Pancreatic cancer circulating tumour cells express a cell motility gene signature that predicts survival after surgery

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Sergeant, Gregory; Eijsden, Rudy van; Roskams, Tania; Van Duppen, Victor; Topal, Baki

2012-01-01

Most cancer deaths are caused by metastases, resulting from circulating tumor cells (CTC) that detach from the primary cancer and survive in distant organs. The aim of the present study was to develop a CTC gene signature and to assess its prognostic relevance after surgery for pancreatic ductal adenocarcinoma (PDAC). Negative depletion fluorescence activated cell sorting (FACS) was developed and validated with spiking experiments using cancer cell lines in whole human blood samples. This FACS-based method was used to enrich for CTC from the blood of 10 patients who underwent surgery for PDAC. Total RNA was isolated from 4 subgroup samples, i.e. CTC, haematological cells (G), original tumour (T), and non-tumoural pancreatic control tissue (P). After RNA quality control, samples of 6 patients were eligible for further analysis. Whole genome microarray analysis was performed after double linear amplification of RNA. ‘Ingenuity Pathway Analysis’ software and AmiGO were used for functional data analyses. A CTC gene signature was developed and validated with the nCounter system on expression data of 78 primary PDAC using Cox regression analysis for disease-free (DFS) and overall survival (OS). Using stringent statistical analysis, we retained 8,152 genes to compare expression profiles of CTC vs. other subgroups, and found 1,059 genes to be differentially expressed. The pathway with the highest expression ratio in CTC was p38 mitogen-activated protein kinase (p38 MAPK) signaling, known to be involved in cancer cell migration. In the p38 MAPK pathway, TGF-β1, cPLA2, and MAX were significantly upregulated. In addition, 9 other genes associated with both p38 MAPK signaling and cell motility were overexpressed in CTC. High co-expression of TGF-β1 and our cell motility panel (≥ 4 out of 9 genes for DFS and ≥ 6 out of 9 genes for OS) in primary PDAC was identified as an independent predictor of DFS (p=0.041, HR (95% CI) = 1.885 (1.025 – 3.559)) and OS (p=0.047, HR

Inhibitor of Apoptosis (IAP) survivin is indispensable for survival of HER2 gene-amplified breast cancer cells with primary resistance to HER1/2-targeted therapies

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Oliveras-Ferraros, Cristina; Vazquez-Martin, Alejandro; Cufi, Silvia; Torres-Garcia, Violeta Zenobia; Sauri-Nadal, Tamara; Barco, Sonia Del; Lopez-Bonet, Eugeni; Brunet, Joan; Martin-Castillo, Begona; Menendez, Javier A.

2011-01-01

Highlights: → Intrinsic trastuzumab resistance occurs in ∼70% of metastatic HER2 + breast carcinomas (BC). → Approximately 15% of early HER2 + BC relapse in spite of treatment with trastuzumab-based therapies. → HER2-independent downstream pro-survival pathways might underlie trastuzumab refractoriness. → Survivin is indispensable for proliferation and survival of HER2 + BC unresponsive to HER2-targeted therapies ab initio. → Survivin antagonists may clinically circumvent the occurrence of de novo resistance to HER2-directed drugs. -- Abstract: Primary resistance of HER2 gene-amplified breast carcinomas (BC) to HER-targeted therapies can be explained in terms of overactive HER2-independent downstream pro-survival pathways. We here confirm that constitutive overexpression of Inhibitor of Apoptosis (IAP) survivin is indispensable for survival of HER2-positive BC cells with intrinsic cross-resistance to multiple HER1/2 inhibitors. The IC 50 values for the HER1/2 Tyrosine Kinase Inhibitors (TKIs) gefitinib, erlotinib and lapatinib were up to 40-fold higher in trastuzumab-unresponsive JIMT-1 cells than in trastuzumab-naive SKBR3 cells. ELISA-based and immunoblotting assays demonstrated that trastuzumab-refractory JIMT-1 cells constitutively expressed ∼4 times more survivin protein than trastuzumab-responsive SKBR3 cells. In response to trastuzumab, JIMT-1 cells accumulated ∼10 times more survivin than SKBR3 cells. HER1/2 TKIs failed to down-regulate survivin expression in JIMT-1 cells whereas equimolar doses of HER1/HER2 TKIs drastically depleted survivin protein in SKBR3 cells. ELISA-based detection of histone-associated DNA fragments confirmed that trastuzumab-refractory JIMT-1 cells were intrinsically protected against the apoptotic effects of HER1/2 TKIs. Of note, when we knocked-down survivin expression using siRNA and then added trastuzumab, cell proliferation and colony formation were completely suppressed in JIMT-1 cells. Our current findings may

