerated cells. Primary cultures of endometrial stromal cell colonies were screened for GEN-mediated alterations of DNA methylation by a high-resolution melting (HRM) method. One out of 20 colony-forming cell clones showed GEN-induced demethylation of SF-1. This clone exhibited a high proliferation capacity with continuous colony formation activity through multiple serial clonings. We propose that only a portion of endometrial cells are capable of receiving epigenetic modulation by GEN.« less

Reelin is involved in transforming growth factor-β1-induced cell migration in esophageal carcinoma cells.

PubMed

Yuan, Yi; Chen, Hongyan; Ma, Gang; Cao, Xiaofeng; Liu, Zhihua

2012-01-01

Reelin (RELN), which is a glycoprotein secreted by Cajal-Retzius cells of the developing cerebral cortex, plays an important role in neuronal migration, but its role in cell migration and cancer metastasis is largely unclear. Here, we showed that cell motility was significantly increased in KYSE-510 cells by TGF-β1 treatment. Moreover, TGF-β1 decreased RELN mRNA expression and overexpression of Reelin at least partly reversed TGF-β1-induced cell migration in KYSE-30 cells. Furthermore, this negative regulation of Reelin expression by TGF-β1 was through Snail, one transcription factor which was induced by TGF-β1 in KYSE-510 cells. RELN promoter activity was reduced in parallel with the induction of Snail after TGF-β1 treatment and Snail suppressed both RELN promoter activity and expression through binding to E-box sequences in the RELN promoter region in ESCC cells. Knockdown of RELN induced cell migration in KYSE-510 cells, together with the increase of mesenchymal markers expression. Taken together, Reelin is an essential negative regulator in the TGF-β1-induced cell migration process, and is suppressed by TGF-β pathway at the transcriptional level through Snail regulation. Therefore, the correlation of Reelin and TGF-β pathway was critical in cancer metastasis, and Reelin could be one potential anti-metastasis target in future clinical practice.

RB1 is the crucial target of the Merkel cell polyomavirus Large T antigen in Merkel cell carcinoma cells.

PubMed

Hesbacher, Sonja; Pfitzer, Lisa; Wiedorfer, Katharina; Angermeyer, Sabrina; Borst, Andreas; Haferkamp, Sebastian; Scholz, Claus-Jürgen; Wobser, Marion; Schrama, David; Houben, Roland

2016-05-31

The pocket protein (PP) family consists of the three members RB1, p107 and p130 all possessing tumor suppressive properties. Indeed, the PPs jointly control the G1/S transition mainly by inhibiting E2F transcription factors. Notably, several viral oncoproteins are capable of binding and inhibiting PPs. Merkel cell polyomavirus (MCPyV) is considered as etiological factor for Merkel cell carcinoma (MCC) with expression of the viral Large T antigen (LT) harboring an intact PP binding domain being required for proliferation of most MCC cells. Therefore, we analyzed the interaction of MCPyV-LT with the PPs. Co-IP experiments indicate that MCPyV-LT binds potently only to RB1. Moreover, MCPyV-LT knockdown-induced growth arrest in MCC cells can be rescued by knockdown of RB1, but not by p107 or p130 knockdown. Accordingly, cell cycle arrest and E2F target gene repression mediated by the single PPs can only in the case of RB1 be significantly reverted by MCPyV-LT expression. Moreover, data from an MCC patient indicate that loss of RB1 rendered the MCPyV-positive MCC cells LT independent. Thus, our results suggest that RB1 is the dominant tumor suppressor PP in MCC, and that inactivation of RB1 by MCPyV-LT is largely sufficient for its growth supporting function in established MCPyV-positive MCC cells.

Expression of insulin-like growth factor-1 and insulin-like growth factor-1 receptors in EL4 lymphoma cells overexpressing growth hormone.

PubMed

Weigent, Douglas A; Arnold, Robyn E

2005-03-01

Almost all of the previous studies with growth hormone (GH) have been done with exogenously supplied GH and, therefore, involve actions of the hormone through its receptor. However, the actions of endogenous or lymphocyte GH are still unclear. In a previous study, we showed that overexpression of GH (GHo) in a lymphoid cell line resulted in protection of the cells to apoptosis mediated by nitric oxide (NO). In the present study, we show that the protection from apoptosis could be transferred to control cells with culture fluids obtained from GHo cells and blocked by antibodies to the insulin-like growth factor-1 (IGF-1) or antibodies to the IGF-1-receptor (IGF-1R). Northern and Western blot analysis detected significantly higher levels of IGF-1 in cells overexpressing GH. An increase in the expression of the IGF-1R in GHo cells was also detected by Western blot analysis, (125)I-IGF-1 binding and analysis of IGF-1R promoter luciferase constructs. Transfection of GHo cells with a dominant negative IGF-1R mutant construct blocked the generation of NO and activation of Akt seen in GHo cells compared to vector alone control EL4 cells. The results suggest that one of the consequences of the overexpression of GH, in cells lacking the GH receptor, is an increase in the expression of IGF-1 and the IGF-1R which mediate the protection of EL4 lymphoma cells from apoptosis.

DMRT1 Is Required for Mouse Spermatogonial Stem Cell Maintenance and Replenishment.

PubMed

Zhang, Teng; Oatley, Jon; Bardwell, Vivian J; Zarkower, David

2016-09-01

Male mammals produce sperm for most of postnatal life and therefore require a robust germ line stem cell system, with precise balance between self-renewal and differentiation. Prior work established doublesex- and mab-3-related transcription factor 1 (Dmrt1) as a conserved transcriptional regulator of male sexual differentiation. Here we investigate the role of Dmrt1 in mouse spermatogonial stem cell (SSC) homeostasis. We find that Dmrt1 maintains SSCs during steady state spermatogenesis, where it regulates expression of Plzf, another transcription factor required for SSC maintenance. We also find that Dmrt1 is required for recovery of spermatogenesis after germ cell depletion. Committed progenitor cells expressing Ngn3 normally do not contribute to SSCs marked by the Id4-Gfp transgene, but do so when spermatogonia are chemically depleted using busulfan. Removal of Dmrt1 from Ngn3-positive germ cells blocks the replenishment of Id4-GFP-positive SSCs and recovery of spermatogenesis after busulfan treatment. Our data therefore reveal that Dmrt1 supports SSC maintenance in two ways: allowing SSCs to remain in the stem cell pool under normal conditions; and enabling progenitor cells to help restore the stem cell pool after germ cell depletion.

17beta-estradiol promotes breast cancer cell proliferation-inducing stromal cell-derived factor-1-mediated epidermal growth factor receptor transactivation: reversal by gefitinib pretreatment.

PubMed

Pattarozzi, Alessandra; Gatti, Monica; Barbieri, Federica; Würth, Roberto; Porcile, Carola; Lunardi, Gianluigi; Ratto, Alessandra; Favoni, Roberto; Bajetto, Adriana; Ferrari, Angelo; Florio, Tullio

2008-01-01

The coordinated activity of estrogens and epidermal growth factor receptor (EGFR) family agonists represents the main determinant of breast cancer cell proliferation. Stromal cell-derived factor-1 (SDF-1) enhances extracellular signal-regulated kinases 1 and 2 (ERK1/2) activity via the transactivation of EGFR and 17beta-estradiol (E2) induces SDF-1 production to exert autocrine proliferative effects. On this basis, we evaluated whether the inhibition of the tyrosine kinase (TK) activity of EGFR may control different mitogenic stimuli in breast tumors using the EGFR-TK inhibitor gefitinib to antagonize the proliferation induced by E2 in T47D human breast cancer cells. EGF, E2, and SDF-1 induced a dose-dependent T47D cell proliferation, that being nonadditive suggested the activation of common intracellular pathways. Gefitinib treatment inhibited not only the EGF-dependent proliferation and ERK1/2 activation but also the effects of SDF-1 and E2, suggesting that these activities were mediated by EGFR transactivation. Indeed, both SDF-1 and E2 caused EGFR tyrosine phosphorylation. The molecular link between E2 and SDF-1 proliferative effects was identified because 1,1'-(1,4-phenylenebis(methylene))-bis-1,4,8,11-tetraazacyclotetradecane octahydrochloride (AMD3100), a CXCR4 antagonist, inhibited SDF-1- and E2-dependent proliferation and EGFR and ERK1/2 phosphorylation. EGFR transactivation was dependent on c-Src activation. E2 treatment caused a powerful SDF-1 release from T47D cells. Finally, in SKBR3, E2-resistant cells, EGFR was constitutively activated, and AMD3100 reduced EGFR phosphorylation and cell proliferation, whereas HER2-neu was transactivated by SDF-1 in SKBR3 but not in T47D cells. In conclusion, we show that activation of CXCR4 transduces proliferative signals from the E2 receptor to EGFR, whose inhibition is able to revert breast cancer cell proliferation induced by multiple receptor activation.

Apoptosis Signal-Regulating Kinase 1 Is Involved in Brain-Derived Neurotrophic Factor (BDNF)-Enhanced Cell Motility and Matrix Metalloproteinase 1 Expression in Human Chondrosarcoma Cells

PubMed Central

Lin, Chih-Yang; Chang, Sunny Li-Yun; Fong, Yi-Chin; Hsu, Chin-Jung; Tang, Chih-Hsin

2013-01-01

Chondrosarcoma is the primary malignancy of bone that is characterized by a potent capacity to invade locally and cause distant metastasis, and is therefore associated with poor prognoses. Chondrosarcoma further shows a predilection for metastasis to the lungs. The brain-derived neurotrophic factor (BDNF) is a small molecule in the neurotrophin family of growth factors that is associated with the disease status and outcome of cancers. However, the effect of BDNF on cell motility in human chondrosarcoma cells is mostly unknown. Here, we found that human chondrosarcoma cell lines had significantly higher cell motility and BDNF expression compared to normal chondrocytes. We also found that BDNF increased cell motility and expression of matrix metalloproteinase-1 (MMP-1) in human chondrosarcoma cells. BDNF-mediated cell motility and MMP-1 up-regulation were attenuated by Trk inhibitor (K252a), ASK1 inhibitor (thioredoxin), JNK inhibitor (SP600125), and p38 inhibitor (SB203580). Furthermore, BDNF also promoted Sp1 activation. Our results indicate that BDNF enhances the migration and invasion activity of chondrosarcoma cells by increasing MMP-1 expression through a signal transduction pathway that involves the TrkB receptor, ASK1, JNK/p38, and Sp1. BDNF thus represents a promising new target for treating chondrosarcoma metastasis. PMID:23892595

Evaluation of aldehyde dehydrogenase 1 and transcription factors in both primary breast cancer and axillary lymph node metastases as a prognostic factor.

PubMed

Ito, Maiko; Shien, Tadahiko; Omori, Masako; Mizoo, Taeko; Iwamoto, Takayuki; Nogami, Tomohiro; Motoki, Takayuki; Taira, Naruto; Doihara, Hiroyoshi; Miyoshi, Shinichiro

2016-05-01

Aldehyde dehydrogenase 1 (ALDH1) is a marker of breast cancer stem cells, and the expression of ALDH1 may be a prognostic factor of poor clinical outcome. The epithelial-mesenchymal transition may produce cells with stem-cell-like properties promoted by transcription factors. We investigated the expression of ALDH1 and transcription factors in both primary and metastatic lesions, and prognostic value of them in breast cancer patients with axillary lymph node metastasis (ALNM). Forty-seven breast cancer patients with ALNM who underwent surgery at Okayama University Hospital from 2002 to 2008 were enrolled. We retrospectively evaluated the levels of ALDH1 and transcription factors, such as Snail, Slug and Twist, in both primary and metastatic lesions by immunohistochemistry. In primary lesions, the positive rate of ALDH1, Snail, Slug and Twist was 19, 49, 40 and 26%, respectively. In lymph nodes, that of ALDH1, Snail, Slug and Twist was 21, 32, 13 and 23%, respectively. The expression of ALDH1 or transcription factors alone was not significantly associated with a poor prognosis. However, co-expression of ALDH1 and Slug in primary lesions was associated with a shorter DFS (P = 0.009). The evaluation of the co-expression of ALDH1 and transcription factors in primary lesions may be useful in prognosis of node-positive breast cancers.

Doublecortin and CaM kinase-like-1 expression in pathological stage I non-small cell lung cancer.

PubMed

Tao, Hiroyuki; Tanaka, Toshiki; Okabe, Kazunori

2017-08-01

Doublecortin and CaM kinase-like-1 (DCLK1) regulates microtubule polymerization in migrating neurons. Recently, DCLK1 has been reported to act as an intestinal tumor stem cell marker and has been shown to be expressed in cancer cells and in the stroma of breast, colon, pancreatic, and prostate cancers. Here, we studied DCLK1 expression in non-small cell lung cancer (NSCLC) by immunohistochemistry in association with clinicopathological factors and patient prognosis. DCLK1 expression was analyzed by immunohistochemical staining of surgical specimens from 232 patients with pathological stage I NSCLC, including 187 adenocarcinomas. Relationships between the expression status of DCLK1 and clinicopathological factors were examined. The impact of DCLK1 expression status and other clinicopathological factors on survival was evaluated by univariate and multivariate analyses. Thirty-three (14.2%) of 232 patients had DCLK1-positive cancer cells. DCLK1 was also expressed in the tumor stroma in most of the specimens and was significantly associated with DCLK1 expression in cancer cells. DCLK1-positive cancer cells were more common in non-adenocarcinoma tissues (44.4%) than in adenocarcinoma tissues (7.0%). Moreover, positive DCLK1 expression in cancer cells and stromal cells was correlated with a worse prognosis. Histological analyses revealed that the presence of DCLK1-positive cancer cells was an independent risk factor for poor prognosis in adenocarcinoma, but was not significantly associated with prognosis in non-adenocarcinoma. DCLK1 expression was observed in tumor cells in patients with pathological stage I NSCLC and was correlated with adverse prognosis, especially in patients with adenocarcinoma. DCLK1 may be a potential new therapeutic target.