Inhibitor of Apoptosis (IAP) survivin is indispensable for survival of HER2 gene-amplified breast cancer cells with primary resistance to HER1/2-targeted therapies

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Oliveras-Ferraros, Cristina; Vazquez-Martin, Alejandro; Cufi, Silvia; Torres-Garcia, Violeta Zenobia [Unit of Translational Research, Catalan Institute of Oncology-Girona, Avenida de Francia S/N, E-17007 Girona, Catalonia (Spain); Girona Biomedical Research Institute, Avenida de Francia S/N, E-17007 Girona, Catalonia (Spain); Sauri-Nadal, Tamara; Barco, Sonia Del [Girona Biomedical Research Institute, Avenida de Francia S/N, E-17007 Girona, Catalonia (Spain); Medical Oncology, Catalan Institute of Oncology-Girona, Avenida de Francia S/N, E-17007 Girona, Catalonia (Spain); Lopez-Bonet, Eugeni [Girona Biomedical Research Institute, Avenida de Francia S/N, E-17007 Girona, Catalonia (Spain); Department of Anatomical Pathology, Dr. Josep Trueta University Hospital, Avenida de Francia S/N, E-17007 Girona, Catalonia (Spain); Brunet, Joan [Girona Biomedical Research Institute, Avenida de Francia S/N, E-17007 Girona, Catalonia (Spain); Medical Oncology, Catalan Institute of Oncology-Girona, Avenida de Francia S/N, E-17007 Girona, Catalonia (Spain); Martin-Castillo, Begona [Girona Biomedical Research Institute, Avenida de Francia S/N, E-17007 Girona, Catalonia (Spain); Unit of Clinical Research, Catalan Institute of Oncology-Girona, Avenida de Francia S/N, E-17007 Girona, Catalonia (Spain); Menendez, Javier A., E-mail: jmenendez@idibgi.org [Unit of Translational Research, Catalan Institute of Oncology-Girona, Avenida de Francia S/N, E-17007 Girona, Catalonia (Spain); Girona Biomedical Research Institute, Avenida de Francia S/N, E-17007 Girona, Catalonia (Spain)

2011-04-08

Highlights: {yields} Intrinsic trastuzumab resistance occurs in {approx}70% of metastatic HER2 + breast carcinomas (BC). {yields} Approximately 15% of early HER2 + BC relapse in spite of treatment with trastuzumab-based therapies. {yields} HER2-independent downstream pro-survival pathways might underlie trastuzumab refractoriness. {yields} Survivin is indispensable for proliferation and survival of HER2 + BC unresponsive to HER2-targeted therapies ab initio. {yields} Survivin antagonists may clinically circumvent the occurrence of de novo resistance to HER2-directed drugs. -- Abstract: Primary resistance of HER2 gene-amplified breast carcinomas (BC) to HER-targeted therapies can be explained in terms of overactive HER2-independent downstream pro-survival pathways. We here confirm that constitutive overexpression of Inhibitor of Apoptosis (IAP) survivin is indispensable for survival of HER2-positive BC cells with intrinsic cross-resistance to multiple HER1/2 inhibitors. The IC{sub 50} values for the HER1/2 Tyrosine Kinase Inhibitors (TKIs) gefitinib, erlotinib and lapatinib were up to 40-fold higher in trastuzumab-unresponsive JIMT-1 cells than in trastuzumab-naive SKBR3 cells. ELISA-based and immunoblotting assays demonstrated that trastuzumab-refractory JIMT-1 cells constitutively expressed {approx}4 times more survivin protein than trastuzumab-responsive SKBR3 cells. In response to trastuzumab, JIMT-1 cells accumulated {approx}10 times more survivin than SKBR3 cells. HER1/2 TKIs failed to down-regulate survivin expression in JIMT-1 cells whereas equimolar doses of HER1/HER2 TKIs drastically depleted survivin protein in SKBR3 cells. ELISA-based detection of histone-associated DNA fragments confirmed that trastuzumab-refractory JIMT-1 cells were intrinsically protected against the apoptotic effects of HER1/2 TKIs. Of note, when we knocked-down survivin expression using siRNA and then added trastuzumab, cell proliferation and colony formation were completely