In vivo dynamics of GFRα1-positive spermatogonia stimulated by GDNF signals using a bead transplantation assay

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

Uchida, Aya; Kishi, Kasane; Aiyama, Yoshimi

In mouse testes, spermatogonial stem cells (SSCs), a subpopulation of GFRα1 (GDNF family receptor-α1)-positive spermatogonia, are widely distributed along the convoluted seminiferous tubules. The proliferation and differentiation of the SSCs are regulated in part by local expression of GDNF (glial cell-derived neurotorphic factor), one of major niche factors for SSCs. However, the in vivo dynamics of the GDNF-stimulated GFRα1-positive spermatogonia remains unclear. Here, we developed a simple method for transplanting DiI-labeled and GDNF-soaked beads into the mouse testicular interstitium. By using this method, we examined the dynamics of GFRα1-positive spermatogonia in the tubular walls close to the transplanted GDNF-soaked beads. Themore » bead-derived GDNF signals were able to induce the stratified aggregate formation of GFRα1-positive undifferentiated spermatogonia by day 3 post-transplantation. Each aggregate consisted of tightly compacted A{sub single} and marginal A{sub paired}–A{sub aligned} GFRα1-positive spermatogonia and was surrounded by A{sub aligned} GFRα1-negative spermatogonia at more advanced stages. These data not only provide in vivo evidence for the inductive roles of GDNF in forming a rapid aggregation of GFRα1-positive spermatogonia but also indicate the usefulness of this in vivo assay system of various growth factors for the stem/progenitor spermatogonia in mammalian spermatogenesis. - Highlights: • A novel bead transplantation assay was developed to examine the in vivo effects of growth factors on spermatogonia. • A rapid aggregation of GFRα1-positive spermatogonia was induced by the transplanted GDNF-soaked beads. • Tightly-compacted A{sub single} and marginal A{sub paired}–A{sub aligned} spermatogonia were formed in each GFRα1-positive aggregate.« less

Glial cell line-derived neurotrophic factor promotes the development of adrenergic neurons in mouse neural crest cultures

PubMed Central

Maxwell, Gerald D.; Reid, Kate; Elefanty, Andrew; Bartlett, Perry F.; Murphy, Mark

1996-01-01

Growth of mouse neural crest cultures in the presence of glial cell line-derived neurotrophic factor (GDNF) resulted in a dramatic dose-dependent increase in the number of tyrosine hydroxylase (TH)-positive cells that developed when 5% chicken embryo extract was present in the medium. In contrast, growth in the presence of bone morphogenetic protein (BMP)-2, BMP-4, BMP-6, transforming growth factor (TGF) β1, TGF-β2, and TGF-β3 elicited no increase in the number of TH-positive cells. The TH-positive cells that developed in the presence of GDNF had neuronal morphology and contained the middle and low molecular weight neurofilament proteins. Numerous TH-negative cells with the morphology of neurons also were observed in GDNF-treated cultures. Analysis revealed that the period from 6 to 12 days in vitro was the critical time for exposure to GDNF to generate the increase in TH-positive cell number. The growth factors neurotrophin-3 and fibroblast growth factor-2 elicited increases in the number of TH-positive cells similar to that seen in response to GDNF. In contrast, nerve growth factor was unable to substitute for GDNF. These findings extend the previously reported biological activities of GDNF by showing that it can act on mouse neural crest cultures to promote the development of neurons. PMID:8917581

Generation of H1 PAX6WT/EGFP reporter cells to purify PAX6 positive neural stem/progenitor cells.

PubMed

Wu, Wei; Liu, Juli; Su, Zhenghui; Li, Zhonghao; Ma, Ning; Huang, Ke; Zhou, Tiancheng; Wang, Linli

2018-08-25

Neural conversion from human pluripotent cells (hPSCs) is a potential therapy to neurological disease in the future. However, this is still limited by efficiency and stability of existed protocols used for neural induction from hPSCs. To overcome this obstacle, we developed a reporter system to screen PAX6 + neural progenitor/stem cells using transcription activator like effector nuclease (TALEN). We found that knock-in 2 A-EGFP cassette into PAX6 exon of human embryonic stem cells H1 with TALEN-based homology recombination could establish PAX6 WT/EGFP H1 reporter cell line fast and efficiently. This reporter cell line could differentiate into PAX6 and EGFP double positive neural progenitor/stem cells (NPCs/NSCs) after neural induction. Those PAX6 WT/EGFP NPCs could be purified, expanded and specified to post-mitotic neurons in vitro efficiently. With this reporter cell line, we also screened out 1 NPC-specific microRNA, hsa-miR-99a-5p, and 3 ESCs-enriched miRNAs, hsa-miR-302c-5p, hsa-miR-512-3p and hsa-miR-518 b. In conclusion, the TALEN-based neural stem cell screening system is safe and efficient and could help researcher to acquire adequate and pure neural progenitor cells for further application. Copyright © 2018 Elsevier Inc. All rights reserved.

Stromal cell-derived factor-1{alpha} (SDF-1{alpha}/CXCL12) stimulates ovarian cancer cell growth through the EGF receptor transactivation

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

Porcile, Carola; Bajetto, Adriana; Barbieri, Federica

2005-08-15

Ovarian cancer (OC) is the leading cause of death in gynecologic diseases in which there is evidence for a complex chemokine network. Chemokines are a family of proteins that play an important role in tumor progression influencing cell proliferation, angiogenic/angiostatic processes, cell migration and metastasis, and, finally, regulating the immune cells recruitment into the tumor mass. We previously demonstrated that astrocytes and glioblastoma cells express both the chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 (SDF-1), and that SDF-1{alpha} treatment induced cell proliferation, supporting the hypothesis that chemokines may play an important role in tumor cells' growth in vitro.more » In the present study, we report that CXCR4 and SDF-1 are expressed in OC cell lines. We demonstrate that SDF-1{alpha} induces a dose-dependent proliferation in OC cells, by the specific interaction with CXCR4 and a biphasic activation of ERK1/2 and Akt kinases. Our results further indicate that CXCR4 activation induces EGF receptor (EGFR) phosphorylation that in turn was linked to the downstream intracellular kinases activation, ERK1/2 and Akt. In addition, we provide evidence for cytoplasmic tyrosine kinase (c-Src) involvement in the SDF-1/CXCR4-EGFR transactivation. These results suggest a possible important 'cross-talk' between SDF-1/CXCR4 and EGFR intracellular pathways that may link signals of cell proliferation in ovarian cancer.« less

Radotinib induces high cytotoxicity in c-KIT positive acute myeloid leukemia cells.

PubMed

Heo, Sook-Kyoung; Noh, Eui-Kyu; Kim, Jeong Yi; Jo, Jae-Cheol; Choi, Yunsuk; Koh, SuJin; Baek, Jin Ho; Min, Young Joo; Kim, Hawk

2017-06-05

Previously, we reported that radotinib, a BCR-ABL1 tyrosine kinase inhibitor, induced cytotoxicity in acute myeloid leukemia (AML) cells. However, the effects of radotinib in the subpopulation of c-KIT-positive AML cells were unclear. We observed that low-concentration radotinib had more potent cytotoxicity in c-KIT-positive cells than c-KIT-negative cells from AML patients. To address this issue, cell lines with high c-KIT expression, HEL92.1.7, and moderate c-KIT expression, H209, were selected. HEL92.1.7 cells were grouped into intermediate and high c-KIT expression populations. The cytotoxicity of radotinib against the HEL92.1.7 cell population with intermediate c-KIT expression was not different from that of the population with high c-KIT expression. When H209 cells were grouped into c-KIT expression-negative and c-KIT expression-positive populations, radotinib induced cytotoxicity in the c-KIT-positive population, but not the c-KIT-negative population. Thus, radotinib induces cytotoxicity in c-KIT-positive cells, regardless of the c-KIT expression intensity. Therefore, radotinib induces significant cytotoxicity in c-KIT-positive AML cells, suggesting that radotinib is a potential target agent for the treatment of c-KIT-positive malignancies including AML. Copyright © 2017 Elsevier B.V. All rights reserved.

GPER1-mediated IGFBP-1 induction modulates IGF-1-dependent signaling in tamoxifen-treated breast cancer cells.

PubMed

Vaziri-Gohar, Ali; Houston, Kevin D

2016-02-15

Tamoxifen, a selective estrogen receptor modulator, is a commonly prescribed adjuvant therapy for estrogen receptor-α (ERα)-positive breast cancer patients. To determine if extracellular factors contribute to the modulation of IGF-1 signaling after tamoxifen treatment, MCF-7 cells were treated with IGF-1 in conditioned medium (CM) obtained from 4-OHT-treated MCF-7 cells and the accumulation of phospho-Akt (S473) was measured. CM inhibited IGF-1-dependent cell signaling and suggesting the involvement of extracellular factors (ie. IGFBPs). A significant increase in IGFBP-1 mRNA and extracellular IGFBP-1 protein was observed in 4-OHT-treated MCF-7 cells. Knockdown experiments demonstrated that both GPER1 and CREB mediate IGFBP-1 induction. Furthermore, experiments showed that 4-OHT-dependent IGFBP-1 transcription is downstream of GPER1-activation in breast cancer cells. Additionally, neutralization and knockdown experiments demonstrated a role for IGFBP-1 in the observed inhibition of IGF-1 signaling. These results suggested that 4-OHT inhibits IGF-1 signaling via GPER1 and CREB mediated extracellular IGFBP-1 accumulation in breast cancer cells. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Overexpression of hypoxia-inducible factor 1 alpha improves immunomodulation by dental mesenchymal stem cells.

PubMed

Martinez, Victor G; Ontoria-Oviedo, Imelda; Ricardo, Carolina P; Harding, Sian E; Sacedon, Rosa; Varas, Alberto; Zapata, Agustin; Sepulveda, Pilar; Vicente, Angeles

2017-09-29

Human dental mesenchymal stem cells (MSCs) are considered as highly accessible and attractive MSCs for use in regenerative medicine, yet some of their features are not as well characterized as other MSCs. Hypoxia-preconditioning and hypoxia-inducible factor 1 (HIF-1) alpha overexpression significantly improves MSC therapeutics, but the mechanisms involved are not fully understood. In the present study, we characterize immunomodulatory properties of dental MSCs and determine changes in their ability to modulate adaptive and innate immune populations after HIF-1 alpha overexpression. Human dental MSCs were stably transduced with green fluorescent protein (GFP-MSCs) or GFP-HIF-1 alpha lentivirus vectors (HIF-MSCs). A hypoxic-like metabolic profile was confirmed by mitochondrial and glycolysis stress test. Capacity of HIF-MSCs to modulate T-cell activation, dendritic cell differentiation, monocyte migration, and polarizations towards macrophages and natural killer (NK) cell lytic activity was assessed by a number of functional assays in co-cultures. The expression of relevant factors were determined by polymerase chain reaction (PCR) analysis and enzyme-linked immunosorbent assay (ELISA). While HIF-1 alpha overexpression did not modify the inhibition of T-cell activation by MSCs, HIF-MSCs impaired dendritic cell differentiation more efficiently. In addition, HIF-MSCs showed a tendency to induce higher attraction of monocytes, which differentiate into suppressor macrophages, and exhibited enhanced resistance to NK cell-mediated lysis, which supports the improved therapeutic capacity of HIF-MSCs. HIF-MSCs also displayed a pro-angiogenic profile characterized by increased expression of CXCL12/SDF1 and CCL5/RANTES and complete loss of CXCL10/IP10 transcription. Immunomodulation and expression of trophic factors by dental MSCs make them perfect candidates for cell therapy. Overexpression of HIF-1 alpha enhances these features and increases their resistance to allogenic NK

Interleukin-1, tumor necrosis factor-alpha, and transforming growth factor-beta 1 and integrative meniscal repair: influences on meniscal cell proliferation and migration

PubMed Central

2011-01-01

Introduction Interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) are up-regulated in injured and osteoarthritic knee joints. IL-1 and TNF-α inhibit integrative meniscal repair; however, the mechanisms by which this inhibition occurs are not fully understood. Transforming growth factor-β1 (TGF-β1) increases meniscal cell proliferation and accumulation, and enhances integrative meniscal repair. An improved understanding of the mechanisms modulating meniscal cell proliferation and migration will help to improve approaches for enhancing intrinsic or tissue-engineered repair of the meniscus. The goal of this study was to examine the hypothesis that IL-1 and TNF-α suppress, while TGF-β1 enhances, cellular proliferation and migration in cell and tissue models of meniscal repair. Methods A micro-wound assay was used to assess meniscal cell migration and proliferation in response to the following treatments for 0, 24, or 48 hours: 0 to 10 ng/mL IL-1, TNF-α, or TGF-β1, in the presence or absence of 10% serum. Proliferated and total cells were fluorescently labeled and imaged using confocal laser scanning microscopy and the number of proliferated, migrated, and total cells was determined in the micro-wound and edges of each image. Meniscal cell proliferation was also assessed throughout meniscal repair model explants treated with 0 or 10 ng/mL IL-1, TNF-α, or TGF-β1 for 14 days. At the end of the culture period, biomechanical testing and histological analyses were also performed. Statistical differences were assessed using an ANOVA and Newman-Keuls post hoc test. Results IL-1 and TNF-α decreased cell proliferation in both cell and tissue models of meniscal repair. In the presence of serum, TGF-β1 increased outer zone cell proliferation in the micro-wound and in the cross section of meniscal repair model explants. Both IL-1 and TNF-α decreased the integrative shear strength of repair and extracellular matrix deposition in the meniscal repair model system

Notch-1 regulates pulmonary neuroendocrine cell differentiation in cell lines and in transgenic mice.

PubMed

Shan, Lin; Aster, Jon C; Sklar, Jeffrey; Sunday, Mary E

2007-02-01

The notch gene family encodes transmembrane receptors that regulate cell differentiation by interacting with surface ligands on adjacent cells. Previously, we demonstrated that tumor necrosis factor-alpha (TNF) induces neuroendocrine (NE) cell differentiation in H82, but not H526, undifferentiated small cell lung carcinoma lines. We now test the hypothesis that TNF mediates NE cell differentiation in part by altering Notch gene expression. First, using RT-PCR, we determined that TNF treatment of H82, but not H526, transiently decreases notch-1 mRNA in parallel with induction of gene expression for the NE-specific marker DOPA decarboxylase (DDC). Second, we treated H82 and H526 with notch-1 antisense vs. sense oligodeoxynucleotides. Using quantitative RT-PCR and Western analyses we demonstrate that DDC mRNA and protein are increased in H82 by notch-1 antisense, whereas notch-1 mRNA and activated Notch-1 protein are decreased. mRNA for Hes1, a transcription factor downstream from activated Notch, is also decreased by Notch-1 antisense in H82 but not H526. After 7 days of Notch-1 antisense treatment, neural cell adhesion molecule (NCAM) immunoreactivity is induced in H82 but not H526. Third, we generated transgenic mice bearing notch-1 driven by the neural/NE-specific calcitonin promoter, which express activated Notch-1 in developing lung epithelium. Newborn NotchCal mouse lungs have high levels of hes1 mRNA, reflecting increased activated Notch, compared with wild-type. NotchCal lungs have decreased CGRP-positive NE cells, decreased protein gene product 9.5 (PGP9.5)-positive NE cells, and decreased gastrin-releasing peptide (GRP), CGRP, and DDC mRNA levels compared with normal littermates. Cumulatively, these observations provide further support for a role for Notch-1 signaling in regulating pulmonary NE cell differentiation.

CSF-1R regulates non-small cell lung cancer cells dissemination through Wnt3a signaling.

PubMed

Yu, Yan Xia; Wu, Hai Jian; Tan, Bing Xu; Qiu, Chen; Liu, Hui Zhong

2017-01-01

Therapeutic antibodies targeting colony stimulating factor 1 receptor (CSF-1R) to block colony stimulating factor-1/colony stimulating factor 1 receptor (CSF-1/CSF-R) signaling axis have exhibit remarkable efficacy in the treatment of malignant tumor. Yet, little is known about the effects of intrinsic CSF-1R in human non-small-cell carcinoma (NSCLC). Here we demonstrated that NSCLC cell-intrinsic CSF-1R promoted cells growth and metastasis both in vitro and in vivo. CSF-1R knocked-down by transfecting with shRNA target CSF-1R suppressed NSCLC cells proliferation and tumor growth in nude mice. Conversely, ectopic expression of CSF-1R promoted cells proliferation and accelerated tumor growth. Mechanistically, the NSCLC CSF-1R modulated downstream effectors of phosphatidylinositol 3-kinase (PI3K) signaling. In addition, CSF-1R overexpression significantly enhanced NSCLC cells mobility, invasion and epithelial-mesenchymal transition (EMT) process, whereas silencing CSF-1R inhibits these phenotypes. Microarray analysis suggested that Wnt family member 3a (Wnt3a) function as a downstream factor of CSF-1R. On account of this, we future identified CSF-1R/Wnt3a a signaling pathway sustained NSCLC cells metastasis. Finally, in patients, CSF-1R and Wnt3a expression positively correlated with the of NSCLC patients. Our results identify NSCLC cell intrinsic functions of CSF-1R/Wnt3a axis in dissemination of NSCLC.

CSF-1R regulates non-small cell lung cancer cells dissemination through Wnt3a signaling

PubMed Central

Yu, Yan Xia; Wu, Hai Jian; Tan, Bing Xu; Qiu, Chen; Liu, Hui Zhong

2017-01-01

Therapeutic antibodies targeting colony stimulating factor 1 receptor (CSF-1R) to block colony stimulating factor-1/colony stimulating factor 1 receptor (CSF-1/CSF-R) signaling axis have exhibit remarkable efficacy in the treatment of malignant tumor. Yet, little is known about the effects of intrinsic CSF-1R in human non-small-cell carcinoma (NSCLC). Here we demonstrated that NSCLC cell-intrinsic CSF-1R promoted cells growth and metastasis both in vitro and in vivo. CSF-1R knocked-down by transfecting with shRNA target CSF-1R suppressed NSCLC cells proliferation and tumor growth in nude mice. Conversely, ectopic expression of CSF-1R promoted cells proliferation and accelerated tumor growth. Mechanistically, the NSCLC CSF-1R modulated downstream effectors of phosphatidylinositol 3-kinase (PI3K) signaling. In addition, CSF-1R overexpression significantly enhanced NSCLC cells mobility, invasion and epithelial-mesenchymal transition (EMT) process, whereas silencing CSF-1R inhibits these phenotypes. Microarray analysis suggested that Wnt family member 3a (Wnt3a) function as a downstream factor of CSF-1R. On account of this, we future identified CSF-1R/Wnt3a a signaling pathway sustained NSCLC cells metastasis. Finally, in patients, CSF-1R and Wnt3a expression positively correlated with the of NSCLC patients. Our results identify NSCLC cell intrinsic functions of CSF-1R/Wnt3a axis in dissemination of NSCLC. PMID:29218239

Differential Regulation of Mouse B Cell Development by Transforming Growth Factor β1

PubMed Central

Kaminski, Denise A.; Letterio, John J.; Burrows, Peter D.

2002-01-01

Transforming growth factor β (TGFβ) can inhibit the in vitro proliferation, survival and differentiation of B cell progenitors, mature B lymphocytes and plasma cells. Here we demonstrate unexpected, age-dependent reductions in the bone marrow (BM) B cell progenitors and immature B cells in TGFβ1-/- mice. To evaluate TGFβ responsiveness during normal B lineage development, cells were cultured in interleukin 7 (IL7)±TGFβ. Picomolar doses of TGFβ1 reduced pro-B cell recoveries at every timepoint. By contrast, the pre-B cells were initially reduced in number, but subsequently increased compared to IL7 alone, resulting in a 4-fold increase in the growth rate for the pre-B cell population. Analysis of purified BM sub-populations indicated that pro-B cells and the earliest BP1- pre-B cells were sensitive to the inhibitory effects of TGFβ1. However, the large BP1+ pre-B cells, although initially reduced, were increased in number at days 5 and 7 of culture. These results indicate that TGFβ1 is important for normal B cell development in vivo, and that B cell progenitors are differentially affected by the cytokine according to their stage of differentiation. PMID:12739785

Two distinct factors are required for induction of T-cell growth.

PubMed

Larsson, E L; Iscove, N N; Coutinho, A

1980-02-14

The molecular and cellular basis of T-lymphocyte activation remains a central question in immunology. The growth of already proliferating T cells is known to depend on T-cell growth factor (TCGF), a physiological mitogen. Noncycling T lymphocytes, however, are not sensitive to TCGF. They require a short contact with mitogenic lectins, such as concanavalin A (Con A) or leukoagglutinin to bind and respond to TCGF, and will thereafter maintain exponential growth for long periods provided that TCGF is not limiting. While the induction of TCGF reactivity results from the direct contact of Con A with resting T cells, the lectin-dependent production of TCGF is known to involve two cell types, both present in mouse spleen. One consists of I-A-negative cells, most of which are Thy-1-positive T lymphocytes, and the other consists of I-A-positive, immunoglobulin-negative, Thy-1-negative cells, most of which are macrophages. The nature of the respective contributions of the two cell types, and in particular the cellular origin of TCGF, has not yet been established. We have now established the I-A-negative population as the source of TCGF and show here that macrophages are required to supply a 20,000-molecular weight factor, chemically and functionally distinct from TCGF, which supports the production of TCGF by the I-A-negative cells.

Clinical features of Bim deletion polymorphism and its relation with crizotinib primary resistance in Chinese patients with ALK/ROS1 fusion-positive non-small cell lung cancer.

PubMed

Zhang, Limin; Jiang, Tao; Li, Xuefei; Wang, Yan; Zhao, Chao; Zhao, Sha; Xi, Lei; Zhang, Shijia; Liu, Xiaozhen; Jia, Yijun; Yang, Hui; Shi, Jinpeng; Su, Chunxia; Ren, Shengxiang; Zhou, Caicun

2017-08-01

The authors' previous study demonstrated that the B-cell chronic lymphocytic leukemia/lymphoma (Bcl-2)-like 11 (BCL2L11) (Bim) deletion polymorphism was associated with poor clinical response to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in patients with non-small cell lung cancer (NSCLC) with EGFR mutations. The objective of the current study was to investigate the impact of the Bim deletion polymorphism among patients with anaplastic lymphoma kinase (ALK)-positive or ROS proto-oncogene 1, receptor tyrosine kinase (ROS1)-positive NSCLC who were treated with crizotinib. A total of 55 patients with ALK-positive NSCLC and 14 patients with ROS1-positive NSCLC who were treated with crizotinib were enrolled into the current study. The Bim deletion polymorphism was analyzed by polymerase chain reaction. The clinical features of the Bim deletion polymorphism and its impact on the effect of crizotinib were investigated. The Bim deletion polymorphism was present in 9 of 69 patients with ALK-positive or ROS1-positive NSCLC (13.0%). There were no differences noted with regard to clinicopathological features between patients with and without the Bim deletion polymorphism. Patients with the Bim deletion polymorphism had a significantly shorter progression-free survival (PFS) and lower objective response rate compared with those without (median PFS, 182 days vs 377 days [P = .008]) (objective response rate, 44.4% vs 81.7% [P =.041]) in all populations. The significant difference in PFS was observed in patients with ALK-positive NSCLC (83 days vs 305 days [P =.0304]) compared with those with ROS1-positive NSCLC (218 days vs not reached [P =.082]). Multivariate analysis indicated that the Bim deletion polymorphism was an independent predictive factor for patients with ALK-positive NSCLC who were treated with crizotinib (hazard ratio, 4.786 [P =.006]). The Bim deletion polymorphism was found to be associated with poor clinical response to crizotinib in

Lgr5-positive cells are cancer stem cells in skin squamous cell carcinoma.

PubMed

Liu, Shunli; Gong, Zhenyu; Chen, Mingrui; Liu, Benli; Bian, Donghui; Wu, Kai

2014-11-01

Cancer stem cells (CSCs) in most human tumors are commonly identified and enriched using similar strategies for identifying normal stem cells, including flow cytometry assays for side population, high aldehyde dehydrogenase (ALDH) activity, and CD133 positivity. Thus, development of a method for isolating a specific cancer using cancer-specific characteristic appears to be potentially important. Here, we reported extremely high Lgr5 levels in the specimen from skin squamous cell carcinoma (SCC) in patients. Using SCC cell line A431, we detected high Lgr5 and CD133 levels in ALDH-high or side population from these cancer cells. To figure out whether Lgr5 is a marker of CSCs in SCC, we transfected A431 cells with a Lgr5-creERT-2A-DTR/Cag-Loxp-GFP-STOP-Loxp-RFP plasmid and purified transfected cells (tA431) based on GFP by flow cytometry. 4-Hydroxytamoxifen (4-OHT) was given to label Lgr5-positive cells with RFP, for comparison to GFP-positive Lgr5-negative cells. Lgr5-positive cells grew significantly faster than Lgr5-negative cells, and the fold increase in growth of Lgr5-positive vs Lgr5-negative cells is significantly higher than SP vs non-SP, or ALDH-high vs ALDH-low, or CD133-positive vs CD133-negative cells. Moreover, in Lgr5-negative population, Lgr5-positive re-appeared in culture with time, suggesting that Lgr5-positive cells can be regenerated from Lgr5-negative cells. Furthermore, the growth of tA431 cells significantly decreased upon a single dose of diphtheria toxin (DT)/4-OHT to eliminate Lgr5-positive cell lineage, while multiple doses of DT/4-OHT nearly completely inhibited tA431 cell growth. Taken together, our data provide compelling data to demonstrate that Lgr5-positive cells are CSCs in skin SCC.

Apoptosis and TRAF-1 cleavage in Epstein-Barr virus-positive nasopharyngeal carcinoma cells treated with doxorubicin combined with a farnesyl-transferase inhibitor.

PubMed

Vicat, Jean Michel; Ardila-Osorio, Hector; Khabir, Abdelmajid; Brezak, Marie Christine; Viossat, Isabelle; Kasprzyk, Philip; Jlidi, Rachid; Opolon, Paule; Ooka, Tadamassa; Prevost, Grégoire; Huang, Dolly P; Busson, Pierre

2003-02-01

Epstein-Barr virus (EBV)-associated nasopharyngeal carcinomas (NPC) are much more sensitive to chemotherapy than other head and neck carcinomas. Spectacular regressions are frequently observed after induction chemotherapy. However, these favorable responses are difficult to predict and often of short duration. So far there have been only few experiments to investigate the mechanisms which underline the cytotoxic effects of anti-neoplastic drugs against NPC cells. In addition, these studies were performed almost entirely on EBV-negative cell lines therefore not truly representative of NPC cells. For the first time, we have used two EBV-positive NPC tumor lines derived from a North African (C15) and a Chinese (C666-1) patient as in vitro targets for a panel of anti-neoplastic agents. Doxorubicin, taxol and in a lesser extent cis-platinum efficiently inhibited NPC cell proliferation at clinically relevant concentrations, but all three agents failed to induce apoptosis. However, massive apoptosis of C15 cells was achieved when doxorubicin (1 microM) was combined with a farnesyl-transferase inhibitor, BIM 2001 (5 microM). Moreover, this apoptotic process was associated with a caspase-dependent early cleavage of the TNF-receptor associated factor 1 (TRAF-1) molecule, a signaling adaptor which is specifically expressed in latently EBV-infected cells. TRAF-1 cleavage might become a useful indicator of chemo-induced apoptosis in EBV-associated NPCs.

Aspirin regulation of c-myc and cyclinD1 proteins to overcome tamoxifen resistance in estrogen receptor-positive breast cancer cells.

PubMed

Cheng, Ran; Liu, Ya-Jing; Cui, Jun-Wei; Yang, Man; Liu, Xiao-Ling; Li, Peng; Wang, Zhan; Zhu, Li-Zhang; Lu, Si-Yi; Zou, Li; Wu, Xiao-Qin; Li, Yu-Xia; Zhou, You; Fang, Zheng-Yu; Wei, Wei

2017-05-02

Tamoxifen is still the most commonly used endocrine therapy drug for estrogen receptor (ER)-positive breast cancer patients and has an excellent outcome, but tamoxifen resistance remains a great impediment to successful treatment. Recent studies have prompted an anti-tumor effect of aspirin. Here, we demonstrated that aspirin not only inhibits the growth of ER-positive breast cancer cell line MCF-7, especially when combined with tamoxifen, but also has a potential function to overcome tamoxifen resistance in MCF-7/TAM. Aspirin combined with tamoxifen can down regulate cyclinD1 and block cell cycle in G0/G1 phase. Besides, tamoxifen alone represses c-myc, progesterone receptor (PR) and cyclinD1 in MCF-7 cell line but not in MCF-7/TAM, while aspirin combined with tamoxifen can inhibit the expression of these proteins in the resistant cell line. When knocking down c-myc in MCF-7/TAM, cells become more sensitive to tamoxifen, cell cycle is blocked as well, indicating that aspirin can regulate c-myc and cyclinD1 proteins to overcome tamoxifen resistance. Our study discovered a novel role of aspirin based on its anti-tumor effect, and put forward some kinds of possible mechanisms of tamoxifen resistance in ER-positive breast cancer cells, providing a new strategy for the treatment of ER-positive breast carcinoma.

A positive circuit of VEGF increases Glut-1 expression by increasing HIF-1α gene expression in human retinal endothelial cells.

PubMed

Choi, Yoon Kyung

2017-12-01

Treatment of human retinal microvascular endothelial cells (HRMECs) with vascular endothelial growth factor 165 (VEGF 165 ) increased hypoxia-inducible factor 1α (HIF-1α), VEGF, and glucose transporter 1 (Glut-1) mRNA expression and Glut-1 protein localization to the membrane. In contrast, treatment of human retinal pigment epithelium cells with VEGF 165 did not induce HIF-1α, VEGF, and Glut-1 gene expression. Microvascular endothelial cells are surrounded by astrocytic end feet in the retina. Astrocyte-derived A-kinase anchor protein 12 overexpression during hypoxia downregulated VEGF secretion, and this conditioned medium reduced VEGF and Glut-1 expression in HRMECs, suggesting that communications between astrocytes and endothelial cells may be the determinants of the blood vessel network. In HRMECs, HIF-1α small interfering RNA transfection blocked the VEGF 165 -mediated increase in VEGF and Glut-1 gene expression. Inhibition of protein kinase C (PKC) with inhibitor GF109203X or with a small interfering RNA targeting PKCζ attenuated the VEGF 165 -induced Glut-1 protein expression and VEGF and Glut-1 mRNA expression. In addition, results of an immunoprecipitation assay imply an interaction between VEGF receptor 2 (VEGFR2) and PKCζ in HRMECs. Therefore, VEGF secretion by hypoxic astrocytes may upregulate HIF-1α gene expression, inducing VEGF and Glut-1 expression via the VEGFR2-PKCζ axis in HRMECs.

Depletion of mitochondrial fission factor DRP1 causes increased apoptosis in human colon cancer cells

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

Inoue-Yamauchi, Akane, E-mail: ainoyama@research.twmu.ac.jp; Oda, Hideaki

2012-04-27

Highlights: Black-Right-Pointing-Pointer DRP1 is required for mitochondrial fission in colon cancer cells. Black-Right-Pointing-Pointer DRP1 participates in inhibition of colon cancer cell apoptosis. Black-Right-Pointing-Pointer DRP1 can inhibit apoptosis through the regulation of cytochrome c release. -- Abstract: Mitochondria play a critical role in regulation of apoptosis, a form of programmed cell death, by releasing apoptogenic factors including cytochrome c. Growing evidence suggests that dynamic changes in mitochondrial morphology are involved in cellular apoptotic response. However, whether DRP1-mediated mitochondrial fission is required for induction of apoptosis remains speculative. Here, we show that siRNA-mediated DRP1 knockdown promoted accumulation of elongated mitochondria in HCT116more » and SW480 human colon cancer cells. Surprisingly, DRP1 down-regulation led to decreased proliferation and increased apoptosis of these cells. A higher rate of cytochrome c release and reductions in mitochondrial membrane potential were also revealed in DRP1-depleted cells. Taken together, our present findings suggest that mitochondrial fission factor DRP1 inhibits colon cancer cell apoptosis through the regulation of cytochrome c release and mitochondrial membrane integrity.« less

Anticancer effects of phenoxazine derivatives revealed by inhibition of cell growth and viability, disregulation of cell cycle, and apoptosis induction in HTLV-1-positive leukemia cells.

PubMed

Miyano-Kurosaki, Naoko; Ikegami, Kou; Kurosaki, Kunihiko; Endo, Takahiko; Aoyagi, Hoshimi; Hanami, Mari; Yasumoto, Jun; Tomoda, Akio

2009-05-01

Adult T-cell leukemia (ATL) is a malignant tumor of human CD4(+) T cells infected with a human retrovirus, T lymphotropic virus type-1 (HTLV-1). The aim of the present study was to investigate the apoptotic effects of phenoxazines, 2-amino-4,4alpha-dihydro-4alpha,7-dimethyl-3H-phenoxazine-3-one (Phx-1), 3-amino-1,4alpha-dihydro-4alpha,8-dimethyl-2H-phenoxazine-2-one (Phx-2), and 2-aminophenoxazine-3-one (Phx-3) on a T cell leukemia cell line from ATL patients, MT-1 cells; HTLV-1 transformed T-cell lines, HUT-102 cells and MT-2 cells; and an HTLV-1-negative rat sarcoma cell line, XC cells. Among these phenoxazines, Phx-3 at concentrations of less than 10 microg/ml extensively inhibited growth and cell viability; arrested cell cycles at sub G(0)/G(1) phase; and augmented apoptosis of MT-1, HUT-102, and MT-2 cells. However, these phenoxazines did not affect the cell viability of an HTLV-1-negative rat sarcoma cell line, XC cells, and phytohemaggutinin-activated human peripheral blood mononuclear cells, although they markedly inhibited the growth of these cells. The transmission of HTLV-1 from HTLV-1-positive cells (MT-2 cells) to HTLV-1-negative cells (XC cells) was considered to be prevented by Phx-1, Phx-2, or Phx-3 because the syncytium formation between these cells was inhibited markedly in the presence of these phenoxazines. The present results suggest that Phx-1, Phx-2, and, in particular, Phx-3 may be useful as therapeutic agents against ATL, which is extremely refractory to current therapies.

Environmental Factors Contribute to β Cell Endoplasmic Reticulum Stress and Neo-Antigen Formation in Type 1 Diabetes

PubMed Central

Marré, Meghan L.; Piganelli, Jon D.

2017-01-01

Type 1 diabetes (T1D) is an autoimmune disease in which immune-mediated targeting and destruction of insulin-producing pancreatic islet β cells leads to chronic hyperglycemia. There are many β cell proteins that are targeted by autoreactive T cells in their native state. However, recent studies have demonstrated that many β cell proteins are recognized as neo-antigens following posttranslational modification (PTM). Although modified neo-antigens are well-established targets of pathology in other autoimmune diseases, the effects of neo-antigens in T1D progression and the mechanisms by which they are generated are not well understood. We have demonstrated that PTM occurs during endoplasmic reticulum (ER) stress, a process to which β cells are uniquely susceptible due to the high rate of insulin production in response to dynamic glucose sensing. In the context of genetic susceptibility to autoimmunity, presentation of these modified neo-antigens may activate autoreactive T cells and cause pathology. However, inherent β cell ER stress and protein PTM do not cause T1D in every genetically susceptible individual, suggesting the contribution of additional factors. Indeed, many environmental factors, such as viral infection, chemicals, or inflammatory cytokines, are associated with T1D onset, but the mechanisms by which these factors lead to disease onset remain unknown. Since these environmental factors also cause ER stress, exposure to these factors may enhance production of neo-antigens, therefore boosting β cell recognition by autoreactive T cells and exacerbating T1D pathogenesis. Therefore, the combined effects of physiological ER stress and the stress that is induced by environmental factors may lead to breaks in peripheral tolerance, contribute to antigen spread, and hasten disease onset. This Hypothesis and Theory article summarizes what is currently known about ER stress and protein PTM in autoimmune diseases including T1D and proposes a role for

Novel digitalis-like factor, marinobufotoxin, isolated from cultured Y-1 cells, and its hypertensive effect in rats.

PubMed

Yoshika, Masamichi; Komiyama, Yutaka; Konishi, Motomi; Akizawa, Toshifumi; Kobayashi, Takahisa; Date, Mutsuhiro; Kobatake, Shinzo; Masuda, Midori; Masaki, Hiroya; Takahashi, Hakuo

2007-01-01

Marinobufagenin and telecinobufagin have been identified as digitalis-like factors in mammals. In toads, marinobufagenin-related compounds, such as marinobufotoxin (MBT), have been isolated in some tissues but not in mammals, and its biological action has not been elucidated. Herein, we aimed to explore the possible production and/or secretion of MBT and the biological action in rats. First, the MBT in culture supernatant of the adrenocortical-originated cell line Y-1 was analyzed by high-performance liquid chromatography and sensitive ELISA for marinobufagenin-like immunoreactivity. Moreover, the structural information was obtained by mass spectrometry. To determine the biological action, MBT (9.6 and 0.96 microg/kg per day) was intraperitoneally infused via an osmotic minipump for 1 week. Blood pressure and renal excretion of marinobufagenin-like immunoreactivity were measured. Marinobufagenin-like immunoreactivity was found in Y-1 cell culture media, and the concentration increased until 24 hours. The structural analysis suggested that marinobufagenin-like immunoreactivities were marinobufagenin and MBT, and tandem mass spectrum analysis revealed them with the specific daughter ions. The highest sensitive ELISA-positive peak of marinobufagenin-like immunoreactivity in the media was MBT. Continuous administration of MBT in rats for 1 week significantly increased systolic blood pressure and renal excretion of marinobufagenin-like immunoreactivity compared with control rats (135+/-3.0 versus 126+/-2.0 mm Hg and 1.41+/-0.286 versus 0.34+/-0.064 ng/day, respectively). These data suggest that MBT, arginine-suberoyl ester of marinobufagenin, can be a novel digitalis-like factor with hypertensive action and is secreted from the adrenocortical cells.

Growth differentiation factor 9 signaling requires ERK1/2 activity in mouse granulosa and cumulus cells.

PubMed

Sasseville, Maxime; Ritter, Lesley J; Nguyen, Thao M; Liu, Fang; Mottershead, David G; Russell, Darryl L; Gilchrist, Robert B

2010-09-15

Ovarian folliculogenesis is driven by the combined action of endocrine cues and paracrine factors. The oocyte secretes powerful mitogens, such as growth differentiation factor 9 (GDF9), that regulate granulosa cell proliferation, metabolism, steroidogenesis and differentiation. This study investigated the role of the epidermal growth factor receptor (EGFR)-extracellular signal-regulated kinase 1 and 2 (ERK1/2; also known as MAPK3/1) signaling pathway on GDF9 action on granulosa cells. Results show that mitogenic action of the oocyte is prevented by pharmacological inhibition of the EGFR-ERK1/2 pathway. Importantly, EGFR-ERK1/2 activity as well as rous sarcoma oncogene family kinases (SFK) are required for signaling through SMADs, mediating GDF9, activin A and TGFbeta1 mitogenic action in granulosa cells. GDF9 could not activate ERK1/2 or affect EGF-stimulated ERK1/2 in granulosa cells. However, induction of the SMAD3-specific CAGA reporter by GDF9 in granulosa cells required active EGFR, SFKs and ERK1/2 as did GDF9-responsive gene expression. Finally, the EGFR-SFKs-ERK1/2 pathway was shown to be required for the maintenance of phosphorylation of the SMAD3 linker region. Together our results suggest that receptivity of granulosa cells to oocyte-secreted factors, including GDF9, is regulated by the level of activation of the EGFR and resulting ERK1/2 activity, through the requisite permissive phosphorylation of SMAD3 in the linker region. Our results indicate that oocyte-secreted TGFbeta-like ligands and EGFR-ERK1/2 signaling are cooperatively required for the unique granulosa cell response to the signal from oocytes mediating granulosa cell survival and proliferation and hence the promotion of follicle growth and ovulation.

Soluble Tumor Necrosis Factor Receptor 1 Released by Skin-Derived Mesenchymal Stem Cells Is Critical for Inhibiting Th17 Cell Differentiation

PubMed Central

Ke, Fang; Zhang, Lingyun; Liu, Zhaoyuan; Yan, Sha; Xu, Zhenyao; Bai, Jing; Zhu, Huiyuan; Lou, Fangzhou; Cai, Wei; Sun, Yang; Gao, Yuanyuan; Wang, Hong

2016-01-01

T helper 17 (Th17) cells play an important role in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Th17 cell differentiation from naïve T cells can be induced in vitro by the cytokines transforming growth factor β1 and interleukin-6. However, it remains unclear whether other regulatory factors control the differentiation of Th17 cells. Mesenchymal stem cells (MSCs) have emerged as a promising candidate for inhibiting Th17 cell differentiation and autoimmune diseases. Despite the fact that several molecules have been linked to the immunomodulatory function of MSCs, many other key MSC-secreted regulators that are involved in inhibiting Th17 cell polarization are ill-defined. In this study, we demonstrated that the intraperitoneal administration of skin-derived MSCs (S-MSCs) substantially ameliorated the development of EAE in mice. We found that the proinflammatory cytokine tumor necrosis factor (TNF)-α, a key mediator in the pathophysiology of MS and EAE, was capable of promoting Th17 cell differentiation. Moreover, under inflammatory conditions, we demonstrated that S-MSCs produced high amounts of soluble TNF receptor 1 (sTNFR1), which binds TNF-α and antagonizes its function. Knockdown of sTNFR1 in S-MSCs decreased their inhibitory effect on Th17 cell differentiation ex vivo and in vivo. Thus, our data identified sTNFR1 and its target TNF-α as critical regulators for Th17 cell differentiation, suggesting a previously unrecognized mechanism for MSC therapy in Th17-mediated autoimmune diseases. Significance This study showed that administration of skin-derived mesenchymal stem cells (S-MSCs) was able to alleviate the clinical score of experimental autoimmune encephalomyelitis by inhibiting the differentiation of T helper 17 (Th17) cells. Tumor necrosis factor (TNF)-α is a critical cytokine for promoting Th17 cell differentiation. It was discovered that activated S-MSCs produced high amount of soluble TNF receptor 1

AMP-activated protein kinase α2 and E2F1 transcription factor mediate doxorubicin-induced cytotoxicity by forming a positive signal loop in mouse embryonic fibroblasts and non-carcinoma cells.

PubMed

Yang, Wookyeom; Park, In-Ja; Yun, Hee; Im, Dong-Uk; Ock, Sangmi; Kim, Jaetaek; Seo, Seon-Mi; Shin, Ha-Yeon; Viollet, Benoit; Kang, Insug; Choe, Wonchae; Kim, Sung-Soo; Ha, Joohun

2014-02-21

Doxorubicin is one of the most widely used anti-cancer drugs, but its clinical application is compromised by severe adverse effects in different organs including cardiotoxicity. In the present study we explored mechanisms of doxorubicin-induced cytotoxicity by revealing a novel role for the AMP-activated protein kinase α2 (AMPKα2) in mouse embryonic fibroblasts (MEFs). Doxorubicin robustly induced the expression of AMPKα2 in MEFs but slightly reduced AMPKα1 expression. Our data support the previous notion that AMPKα1 harbors survival properties under doxorubicin treatment. In contrast, analyses of Ampkα2(-/-) MEFs, gene knockdown of AMPKα2 by shRNA, and inhibition of AMPKα2 activity with an AMPK inhibitor indicated that AMPKα2 functions as a pro-apoptotic molecule under doxorubicin treatment. Doxorubicin induced AMPKα2 at the transcription level via E2F1, a transcription factor that regulates apoptosis in response to DNA damage. E2F1 directly transactivated the Ampkα2 gene promoter. In turn, AMPKα2 significantly contributed to stabilization and activation of E2F1 by doxorubicin, forming a positive signal amplification loop. AMPKα2 directly interacted with and phosphorylated E2F1. This signal loop was also detected in H9c2, C2C12, and ECV (human epithelial cells) cells as well as mouse liver under doxorubicin treatment. Resveratrol, which has been suggested to attenuate doxorubicin-induced cytotoxicity, significantly blocked induction of AMPKα2 and E2F1 by doxorubicin, leading to protection of these cells. This signal loop appears to be non-carcinoma-specific because AMPKα2 was not induced by doxorubicin in five different tested cancer cell lines. These results suggest that AMPKα2 may serve as a novel target for alleviating the cytotoxicity of doxorubicin.

Expansion of natural (NK1+) T cells that express alpha beta T cell receptors in transporters associated with antigen presentation-1 null and thymus leukemia antigen positive mice

PubMed Central

1996-01-01

Thymic selection of natural killer-1+ natural T cells that express alpha beta T cell receptors requires a conserved beta 2-microglobulin- associated molecule, presumably CD1d, displayed by CD4+8+ thymocytes. Here we demonstrate that positive selection of natural T cells occurs independent of transporters associated with antigen presentation-1 (TAP- 1) function. Moreover, natural T cells in TAP-1o/o mice are numerically expanded. Several H-2 class Ib molecules function in a TAP-independent manner, suggesting that if expressed in TAP-1o/o thymocytes, they could play a role in natural T cell development. Of these class Ib molecules, H-2TL is expressed by TAP-1o/o thymocytes. Moreover, we find that thymi of TL+ mice congenic or transgenic for H-2T18 also have a numerically expanded natural T cell repertoire compared with TL- mice. This expansion, as in TAP-1o/o thymi, is evident in each of the limited T cell receptor V beta chains expressed by natural T cells, suggesting that TL and CD1d impact similar repertoires. Thus TL, in addition to CD1d, plays a role in natural T cell development. PMID:8879233

Role of stromal cell-derived factor 1 (SDF1/CXCL12) in regulating anterior pituitary function.

PubMed

Barbieri, Federica; Bajetto, Adriana; Porcile, Carola; Pattarozzi, Alessandra; Schettini, Gennaro; Florio, Tullio

2007-03-01

Chemokines are key factors involved in the regulation of immune response, through the activation and control of leukocyte traffic, lymphopoiesis and immune surveillance. However, a large number of chemokines and their receptors are expressed in central nervous system (CNS) cells, either constitutively or induced by inflammatory stimuli, playing a role in many neuropathological processes. Stromal cell-derived factor 1 (SDF1) is a chemokine whose extra-immunological localization and functions have been extensively studied. SDF1 and its receptor CXCR4 were identified in both neurons and glia of many brain areas, including the hypothalamus, as well as at the pituitary level. Importantly, SDF1 and CXCR4 expression is increased in brain tumors in which their activity induced tumor cell proliferation and brain parenchyma invasion. Despite their localization, to date very few reports addressed the role of CXCR4 and SDF1 in the modulation of the hypothalamus/pituitary axis and their possible involvement in the development of pituitary adenomas. In this review, we discuss previous literature data on the role of chemokines in normal and adenomatous pituitary cells, focusing on recent data from our group showing that CXCR4 activation controls proliferation and both prolactin and GH release in the pituitary adenoma cell line GH4C1 through a complex network of intracellular signals. Thus, the SDF1/CXCR4 system together with other chemokinergic ligand-receptor pairs, may represent a novel regulatory pathway for pituitary function and, possibly, be involved in pituitary adenoma development. These lines of evidence suggest that the inhibition of chemokine receptors may represent a novel pharmacological target for the treatment of pituitary adenomas.

Breast milk from Tanzanian women has divergent effects on cell-free and cell-associated HIV-1 infection in vitro.

PubMed

Lyimo, Magdalena A; Mosi, Matilda Ngarina; Housman, Molly L; Zain-Ul-Abideen, Muhammad; Lee, Frederick V; Howell, Alexandra L; Connor, Ruth I

2012-01-01

Transmission of HIV-1 during breastfeeding is a significant source of new pediatric infections in sub-Saharan Africa. Breast milk from HIV-positive mothers contains both cell-free and cell-associated virus; however, the impact of breast milk on HIV-1 infectivity remains poorly understood. In the present study, breast milk was collected from HIV-positive and HIV-negative Tanzanian women attending antenatal clinics in Dar es Salaam. Milk was analyzed for activity in vitro against both cell-free and cell-associated HIV-1. Potent inhibition of cell-free R5 and X4 HIV-1 occurred in the presence of milk from all donors regardless of HIV-1 serostatus. Inhibition of cell-free HIV-1 infection positively correlated with milk levels of sialyl-Lewis(X) from HIV-positive donors. In contrast, milk from 8 of 16 subjects enhanced infection with cell-associated HIV-1 regardless of donor serostatus. Milk from two of these subjects contained high levels of multiple pro-inflammatory cytokines including TNFα, IL-1β, IL-6, IL-8, MIP-1α, MIP-1β, MCP-1 and IP-10, and enhanced cell-associated HIV-1 infection at dilutions as high as 1∶500. These findings indicate that breast milk contains innate factors with divergent activity against cell-free and cell-associated HIV-1 in vitro. Enhancement of cell-associated HIV-1 infection by breast milk may be associated with inflammatory conditions in the mother and may contribute to infant infection during breastfeeding.

Pluripotency transcription factors and Tet1/2 maintain Brd4-independent stem cell identity.

PubMed

Finley, Lydia W S; Vardhana, Santosha A; Carey, Bryce W; Alonso-Curbelo, Direna; Koche, Richard; Chen, Yanyang; Wen, Duancheng; King, Bryan; Radler, Megan R; Rafii, Shahin; Lowe, Scott W; Allis, C David; Thompson, Craig B

2018-05-01

A robust network of transcription factors and an open chromatin landscape are hallmarks of the naive pluripotent state. Recently, the acetyllysine reader Brd4 has been implicated in stem cell maintenance, but the relative contribution of Brd4 to pluripotency remains unclear. Here, we show that Brd4 is dispensable for self-renewal and pluripotency of embryonic stem cells (ESCs). When maintained in their ground state, ESCs retain transcription factor binding and chromatin accessibility independent of Brd4 function or expression. In metastable ESCs, Brd4 independence can be achieved by increased expression of pluripotency transcription factors, including STAT3, Nanog or Klf4, so long as the DNA methylcytosine oxidases Tet1 and Tet2 are present. These data reveal that Brd4 is not essential for ESC self-renewal. Rather, the levels of pluripotency transcription factor abundance and Tet1/2 function determine the extent to which bromodomain recognition of protein acetylation contributes to the maintenance of gene expression and cell identity.

A Cyclin T1 point mutation that abolishes positive transcription elongation factor (P-TEFb) binding to Hexim1 and HIV tat

PubMed Central

2014-01-01

Background The positive transcription elongation factor b (P-TEFb) plays an essential role in activating HIV genome transcription. It is recruited to the HIV LTR promoter through an interaction between the Tat viral protein and its Cyclin T1 subunit. P-TEFb activity is inhibited by direct binding of its subunit Cyclin T (1 or 2) with Hexim (1 or 2), a cellular protein, bound to the 7SK small nuclear RNA. Hexim1 competes with Tat for P-TEFb binding. Results Mutations that impair human Cyclin T1/Hexim1 interaction were searched using systematic mutagenesis of these proteins coupled with a yeast two-hybrid screen for loss of protein interaction. Evolutionary conserved Hexim1 residues belonging to an unstructured peptide located N-terminal of the dimerization domain, were found to be critical for P-TEFb binding. Random mutagenesis of the N-terminal region of Cyclin T1 provided identification of single amino-acid mutations that impair Hexim1 binding in human cells. Furthermore, conservation of critical residues supported the existence of a functional Hexim1 homologue in nematodes. Conclusions Single Cyclin T1 amino-acid mutations that impair Hexim1 binding are located on a groove between the two cyclin folds and define a surface overlapping the HIV-1 Tat protein binding surface. One residue, Y175, in the centre of this groove was identified as essential for both Hexim1 and Tat binding to P-TEFb as well as for HIV transcription. PMID:24985203

Anti-ATLA (antibody to adult T-cell leukemia virus-associated antigen), highly positive in OKT4-positive mature T-cell malignancies.

PubMed

Tobinai, K; Nagai, M; Setoya, T; Shibata, T; Minato, K; Shimoyama, M

1983-01-01

Serum or plasma specimens from 252 patients with lymphoid malignancies were screened for reactivity with adult T-cell leukemia virus-associated antigen (ATLA), and the relationship between the immunologic phenotype of the tumor cells and ATLA reactivity was determined. Anti-ATLA antibodies were found in 24 (29.3%) of 82 patients with T-cell malignancy. In contrast, the antibodies were found in none of the 106 patients with B-cell malignancy and only rarely in patients with other lymphoid malignancies without blood transfusions. Among the patients with T-cell malignancy, anti-ATLA antibodies were found in 23 (45.1%) of the 51 patients with OKT4-positive mature T-cell (inducer/helper T-cell) malignancy, but in none of the patients with T-cell malignancy of pre-T, thymic T-cell or OKT8-positive mature T-cell (suppressor/cytotoxic T-cell) phenotype. Furthermore, among the OKT4-positive mature T-cell malignancies, the antibodies were found in 16 (84.2%) of 19 patients with ATL and in 5 (27.8%) of 18 patients with mature (peripheral) T-cell lymphoma, in none of four with typical T-chronic lymphocytic leukemia, in one of nine with mycosis fungoides and in the one patient with small-cell variant of Sézary's syndrome. These results suggest that anti-ATLA positive T-cell malignancies with OKT4-positive mature T-cell phenotype must be the same disease, because it is highly possible that they have the same etiology and the same cellular origin. In the atypical cases, it seems necessary to demonstrate monoclonal integration of proviral DNA of ATLV or HTLV into the tumor cells in order to establish the final diagnosis of ATL.

Involvement of matrix metalloproteinase-13 in stromal-cell-derived factor 1 alpha-directed invasion of human basal cell carcinoma cells.

PubMed

Chu, C-Y; Cha, S-T; Chang, C-C; Hsiao, C-H; Tan, C-T; Lu, Y-C; Jee, S-H; Kuo, M-L

2007-04-12

Basal cell carcinoma (BCC) is one of the most common skin neoplasms in humans and is usually characterized by local aggressiveness with little metastatic potential, although deep invasion, recurrence, and regional and distant metastases may occur. Here, we studied the mechanism of BCC invasion. We found that human BCC tissues and a BCC cell line had significant expression of CXCR4, which was higher in invasive than non-invasive BCC types. Further, of 19 recurrent tumors among 390 BCCs diagnosed during the past 12 years, 17/19 (89.5%) had high CXCR4 expression. We found that the CXCR4 ligand, stromal-cell-derived factor 1alpha (SDF-1alpha), directed BCC invasion and that this was mediated by time-dependent upregulation of mRNA expression and gelatinase activity of matrix metalloproteinase-13 (MMP-13). The transcriptional regulation of MMP-13 by SDF-1alpha was mediated by phosphorylation of extracellular signal-related kinase 1/2 and activation of the AP-1 component c-Jun. Finally, CXCR4-transfected BCC cells injected into nude mice induced aggressive BCCs that co-expressed CXCR4 and MMP-13. The identification of SDF-1alpha/CXCR4 as an important factor in BCC invasiveness may contribute insight into mechanisms involved in the aggressive potential of human BCC and may improve therapy for invasive BCCs.

Increased T cell recruitment to the CNS after amyloid beta 1-42 immunization in Alzheimer's mice overproducing transforming growth factor-beta 1.

PubMed

Buckwalter, Marion S; Coleman, Bronwen S; Buttini, Manuel; Barbour, Robin; Schenk, Dale; Games, Dora; Seubert, Peter; Wyss-Coray, Tony

2006-11-01

Immunotherapy targeting the amyloid beta (Abeta) peptide is a novel therapy under investigation for the treatment of Alzheimer's disease (AD). A clinical trial using Abeta(1-42) (AN1792) as the immunogen was halted as a result of development of meningoencephalitis in a small number of patients. The cytokine TGF-beta1 is a key modulator of immune responses that is increased in the brain in AD. We show here that local overexpression of TGF-beta1 in the brain increases both meningeal and parenchymal T lymphocyte number. Furthermore, TGF-beta1 overexpression in a mouse model for AD [amyloid precursor protein (APP) mice] leads to development of additional T cell infiltrates when mice were immunized at a young but not old age with AN1792. Notably, only mice overproducing both Abeta (APP mice) and TGF-beta1 experienced a rise in T lymphocyte number after immunization. One-third of infiltrating T cells were CD4 positive. We did not observe significant differences in B lymphocyte numbers in any of the genotypes or treatment groups. These results demonstrate that TGF-beta1 overproduction in the brain can promote T cell infiltration, in particular after Abeta(1-42) immunization. Likewise, levels of TGF-beta1 or other immune factors in brains of AD patients may influence the response to Abeta(1-42) immunization.

Zyxin regulates migration of renal epithelial cells through activation of hepatocyte nuclear factor-1β.

PubMed

Choi, Yun-Hee; McNally, Brian T; Igarashi, Peter

2013-07-01

Hepatocyte nuclear factor-1β (HNF-1β) is an epithelial tissue-specific transcription factor that regulates gene expression in the kidney, liver, pancreas, intestine, and other organs. Mutations of HNF-1β in humans produce renal cysts and congenital kidney anomalies. Here, we identify the LIM-domain protein zyxin as a novel binding partner of HNF-1β in renal epithelial cells. Zyxin shuttles to the nucleus where it colocalizes with HNF-1β. Immunoprecipitation of zyxin in leptomycin B-treated cells results in coprecipitation of HNF-1β. The protein interaction requires the second LIM domain of zyxin and two distinct domains of HNF-1β. Overexpression of zyxin stimulates the transcriptional activity of HNF-1β, whereas small interfering RNA silencing of zyxin inhibits HNF-1β-dependent transcription. Epidermal growth factor (EGF) induces translocation of zyxin into the nucleus and stimulates HNF-1β-dependent promoter activity. The EGF-mediated nuclear translocation of zyxin requires activation of Akt. Expression of dominant-negative mutant HNF-1β, knockdown of zyxin, or inhibition of Akt inhibits EGF-stimulated cell migration. These findings reveal a novel pathway by which extracellular signals are transmitted to the nucleus to regulate the activity of a transcription factor that is essential for renal epithelial differentiation.

Overexpression of Annexin A1 Suppresses Pro-Inflammatory Factors in PC12 Cells Induced by 1-Methyl-4-Phenylpyridinium

PubMed Central

Kiani-Esfahani, Abbas; Kazemi Sheykhshabani, Sedigheh; Peymani, Maryam; Hashemi, Motahare-Sadat; Ghaedi, Kamran; Nasr-Esfahani, Mohammad Hossein

2016-01-01

Objective Annexin A1 (ANXA1) is suggested to have anti-inflammatory function. However, the precise function of ANXA1 has remained unclear. In this study, we therefore examined the potency of ANXA1 in regulating reactive oxygen species (ROS) production and suppressing pro-inflammatory responses in PC12 cells induced by 1-methyl-4-phenylpyridinium (MPP+). Materials and Methods In this experimental study, cDNA of ANXA1 was cloned and inserted to the PGL268 pEpi-FGM18F vector to produce a recombinant PGL/ANXA1 vector for transfection into the PC12 cells. ANXA1 transfected cells were then treated with MPP+. Apoptosis and the content of pro-inflammatory factors including ROS, Interlukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and nuclear factor-kappa B (NF-κB) were assessed by flow-cytometry, real-time quantitative polymerase chain reaction (RT-qPCR) and western blot in ANXA1-transfected cells and the data were compared with those obtained from mock and control cells. Results Data revealed that overexpression of ANXA1 is associated with decreased levels of ROS and expression level of IL-6 and iNOS transcripts, and NF-κB protein in MPP+ treated PC12 cells. Conclusion ANXA1 may be considered as an agent for prevention of neurodegenerative or inflammatory conditions. PMID:27540524

Overexpression of Annexin A1 Suppresses Pro-Inflammatory Factors in PC12 Cells Induced by 1-Methyl-4-Phenylpyridinium.

PubMed

Kiani-Esfahani, Abbas; Kazemi Sheykhshabani, Sedigheh; Peymani, Maryam; Hashemi, Motahare-Sadat; Ghaedi, Kamran; Nasr-Esfahani, Mohammad Hossein

2016-01-01

Annexin A1 (ANXA1) is suggested to have anti-inflammatory function. However, the precise function of ANXA1 has remained unclear. In this study, we therefore examined the potency of ANXA1 in regulating reactive oxygen species (ROS) production and suppressing pro-inflammatory responses in PC12 cells induced by 1-methyl-4-phenylpyridinium (MPP+). In this experimental study, cDNA of ANXA1 was cloned and inserted to the PGL268 pEpi-FGM18F vector to produce a recombinant PGL/ANXA1 vector for transfection into the PC12 cells. ANXA1 transfected cells were then treated with MPP+. Apoptosis and the content of pro-inflammatory factors including ROS, Interlukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and nuclear factor-kappa B (NF-κB) were assessed by flow-cytometry, real-time quantitative polymerase chain reaction (RT-qPCR) and western blot in ANXA1-transfected cells and the data were compared with those obtained from mock and control cells. Data revealed that overexpression of ANXA1 is associated with decreased levels of ROS and expression level of IL-6 and iNOS transcripts, and NF-κB protein in MPP+ treated PC12 cells. ANXA1 may be considered as an agent for prevention of neurodegenerative or inflammatory conditions.

The granulocyte-macrophage colony-stimulating factor promoter cis-acting element CLE0 mediates induction signals in T cells and is recognized by factors related to AP1 and NFAT.

PubMed Central

Masuda, E S; Tokumitsu, H; Tsuboi, A; Shlomai, J; Hung, P; Arai, K; Arai, N

1993-01-01

Expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene in T cells is activated by the combination of phorbol ester (phorbol myristate acetate) and calcium ionophore (A23187), which mimic antigen stimulation through the T-cell receptor. We have previously shown that a fragment containing bp -95 to +27 of the mouse GM-CSF promoter can confer inducibility to reporter genes in the human Jurkat T-cell line. Here we use an in vitro transcription system to demonstrate that a cis-acting element (positions -54 to -40), referred to as CLE0, is a target for the induction signals. We observed induction with templates containing intact CLE0 but not with templates with deleted or mutated CLE0. We also observed that two distinct signals were required for the stimulation through CLE0, since only extracts from cells treated with both phorbol myristate acetate and A23187 supported optimal induction. Stimulation probably was mediated by CLE0-binding proteins because depletion of these proteins specifically reduced GM-CSF transcription. One of the binding factors possessed biochemical and immunological features identical to those of the transcription factor AP1. Another factor resembled the T-cell-specific factor NFAT. The characteristics of these two factors are consistent with their involvement in GM-CSF induction. The presence of CLE0-like elements in the promoters of interleukin-3 (IL-3), IL-4, IL-5, GM-CSF, and NFAT sites in the IL-2 promoter suggests that the factors we detected, or related factors that recognize these sites, may account for the coordinate induction of these genes during T-cell activation. Images PMID:8246960

Transcription factors YY1, Sp1 and Sp3 modulate dystrophin Dp71 gene expression in hepatic cells.

PubMed

Peñuelas-Urquides, Katia; Becerril-Esquivel, Carolina; Mendoza-de-León, Laura C; Silva-Ramírez, Beatriz; Dávila-Velderrain, José; Cisneros, Bulmaro; de León, Mario Bermúdez

2016-07-01

Dystrophin Dp71, the smallest product encoded by the Duchenne muscular dystrophy gene, is ubiquitously expressed in all non-muscle cells. Although Dp71 is involved in various cellular processes, the mechanisms underlying its expression have been little studied. In hepatic cells, Dp71 expression is down-regulated by the xenobiotic β-naphthoflavone. However, the effectors of this regulation remain unknown. In the present study we aimed at identifying DNA elements and transcription factors involved in Dp71 expression in hepatic cells. Relevant DNA elements on the Dp71 promoter were identified by comparing Dp71 5'-end flanking regions between species. The functionality of these elements was demonstrated by site-directed mutagenesis. Using EMSAs and ChIP, we showed that the Sp1 (specificity protein 1), Sp3 (specificity protein 3) and YY1 (Yin and Yang 1) transcription factors bind to the Dp71 promoter region. Knockdown of Sp1, Sp3 and YY1 in hepatic cells increased endogenous Dp71 expression, but reduced Dp71 promoter activity. In summary, Dp71 expression in hepatic cells is carried out, in part, by YY1-, Sp1- and Sp3-mediated transcription from the Dp71 promoter. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

Soybean (Glycine max) WRINKLED1 transcription factor, GmWRI1a, positively regulates seed oil accumulation.

PubMed

Chen, Liang; Zheng, Yuhong; Dong, Zhimin; Meng, Fanfan; Sun, Xingmiao; Fan, Xuhong; Zhang, Yunfeng; Wang, Mingliang; Wang, Shuming

2018-04-01

Soybean is the world's most important leguminous crop producing high-quality protein and oil. Elevating oil accumulation in soybean seed is always many researchers' goal. WRINKLED1 (WRI1) encodes a transcription factor of the APETALA2/ethylene responsive element-binding protein (AP2/EREBP) family that plays important roles during plant seed oil accumulation. In this study, we isolated and characterized three distinct orthologues of WRI1 in soybean (Glycine max) that display different organ-specific expression patterns, among which GmWRI1a was highly expressed in maturing soybean seed. Electrophoretic mobility shift assays and yeast one-hybrid experiments demonstrated that the GmWRI1a protein was capable of binding to AW-box, a conserved sequence in the proximal upstream regions of many genes involved in various steps of oil biosynthesis. Transgenic soybean seeds overexpressing GmWRI1a under the control of the seed-specific napin promoter showed the increased total oil and fatty acid content and the changed fatty acid composition. Furthermore, basing on the activated expressions in transgenic soybean seeds and existence of AW-box element in the promoter regions, direct downstream genes of GmWRI1a were identified, and their products were responsible for fatty acid production, elongation, desaturation and export from plastid. We conclude that GmWRI1a transcription factor can positively regulate oil accumulation in soybean seed by a complex gene expression network related to fatty acid biosynthesis.

The ZO-1–associated Y-box factor ZONAB regulates epithelial cell proliferation and cell density

PubMed Central

Balda, Maria S.; Garrett, Michelle D.; Matter, Karl

2003-01-01

Epithelial tight junctions regulate paracellular permeability, restrict apical/basolateral intramembrane diffusion of lipids, and have been proposed to participate in the control of epithelial cell proliferation and differentiation. Previously, we have identified ZO-1–associated nucleic acid binding proteins (ZONAB), a Y-box transcription factor whose nuclear localization and transcriptional activity is regulated by the tight junction–associated candidate tumor suppressor ZO-1. Now, we found that reduction of ZONAB expression using an antisense approach or by RNA interference strongly reduced proliferation of MDCK cells. Transfection of wild-type or ZONAB-binding fragments of ZO-1 reduced proliferation as well as nuclear ZONAB pools, indicating that promotion of proliferation by ZONAB requires its nuclear accumulation. Overexpression of ZONAB resulted in increased cell density in mature monolayers, and depletion of ZONAB or overexpression of ZO-1 reduced cell density. ZONAB was found to associate with cell division kinase (CDK) 4, and reduction of nuclear ZONAB levels resulted in reduced nuclear CDK4. Thus, our data indicate that tight junctions can regulate epithelial cell proliferation and cell density via a ZONAB/ZO-1–based pathway. Although this regulatory process may also involve regulation of transcription by ZONAB, our data suggest that one mechanism by which ZONAB and ZO-1 influence proliferation is by regulating the nuclear accumulation of CDK4. PMID:12566432

Lack of differences in radiation-induced immunogenicity parameters between HPV-positive and HPV-negative human HNSCC cell lines.

PubMed

Schneider, Karolin; Bol, Vanesa; Grégoire, Vincent

2017-09-01

Clinical studies indicate that patients with HPV/p16-associated head & neck squamous cell carcinoma (HNSCC) represent a subgroup with a better prognosis and improved response to conventional radiotherapy. Involvement of immune-based factors has been hypothesized. In the present study, we investigated radiation-induced differences in release of damage associated molecular patterns (DAMPs), cytokines and activation of dendritic cells (DCs) in HPV-positive and negative HNSCC cancer cell lines. Calreticulin (CRT) exposure was detected on cancer cell surface. ATP, HMGB1 and cytokines were measured in culture supernatants. Maturation marker CD83 surface exposure was determined on DCs after co-incubation with irradiated tumor cells. There was no increase in DAMPs and cytokine profiles after radiation treatment and no difference between HPV+ and HPV- cell lines. The HPV/p16-positive SCC90 cells showed a trend for increased total CRT, HMGB1, and number of cytokines compared to all other cell lines. None of the irradiated cancer cell lines could affect DC maturation. Radiation treatment did not increase immunogenicity of HNSCC cell lines assessed by membrane CRT, ATP, HMGB1, cytokines production, and by activation of immature DCs. There was no difference between HPV-positive and HPV-negative cell lines. Copyright © 2017 Elsevier B.V. All rights reserved.

Transforming growth factor-beta controls T helper type 1 cell development through regulation of natural killer cell interferon-gamma.

PubMed

Laouar, Yasmina; Sutterwala, Fayyaz S; Gorelik, Leonid; Flavell, Richard A

2005-06-01

Interferon-gamma and interleukin 12 produced by the innate arm of the immune system are important regulators of T helper type 1 (T(H)1) cell development, but signals that negatively regulate their expression remain controversial. Here we show that transforming growth factor-beta (TGF-beta) controlled T(H)1 differentiation through the regulation of interferon-gamma produced by natural killer (NK) cells. Blockade of TGF-beta signaling in NK cells caused the accumulation of a large pool of NK cells secreting copious interferon-gamma, responsible for T(H)1 differentiation and protection from leishmania infection. In contrast, blockade of TGF-beta signaling in dendritic cells did not affect dendritic cell homeostasis or interleukin 12 production, thus indicating a previously undescribed demarcation of the function of TGF-beta in NK cells versus dendritic cells.

Trends in baseline CD4 cell counts and risk factors for late antiretroviral therapy initiation among HIV-positive patients in Shanghai, a retrospective cross-sectional study.

PubMed

Sun, Jianjun; Liu, Li; Shen, Jiayin; Chen, Panpan; Lu, Hongzhou

2017-04-19

There are few studies focus on the factors underlying the late initiation of ART in China. We analyzed the trends in the median CD4 cell counts among different patient groups over time and the risk factors for the late initiation of ART in Shanghai, China. A retrospective cross-sectional survey was made in the Department of Infectious Disease of Shanghai Public Health Clinical Center which is a designated diagnosis and treatment center for HIV-positive patients in Shanghai during the period of January 1st, 2008--June 30th, 2014. Late ART initiation was defined as a CD4 cell count <200 cells/mm 3 or having a clinical AIDS diagnosis prior to ART initiation. Trends in the median CD4 cell count at ART initiation and the proportion of late ART initiation by year were evaluated using Spearman's correlations and Chi-squared methods, respectively. We used a logistic regression model to analyze the risk factors for late ART initiation. The related factors collected in the multivariate model were the patient's age, gender, infection routes and marital status. A total of 3796 patients were analyzed in this study, with a median baseline CD4 cell count of 205 cells/mm 3 [interquartile range: 75-287]. The median CD4 cell counts of patients initiating ART late increased from 76 cells/mm 3 in 2008 to 103 cells/mm 3 in 2014 (p < 0.001), and the proportion of late ART initiation decreased from 80% to 45% (p < 0.001). The risk factors for late ART initiation were male gender, heterosexual transmission and older age (>30 years) (p < 0.001). Notable improvements were made in the median CD4 cell count at ART initiation and the proportion of late ART initiation from 2008 to 2014. However, persons with high risk of HIV exposure who are male, older even heterosexual orientation should be given more opportunities to receive frequently screening, earlier diagnoses and timely treatment.

Prevention of Breast Cell Transformation by Blockade of the AP-1 Transcription Factor

DTIC Science & Technology

2000-10-01

Distribution Unlimited 13. ABSTRACT (Maximum 200 Words) In this study, we are investigating the role of AP- M in controlling breast cell growth and...serum and these growth factors depend on AP-1 to transduce proliferative signal. AP- M blockade induced by the expression of TAM67 inhibits breast...demonstrated that TAM67 inhibits basal AP-1 activity and AP- M activity stimulated by several different growth factors. We have also discovered that AP-1

Distribution of CD163-positive cell and MHC class II-positive cell in the normal equine uveal tract.

PubMed

Sano, Yuto; Matsuda, Kazuya; Okamoto, Minoru; Takehana, Kazushige; Hirayama, Kazuko; Taniyama, Hiroyuki

2016-02-01

Antigen-presenting cells (APCs) in the uveal tract participate in ocular immunity including immune homeostasis and the pathogenesis of uveitis. In horses, although uveitis is the most common ocular disorder, little is known about ocular immunity, such as the distribution of APCs. In this study, we investigated the distribution of CD163-positive and MHC II-positive cells in the normal equine uveal tract using an immunofluorescence technique. Eleven eyes from 10 Thoroughbred horses aged 1 to 24 years old were used. Indirect immunofluorescence was performed using the primary antibodies CD163, MHC class II (MHC II) and CD20. To demonstrate the site of their greatest distribution, positive cells were manually counted in 3 different parts of the uveal tract (ciliary body, iris and choroid), and their average number was assessed by statistical analysis. The distribution of pleomorphic CD163- and MHC II-expressed cells was detected throughout the equine uveal tract, but no CD20-expressed cells were detected. The statistical analysis demonstrated the distribution of CD163- and MHC II-positive cells focusing on the ciliary body. These results demonstrated that the ciliary body is the largest site of their distribution in the normal equine uveal tract, and the ciliary body is considered to play important roles in uveal and/or ocular immune homeostasis. The data provided in this study will help further understanding of equine ocular immunity in the normal state and might be beneficial for understanding of mechanisms of ocular disorders, such as equine uveitis.

The burden of anaemia and associated factors in HIV positive Nigerian women.

PubMed

Ezechi, O C; Kalejaiye, O O; Gab-Okafor, C V; Oladele, D A; Oke, B; Ekama, S O; Odunukwe, N N; Ujah, I A O

2013-02-01

Anaemia is the most common complication of pregnancy and a predictor of poor maternal and foetal outcomes. HIV infection is now recognized as one of the major contributors to anaemia in pregnancy. It is therefore important to determine the burden and risk factors of anaemia in maternal HIV infection in others to plan effective prevention strategies as well as optimize management outcomes. To determine the prevalence and risk factors of anaemia in pregnant HIV positive Nigerians. The prevalence and possible risk factors of anaemia were investigated in HIV positive pregnant Nigerian women at a large HIV treatment clinic in southwestern Nigeria using a cross-sectional design between January 2006 and December 2011. Nine hundred and eighty-five (42.5 %) women of 2,318 HIV positive pregnant women seen during the period were anaemic by WHO standard defined by haemoglobin <11 g/dl. Majority were of mild to moderate severity (97.9 %). Short inter birth interval (p = 0.002), presence of opportunistic infections (OIs), (p = 0.001), use of zidovudine containing regimen (p = 0.0005) and CD4 cell count <200 cells/mm(3) (p = 0.001) were found to be independently associated with anaemia in HIV positive pregnant women after controlling for confounding variables. Anaemia was found to be high at 42.5 % among the HIV positive women studied and was found to be independently associated with short inter birth interval, presence of OIs, advanced HIV disease and use of zidovudine containing HAART regimen.

Tubular epithelial cells in renal clear cell carcinoma express high RIPK1/3 and show increased susceptibility to TNF receptor 1-induced necroptosis

PubMed Central

Al-Lamki, R S; Lu, W; Manalo, P; Wang, J; Warren, A Y; Tolkovsky, A M; Pober, J S; Bradley, J R

2016-01-01

We previously reported that renal clear cell carcinoma cells (RCC) express both tumor necrosis factor receptor (TNFR)-1 and -2, but that, in organ culture, a TNF mutein that only engages TNFR1, but not TNFR2, causes extensive cell death. Some RCC died by apoptosis based on detection of cleaved caspase 3 in a minority TUNEL-positive cells but the mechanism of death in the remaining cells was unexplained. Here, we underpin the mechanism of TNFR1-induced cell death in the majority of TUNEL-positive RCC cells, and show that they die by necroptosis. Malignant cells in high-grade tumors displayed threefold to four fold higher expression of both receptor-interacting protein kinase (RIPK)1 and RIPK3 compared with non-tumor kidney tubular epithelium and low-grade tumors, but expression of both enzymes was induced in lower grade tumors in organ culture in response to TNFR1 stimulation. Furthermore, TNFR1 activation induced significant MLKLSer358 and Drp1Ser616 phosphorylation, physical interactions in RCC between RIPK1-RIPK3 and RIPK3-phospho-MLKLSer358, and coincidence of phospho-MLKLser358 and phospho-Drp1Ser616 at mitochondria in TUNEL-positive RCC. A caspase inhibitor only partially reduced the extent of cell death following TNFR1 engagement in RCC cells, whereas three inhibitors, each targeting a different step in the necroptotic pathway, were much more protective. Combined inhibition of caspases and necroptosis provided additive protection, implying that different subsets of cells respond differently to TNF-α, the majority dying by necroptosis. We conclude that most high-grade RCC cells express increased amounts of RIPK1 and RIPK3 and are poised to undergo necroptosis in response to TNFR1 signaling. PMID:27362805

Real-World Data on Prognostic Factors for Overall Survival in EGFR Mutation-Positive Advanced Non-Small Cell Lung Cancer Patients Treated with First-Line Gefitinib.

PubMed

Yao, Zong-Han; Liao, Wei-Yu; Ho, Chao-Chi; Chen, Kuan-Yu; Shih, Jin-Yuan; Chen, Jin-Shing; Lin, Zhong-Zhe; Lin, Chia-Chi; Chih-Hsin Yang, James; Yu, Chong-Jen

2017-09-01

This study aimed to identify independent prognostic factors for overall survival (OS) of patients with advanced non-small cell lung cancer (NSCLC) harboring an activating epidermal growth factor receptor (EGFR) mutation and receiving gefitinib as first-line treatment in real-world practice. We enrolled 226 patients from June 2011 to May 2013. During this period, gefitinib was the only EGFR-tyrosine kinase inhibitor reimbursed by the Bureau of National Health Insurance of Taiwan. The median progression-free survival and median OS were 11.9 months (95% confidence interval [CI]: 9.7-14.2) and 26.9 months (21.2-32.5), respectively. The Cox proportional hazards regression model revealed that postoperative recurrence, performance status (Eastern Cooperative Oncology Grade [ECOG] ≥2), smoking index (≥20 pack-years), liver metastasis at initial diagnosis, and chronic hepatitis C virus (HCV) infection were independent prognostic factors for OS (hazard ratio [95% CI] 0.3 [0.11-0.83], p  = .02; 2.69 [1.60-4.51], p  < .001; 1.92 [1.24-2.97], p  = .003; 2.26 [1.34-3.82], p  = .002; 3.38 [1.85-7.78], p  < .001, respectively). However, brain metastasis (BM) at initial diagnosis or intracranial progression during gefitinib treatment had no impact on OS (1.266 [0.83-1.93], p  = .275 and 0.75 [0.48-1.19], p  = .211, respectively). HCV infection, performance status (ECOG ≥2), newly diagnosed advanced NSCLC without prior operation, and liver metastasis predicted poor OS in EGFR mutation-positive advanced NSCLC patients treated with first-line gefitinib; however, neither BM at initial diagnosis nor intracranial progression during gefitinib treatment had an impact on OS. The finding that chronic hepatitis C virus (HCV) infection might predict poor overall survival (OS) in epidermal growth factor receptor mutation-positive advanced non-small cell lung cancer (NSCLC) patients treated with first-line gefitinib may raise awareness of benefit from anti

The transcription elongation factor ELL2 is specifically upregulated in HTLV-1-infected T-cells and is dependent on the viral oncoprotein Tax

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

Mann, Melanie C., E-mail: melanie.mann@viro.med.uni-erlangen.de; Strobel, Sarah, E-mail: sarah.strobel@viro.med.uni-erlangen.de; Fleckenstein, Bernhard, E-mail: bernhard.fleckenstein@viro.med.uni-erlangen.de

The oncoprotein Tax of human T-cell leukemia virus type 1 (HTLV-1) is a potent transactivator of viral and cellular transcription. Here, we identified ELL2 as the sole transcription elongation factor to be specifically upregulated in HTLV-1-/Tax-transformed T-cells. Tax contributes to regulation of ELL2, since transient transfection of Tax increases ELL2 mRNA, Tax transactivates the ELL2 promoter, and repression of Tax results in decrease of ELL2 in transformed T-lymphocytes. However, we also measured upregulation of ELL2 in HTLV-1-transformed cells exhibiting undetectable amounts of Tax, suggesting that ELL2 can still be maintained independent of continuous Tax expression. We further show that Taxmore » and ELL2 synergistically activate the HTLV-1 promoter, indicating that ELL2 cooperates with Tax in viral transactivation. This is supported by our findings that Tax and ELL2 accumulate in nuclear fractions and that they co-precipitate upon co-expression in transiently-transfected cells. Thus, upregulation of ELL2 could contribute to HTLV-1 gene regulation. - Highlights: • ELL2, a transcription elongation factor, is upregulated in HTLV-1-positive T-cells. • Tax transactivates the ELL2 promoter. • Tax and ELL2 synergistically activate the HTLV-1 promoter. • Tax and ELL2 interact in vivo.« less

Stromal cell-derived factor 1 protects human periodontal ligament stem cells against hydrogen peroxide-induced apoptosis.

PubMed

Feng, Yimiao; Fu, Xiaohui; Lou, Xintian; Fu, Baiping

2017-10-01

Periodontal ligament stem cells (PDLSCs) are considered a promising cell source for dental tissue regeneration. Stromal cell-derived factor 1 [SDF‑1, also known as chemokine (C‑X‑C motif) ligand 12] is regarded as a critical cytokine involved in stem/progenitor cell chemotaxis and homing during tissue regeneration. The present study described a previously unsuspected role for SDF‑1 in the protection of PDLSCs against oxidative stress‑induced apoptosis. In the present study, apoptosis was induced by exposure of PDLSCs to various concentrations of H2O2 for 12 h, following which cell viability was assessed, and cleaved caspase‑3 and ‑9 expression levels were evaluated. To investigate the potential mechanism underlying this protection, the protein expression levels of total and phosphorylated extracellular signal‑regulated kinase (ERK), a key protein of the mitogen‑activated protein kinase (MAPK) signaling pathway, were examined. The results of the present study revealed that SDF‑1 pretreatment increased cell viability following H2O2 administration, and downregulated protein expression levels of activated caspase‑3 and ‑9. Furthermore, treatment with SDF‑1 increased the phosphorylation of ERK. The protective effect of SDF‑1 was partially inhibited by treatment with PD98059, a MAPK/ERK inhibitor, which decreased cell viability. The results of the present study suggested that SDF‑1 treatment is a potential strategy to improve the survival of PDLSCs, which may be beneficial for dental tissue regeneration.

Fgf10-positive cells represent a progenitor cell population during lung development and postnatally

PubMed Central

El Agha, Elie; Herold, Susanne; Alam, Denise Al; Quantius, Jennifer; MacKenzie, BreAnne; Carraro, Gianni; Moiseenko, Alena; Chao, Cho-Ming; Minoo, Parviz; Seeger, Werner; Bellusci, Saverio

2014-01-01

The lung mesenchyme consists of a widely heterogeneous population of cells that play crucial roles during development and homeostasis after birth. These cells belong to myogenic, adipogenic, chondrogenic, neuronal and other lineages. Yet, no clear hierarchy for these lineages has been established. We have previously generated a novel Fgf10iCre knock-in mouse line that allows lineage tracing of Fgf10-positive cells during development and postnatally. Using these mice, we hereby demonstrate the presence of two waves of Fgf10 expression during embryonic lung development: the first wave, comprising Fgf10-positive cells residing in the submesothelial mesenchyme at early pseudoglandular stage (as well as their descendants); and the second wave, comprising Fgf10-positive cells from late pseudoglandular stage (as well as their descendants). Our lineage-tracing data reveal that the first wave contributes to the formation of parabronchial and vascular smooth muscle cells as well as lipofibroblasts at later developmental stages, whereas the second wave does not give rise to smooth muscle cells but to lipofibroblasts as well as an Nkx2.1- E-Cad- Epcam+ Pro-Spc+ lineage that requires further in-depth analysis. During alveologenesis, Fgf10-positive cells give rise to lipofibroblasts rather than alveolar myofibroblasts, and during adult life, a subpopulation of Fgf10-expressing cells represents a pool of resident mesenchymal stromal (stem) cells (MSCs) (Cd45- Cd31- Sca-1+). Taken together, we show for the first time that Fgf10-expressing cells represent a pool of mesenchymal progenitors in the embryonic and postnatal lung. Our findings suggest that Fgf10-positive cells could be useful for developing stem cell-based therapies for treating interstitial lung diseases. PMID:24353064

Cytoplasmic expression of Twist1, an EMT-related transcription factor, is associated with higher grades renal cell carcinomas and worse progression-free survival in clear cell renal cell carcinoma.

PubMed

Rasti, Arezoo; Madjd, Zahra; Abolhasani, Maryam; Mehrazma, Mitra; Janani, Leila; Saeednejad Zanjani, Leili; Asgari, Mojgan

2018-05-01

Twist1 is a key transcription factor, which confers tumor cells with cancer stem cell (CSC)-like characteristics and enhances epithelial-mesenchymal transition in pathological conditions including tumor malignancy and metastasis. This study aimed to evaluate the expression patterns and clinical significance of Twist1 in renal cell carcinoma (RCC). The cytoplasmic and nuclear expression of Twist1 were examined in 252 well-defined renal tumor tissues, including 173 (68.7%) clear cell renal cell carcinomas (ccRCC), 45 (17.9%) papillary renal cell carcinomas (pRCC) and 34 (13.5%) chromophobe renal cell carcinoma, by immunohistochemistry on a tissue microarray. The association between expression of this marker and clinicopathologic parameters and survival outcomes were then analyzed. Twist1 was mainly localized to the cytoplasm of tumor cells (98.8%). Increased cytoplasmic expression of Twist1 was associated with higher grade tumors (P = 0.045), renal vein invasion (P = 0.031) and microvascular invasion (P = 0.044) in RCC. It was positively correlated with higher grade tumors (P = 0.026), shorter progression-free survival time (P = 0.027) in patients with ccRCC, and also with higher stage in pRCC patients (P = 0.036). Significantly higher cytoplasmic expression levels of Twist1 were found in ccRCC and pRCC subtypes, due to their more aggressive tumor behavior. Increased cytoplasmic expression of Twist1 had a critical role in worse prognosis in ccRCC. These findings suggest that cytoplasmic, rather than nuclear expression of Twist1 can be considered as a prognostic and therapeutic marker for targeted therapy of RCC, especially for ccRCC patients.

Factors associated with a primary surgical approach for sinonasal squamous cell carcinoma.

PubMed

Cracchiolo, Jennifer R; Patel, Krupa; Migliacci, Jocelyn C; Morris, Luc T; Ganly, Ian; Roman, Benjamin R; McBride, Sean M; Tabar, Viviane S; Cohen, Marc A

2018-03-01

Primary surgery is the preferred treatment of T1-T4a sinonasal squamous cell carcinoma (SNSCC). Patients with SNSCC in the National Cancer Data Base (NCDB) were analyzed. Factors that contributed to selecting primary surgical treatment were examined. Overall survival (OS) in surgical patients was analyzed. Four-thousand seven hundred and seventy patients with SNSCC were included. In T1-T4a tumors, lymph node metastases, maxillary sinus location, and treatment at high-volume centers were associated with selecting primary surgery. When primary surgery was utilized, tumor factors and positive margin guided worse OS. Adjuvant therapy improved OS in positive margin resection and advanced T stage cases. Tumor and non-tumor factors are associated with selecting surgery for the treatment of SNSCC. When surgery is selected, tumor factors drive OS. Negative margin resection should be the goal of a primary surgical approach. When a positive margin resection ensues, adjuvant therapy may improve OS. © 2017 Wiley Periodicals, Inc.

The pioneer factor Smed-gata456-1 is required for gut cell differentiation and maintenance in planarians.

PubMed

González-Sastre, Alejandro; De Sousa, Nídia; Adell, Teresa; Saló, Emili

2017-01-01

How adult stem cells differentiate into different cell types remains one of the most intriguing questions in regenerative medicine. Pioneer factors are transcription factors that can bind to and open chromatin, and are among the first elements involved in cell differentiation. We used the freshwater planarian Schmidtea mediterranea as a model system to study the role of the gata456 family of pioneer factors in gut cell differentiation during both regeneration and maintenance of the digestive system. Our findings reveal the presence of two members of the gata456 family in the Schmidtea mediterranea genome; Smed-gata456-1 and Smed-gata456-2. Our results show that Smed-gata456-1 is the only ortholog with a gut cell-related function. Smed-gata456-1 is essential for the differentiation of precursors into intestinal cells and for the survival of these differentiated cells, indicating a key role in gut regeneration and maintenance. Furthermore, tissues other than the gut appear normal following Smed-gata456-1 RNA interference (RNAi), indicating a gut-specific function. Importantly, different neoblast subtypes are unaffected by Smed-gata456-1(RNAi), suggesting that 1) Smed-gata456-1 is involved in the differentiation and maintenance, but not in the early determination, of gut cells; and 2) that the stem cell compartment is not dependent on a functional gut.

Immunohistochemical study of the neural development transcription factors (TTF1, ASCL1 and BRN2) in neuroendocrine prostate tumours.

PubMed

Rodríguez-Zarco, E; Vallejo-Benítez, A; Umbría-Jiménez, S; Pereira-Gallardo, S; Pabón-Carrasco, S; Azueta, A; González-Cámpora, R; Espinal, P S; García-Escudero, A

2017-10-01

Prostatic small-cell neuroendocrine carcinoma is an uncommon malignancy that constitutes 0.5-1% of all prostate malignancies. The median cancer-specific survival of patients with prostatic small-cell neuroendocrine carcinoma is 19 months, and 60.5% of the patients have metastatic disease. Neural development transcription factors are molecules involved in the organogenesis of the central nervous system and of neuroendocrine precursors of various tissues, including the suprarenal gland, thyroid glands, lungs and prostate. We present 3 cases of this uncommon condition, applying the new World Health Organisation criteria. We conducted studies through haematoxylin and eosin staining and analysed the expression of the neural development transcription factors achaete-scute homolog like 1, thyroid transcription factor 1 and the class III/IV POU transcription factors, as a new research line in the carcinogenesis of prostatic neuroendocrine tumours. In case 1, there was no TTF1 immunoexpression. Cases 2 and 3 had positive immunostaining for ASCL1, and Case 1 had negative immunostaining. BRN2 immunostaining was negative in case 1 and positive in cases 2 and 3. The World Health Organisation does not recognise any molecular or genetic marker with prognostic value. ASCL-1 is related to the NOTCH and WNT signalling pathways. ASCL-1, TTF1 and BRN2 could be used for early diagnosis and as prognostic factors and therapeutic targets. Copyright © 2017 AEU. All rights reserved.

Exposure to transforming growth factor-β1 after basic fibroblast growth factor promotes the fibroblastic differentiation of human periodontal ligament stem/progenitor cell lines.

PubMed

Kono, Kiyomi; Maeda, Hidefumi; Fujii, Shinsuke; Tomokiyo, Atsushi; Yamamoto, Naohide; Wada, Naohisa; Monnouchi, Satoshi; Teramatsu, Yoko; Hamano, Sayuri; Koori, Katsuaki; Akamine, Akifumi

2013-05-01

Basic fibroblast growth factor (bFGF) is a cytokine that promotes the regeneration of the periodontium, the specialized tissues supporting the teeth. bFGF, does not, however, induce the synthesis of smooth muscle actin alpha 2 (ACTA2), type I collagen (COL1), or COL3, which are principal molecules in periodontal ligament (PDL) tissue, a component of the periodontium. We have suggested the feasibility of using transforming growth factor-β1 (TGFβ1) to induce fibroblastic differentiation of PDL stem/progenitor cells (PDLSCs). Here, we investigated the effect of the subsequent application of TGFβ1 after bFGF (bFGF/TGFβ1) on the differentiation of PDLSCs into fibroblastic cells. We first confirmed the expression of bFGF and TGFβ1 in rat PDL tissue and primary human PDL cells. Receptors for both bFGF and TGFβ1 were expressed in the human PDLSC lines 1-11 and 1-17. Exposure to bFGF for 2 days promoted vascular endothelial growth factor gene and protein expression in both cell lines and down-regulated the expression of ACTA2, COL1, and COL3 mRNA in both cell lines and the gene fibrillin 1 (FBN1) in cell line 1-11 alone. Furthermore, bFGF stimulated cell proliferation of these cell lines and significantly increased the number of cells in phase G2/M in the cell lines. Exposure to TGFβ1 for 2 days induced gene expression of ACTA2 and COL1 in both cell lines and FBN1 in cell line 1-11 alone. BFGF/TGFβ1 treatment significantly up-regulated ACTA2, COL1, and FBN1 expression as compared with the group treated with bFGF alone or the untreated control. This method might thus be useful for accelerating the generation and regeneration of functional periodontium.

Dexamethasone inhibits inflammatory response via down regulation of AP-1 transcription factor in human lung epithelial cells.

PubMed

Patil, Rajeshwari H; Naveen Kumar, M; Kiran Kumar, K M; Nagesh, Rashmi; Kavya, K; Babu, R L; Ramesh, Govindarajan T; Chidananda Sharma, S

2018-03-01

The production of inflammatory mediators by epithelial cells in inflammatory lung diseases may represent an important target for the anti-inflammatory effects of glucocorticoids. Activator protein-1 is a major activator of inflammatory genes and has been proposed as a target for inhibition by glucocorticoids. We have used human pulmonary type-II A549 cells to examine the effect of dexamethasone on the phorbol ester (PMA)/Lipopolysaccharide (LPS) induced pro-inflammatory cytokines and AP-1 factors. A549 cells were treated with and without PMA or LPS or dexamethasone and the cell viability and nitric oxide production was measured by MTT assay and Griess reagent respectively. Expression of pro-inflammatory cytokines and AP-1 factors mRNA were measured using semi quantitative RT-PCR. The PMA/LPS treated cells show significant 2-3 fold increase in the mRNA levels of pro-inflammatory cytokines (IL-1β, IL-2, IL-6, IL-8 and TNF-α), cyclo‑oxygenase-2 (COX-2) and specific AP-1 factors (c-Jun, c-Fos and Jun-D). Whereas, pretreatment of cells with dexamethasone significantly inhibited the LPS induced nitric oxide production and PMA/LPS induced mRNAs expression of above pro-inflammatory cytokines, COX-2 and AP-1 factors. Cells treated with dexamethasone alone at both the concentrations inhibit the mRNAs expression of IL-1β, IL-6 and TNF-α compared to control. Our study reveals that dexamethasone decreased the mRNAs expression of c-Jun and c-Fos available for AP-1 formation suggested that AP-1 is the probable key transcription factor involved in the anti-inflammatory activity of dexamethasone. This may be an important molecular mechanism of steroid action in asthma and other chronic inflammatory lung diseases which may be useful for treatment of lung inflammatory diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Prognostic impact of programmed cell death 1 ligand 1 expression in human leukocyte antigen class I-positive hepatocellular carcinoma after curative hepatectomy.

PubMed

Umemoto, Yuichiroh; Okano, Shinji; Matsumoto, Yoshihiro; Nakagawara, Hidekazu; Matono, Rumi; Yoshiya, Shohei; Yamashita, Yo-Ichi; Yoshizumi, Tomoharu; Ikegami, Toru; Soejima, Yuji; Harada, Mamoru; Aishima, Shinichi; Oda, Yoshinao; Shirabe, Ken; Maehara, Yoshihiko

2015-01-01

Hepatocellular carcinoma (HCC) is one of the most common solid tumors worldwide. Surgery is potentially curative, but high recurrence rates worsen patient prognosis. The interaction between the proteins programmed cell death 1 (PD-1) and programmed cell death 1 ligand 1 (PD-L1) is an important immune checkpoint. The significance of PD-L1 expression and human leukocyte antigen class I (HLA class I), recognized by CD8 T cells, in the prognosis of patients with HCC remains to be determined. We assessed the levels of PD-L1 and HLA class I expression on HCC samples from 80 patients who had undergone hepatectomy at our institution, and evaluated the correlations between PD-L1 and HLA class I expression and patient prognosis. High HLA class I expression was correlated with significantly better recurrence-free survival (RFS), but not overall survival (OS). Multivariate analysis showed that high HLA class I expression was an independent predictor of improved RFS. Low expression of PD-L1 on HCC tended to predict better OS, but the difference was not statistically significant. PD-L1 expression on HCC correlated with the number of CD163-positive macrophages and HLA class I expression with CD3-positive cell infiltration. Univariable and multivariable analyses showed that combined PD-L1 low/HLA class I high expression on HCCs was prognostic for improved OS and RFS. PD-L1 status may be a good predictor of prognosis in HCC patients with high HLA class I expression. Novel therapies targeting the PD-L1/PD-1 pathway may improve the prognosis of patients with HCC.

Transcription factor E3 protein-positive perivascular epithelioid cell tumor of the appendix presenting as acute appendicitis: a case report and review of the literature.

PubMed

Matkowskyj, Kristina A; Rao, M Sambasiva; Raparia, Kirtee

2013-03-01

Perivascular epithelioid cell tumors (PEComas) are a group of mesenchymal tumors that coexpress melanocytic and smooth muscle markers; their exact origin remains unknown. This group includes renal angiomyolipoma, clear cell sugar tumor, and lymphangioleiomyomatosis, although the term perivascular epithelioid cell tumors is currently used for lesions that exhibit a similar morphologic and immunohistochemical profile throughout the human body. Recently, a distinct subset of PEComas has been shown to harbor transcription factor E3 gene (TFE3) fusions. We report, for the first time, a unique case of TFE3-positive PEComa presenting as acute appendicitis in a 24-year-old woman. Microscopically, the tumor was composed of benign-appearing epithelioid cells with clear and eosinophilic cytoplasm, and arranged in nested and alveolar patterns. Immunohistochemical studies showed diffuse strong positivity for neuron-specific enolase, TFE3, and progesterone receptor and focal strong positivity for human melanoma black-45 (HMB-45) and melanocyte differentiation antigen (Melan-A) in the tumor cells. Although rare, PEComa should be included in the differential diagnosis of mesenchymal tumors of the appendix.

Developmental potential of muscle cell progenitors and the myogenic factor SUM-1 in the sea urchin embryo.

PubMed

Venuti, J M; Gan, L; Kozlowski, M T; Klein, W H

1993-04-01

During sea urchin development, esophageal muscle arises from secondary mesenchyme cells, descendants of the vegetal plate that delaminate from the coelomic epithelium at the end of gastrulation. In lithium-induced exogastrulae, where vegetal plate descendants evert rather than invaginate, myogenesis occurs normally, indicating that myocyte progenitors do not have to be near the future stomodeum for differentiation to occur. Vegetal plate descendants isolated along with the extracellular matrix at different times during gastrulation produce differentiated myocytes in culture as monitored by staining with a myosin heavy chain antibody. Vegetal isolates prepared at mid-gastrulation or later consistently produce differentiated myocytes whose form and position resembled their counterparts in the intact embryo, whereas vegetal isolates prepared a few hours earlier while capable of gut differentiation, as evidenced by the de novo synthesis of the endodermal surface marker Endo 1, did not produce differentiated myocytes. These results suggest that sometime after early gastrulation, a subset of secondary mesenchyme cells are competent to differentiate into muscle cells. RNase protection assays showed that the accumulation of sea urchin myogenic factor (SUM-1) mRNA is likely to be coincident with the earliest demonstrable commitment of myogenic precursors. Premature expression of SUM-1 coding sequences in mesenchyme blastulae resulted in the activation of muscle-specific enhancer elements, demonstrating that SUM-1 can function precociously in the early embryo. However, SUM-1 expressed in this manner did not activate the endogenous MHC gene, nor induce premature or ectopic production of muscle cells.

Hyperproliferation of PKD1 cystic cells is induced by insulin-like growth factor-1 activation of the Ras/Raf signalling system.

PubMed

Parker, E; Newby, L J; Sharpe, C C; Rossetti, S; Streets, A J; Harris, P C; O'Hare, M J; Ong, A C M

2007-07-01

Autosomal dominant polycystic kidney disease (ADPKD) largely results from mutations in the PKD1 gene leading to hyperproliferation of renal tubular epithelial cells and consequent cyst formation. Rodent models of PKD suggest that the multifunctional hormone insulin-like growth factor-1 (IGF-1) could play a pathogenic role in renal cyst formation. In order to test this possibility, conditionally immortalized renal epithelial cells were prepared from normal individuals and from ADPKD patients with known germline mutations in PKD1. All patient cell lines had a decreased or absence of polycystin-1 but not polycystin-2. These cells had an increased sensitivity to IGF-1 and to cyclic AMP, which required phosphatidylinositol-3 (PI3)-kinase and the mitogen-activated protein kinase, extracellular signal-regulated protein kinase (ERK) for enhanced growth. Inhibition of Ras or Raf abolished the stimulated cell proliferation. Our results suggest that haploinsufficiency of polycystin-1 lowers the activation threshold of the Ras/Raf signalling system leading to growth factor-induced hyperproliferation. Inhibition of Ras or Raf activity may be a therapeutic option for decreasing tubular cell proliferation in ADPKD.

The grape berry-specific basic helix-loop-helix transcription factor VvCEB1 affects cell size.

PubMed

Nicolas, Philippe; Lecourieux, David; Gomès, Eric; Delrot, Serge; Lecourieux, Fatma

2013-02-01

The development of fleshy fruits involves complex physiological and biochemical changes. After fertilization, fruit growth usually begins with cell division, continues with both cell division and expansion, allowing fruit set to occur, and ends with cell expansion only. In spite of the economical importance of grapevine, the molecular mechanisms controlling berry growth are not fully understood. The present work identified and characterized Vitis vinifera cell elongation bHLH protein (VvCEB1), a basic helix-loop-helix (bHLH) transcription factor controlling cell expansion in grape. VvCEB1 was expressed specifically in berry-expanding tissues with a maximum around veraison. The study of VvCEB1 promoter activity in tomato confirmed its specific fruit expression during the expansion phase. Overexpression of VvCEB1 in grape embryos showed that this protein stimulates cell expansion and affects the expression of genes involved in cell expansion, including genes of auxin metabolism and signalling. Taken together, these data show that VvCEB1 is a fruit-specific bHLH transcription factor involved in grape berry development.

The grape berry-specific basic helix–loop–helix transcription factor VvCEB1 affects cell size

PubMed Central

Lecourieux, Fatma

2013-01-01

The development of fleshy fruits involves complex physiological and biochemical changes. After fertilization, fruit growth usually begins with cell division, continues with both cell division and expansion, allowing fruit set to occur, and ends with cell expansion only. In spite of the economical importance of grapevine, the molecular mechanisms controlling berry growth are not fully understood. The present work identified and characterized Vitis vinifera cell elongation bHLH protein (VvCEB1), a basic helix–loop–helix (bHLH) transcription factor controlling cell expansion in grape. VvCEB1 was expressed specifically in berry-expanding tissues with a maximum around veraison. The study of VvCEB1 promoter activity in tomato confirmed its specific fruit expression during the expansion phase. Overexpression of VvCEB1 in grape embryos showed that this protein stimulates cell expansion and affects the expression of genes involved in cell expansion, including genes of auxin metabolism and signalling. Taken together, these data show that VvCEB1 is a fruit-specific bHLH transcription factor involved in grape berry development. PMID:23314819

Differential roles of vascular endothelial growth factor receptors 1 and 2 in dendritic cell differentiation.

PubMed

Dikov, Mikhail M; Ohm, Joyce E; Ray, Neelanjan; Tchekneva, Elena E; Burlison, Jared; Moghanaki, Drew; Nadaf, Sorena; Carbone, David P

2005-01-01

Impaired Ag-presenting function in dendritic cells (DCs) due to abnormal differentiation is an important mechanism of tumor escape from immune control. A major role for vascular endothelial growth factor (VEGF) and its receptors, VEGFR1/Flt-1 and VEGFR2/KDR/Flk-1, has been documented in hemopoietic development. To study the roles of each of these receptors in DC differentiation, we used an in vitro system of myeloid DC differentiation from murine embryonic stem cells. Exposure of wild-type, VEGFR1(-/-), or VEGFR2(-/-) embryonic stem cells to exogenous VEGF or the VEGFR1-specific ligand, placental growth factor, revealed distinct roles of VEGF receptors. VEGFR1 is the primary mediator of the VEGF inhibition of DC maturation, whereas VEGFR2 tyrosine kinase signaling is essential for early hemopoietic differentiation, but only marginally affects final DC maturation. SU5416, a VEGF receptor tyrosine kinase inhibitor, only partially rescued the mature DC phenotype in the presence of VEGF, suggesting the involvement of both tyrosine kinase-dependent and independent inhibitory mechanisms. VEGFR1 signaling was sufficient for blocking NF-kappaB activation in bone marrow hemopoietic progenitor cells. VEGF and placental growth factor affect the early stages of myeloid/DC differentiation. The data suggest that therapeutic strategies attempting to reverse the immunosuppressive effects of VEGF in cancer patients might be more effective if they specifically targeted VEGFR1.

Cell wall integrity modulates RHO1 activity via the exchange factor ROM2.

PubMed Central

Bickle, M; Delley, P A; Schmidt, A; Hall, M N

1998-01-01

The essential phosphatidylinositol kinase homologue TOR2 of Saccharomyces cerevisiae controls the actin cytoskeleton by activating a GTPase switch consisting of RHO1 (GTPase), ROM2 (GEF) and SAC7 (GAP). We have identified two mutations, rot1-1 and rot2-1, that suppress the loss of TOR2 and are synthetic-lethal. The wild-type ROT1 and ROT2 genes and a multicopy suppressor, BIG1, were isolated by their ability to rescue the rot1-1 rot2-1 double mutant. ROT2 encodes glucosidase II, and ROT1 and BIG1 encode novel proteins. We present evidence that cell wall defects activate RHO1. First, rot1, rot2, big1, cwh41, gas1 and fks1 mutations all confer cell wall defects and suppress tor2(ts). Second, destabilizing the cell wall by supplementing the growth medium with 0.005% SDS also suppresses a tor2(ts) mutation. Third, disturbing the cell wall with SDS or a rot1, rot2, big1, cwh41, gas1 or fks1 mutation increases GDP/GTP exchange activity toward RHO1. These results suggest that cell wall defects suppress a tor2 mutation by activating RHO1 independently of TOR2, thereby inducing TOR2-independent polarization of the actin cytoskeleton and cell wall synthesis. Activation of RHO1, a subunit of the cell wall synthesis enzyme glucan synthase, by a cell wall alteration would ensure that cell wall synthesis occurs only when and where needed. The mechanism of RHO1 activation by a cell wall alteration is via the exchange factor ROM2 and could be analogous to signalling by integrin receptors in mammalian cells. PMID:9545237

Cloning of a human hepatocyte growth factor/scatter factor transcription variant from a gastric cancer cell line HSC-39.

PubMed

Yokozaki, H; Tahara, H; Oue, N; Tahara, E

2000-01-01

A new transcription variant of hepatocyte growth factor/scatter factor (HGF/SF) was cloned from human gastric cancer cell line HSC-39. Northern blot analysis of eight human gastric cancer cell lines (TMK-1, MKN-1, MKN-7, MKN-28, MKN-45, MKN-74, KATO-III and HSC-39) demonstrated that HSC-39 cells expressed a 1.3 kb abnormal HGF/SF transcript. Screening of 1 x 10(6) colonies of cDNA library from HSC-39 constructed in pAP3neo mammalian expression vector selected four positive clones containing HGF/SF transcript. Among them, two contained a 1.3 kbp insert detecting the identical transcript to that obtained with HGF/SF probe by Northern blotting. Deoxynucleotide sequencing of the 1.3 kbp insert revealed that it was composed of a part of HGF/SF cDNA from exon 14 to exon 18, corresponding to the whole sequence of HGF/SF light chain, with 5' 75 nucleotides unrelated to any sequence involved in HGF/SF.