ROCK inhibitor Y-27632 enhances the survivability of dissociated buffalo (Bubalus bubalis) embryonic stem cell-like cells.

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Sharma, Ruchi; George, Aman; Chauhan, Manmohan S; Singla, Suresh; Manik, Radhey S; Palta, Prabhat

2013-01-01

This study investigated the effects of supplementation of culture medium with 10 μM Y-27632, a specific inhibitor of Rho kinase activity, for 6 days on self-renewal of buffalo embryonic stem (ES) cell-like cells at Passage 50-80. Y-27632 increased mean colony area (P<0.05) although it did not improve their survival. It decreased OCT4 expression (P<0.05), increased NANOG expression (P<0.05), but had no effect on SOX2 expression. It also increased expression of anti-apoptotic gene BCL-2 (P<0.05) and decreased that of pro-apoptotic genes BAX and BID (P<0.05). It increased plating efficiency of single-cell suspensions of ES cells (P<0.05). Following vitrification, the presence of Y-27632 in the vitrification solution or thawing medium or both did not improve ES cell colony survival. However, following seeding of clumps of ES cells transfected with pAcGFP1N1 carrying green fluorescent protein (GFP), Y-27632 increased colony formation rate (P<0.01). ES cell colonies that formed in all Y-27632-supplemented groups were confirmed for expression of pluripotency markers alkaline phosphatase, SSEA-4 and TRA-1-60, and for their ability to generate embryoid bodies containing cells that expressed markers of ectoderm, mesoderm and endoderm. In conclusion, Y-27632 improves survival of buffalo ES cells under unfavourable conditions such as enzymatic dissociation to single cells or antibiotic-assisted selection after transfection, without compromising their pluripotency.

Pro-inflammatory activated Kupffer cells by lipids induce hepatic NKT cells deficiency through activation-induced cell death.

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Tongfang Tang

Full Text Available BACKGROUND: Dietary lipids play an important role in the progression of non-alcoholic fatty liver disease (NAFLD through alternation of liver innate immune response. AIMS: The present study was to investigate the effect of lipid on Kupffer cells phenotype and function in vivo and in vitro. And further to investigate the impact of lipid on ability of Kupffer cell lipid antigen presentation to activate NKT cells. METHODS: Wild type male C57BL/6 mice were fed either normal or high-fat diet. Hepatic steatosis, Kupffer cell abundance, NKT cell number and cytokine gene expression were evaluated. Antigen presentation assay was performed with Kupffer cells treated with certain fatty acids in vitro and co-cultured with NKT cells. RESULTS: High-fat diet induced hepatosteatosis, significantly increased Kupffer cells and decreased hepatic NKT cells. Lipid treatment in vivo or in vitro induced increase of pro-inflammatory cytokines gene expression and toll-like receptor 4 (TLR4 expression in Kupffer cells. Kupffer cells expressed high levels of CD1d on cell surface and only presented exogenous lipid antigen to activate NKT cells. Ability of Kupffer cells to present antigen and activate NKT cells was enhanced after lipid treatment. In addition, pro-inflammatory activated Kupffer cells by lipid treatment induced hepatic NKT cells activation-induced apoptosis and necrosis. CONCLUSION: High-fat diet increase Kupffer cells number and induce their pro-inflammatory status. Pro-inflammatory activated Kupfffer cells by lipid promote hepatic NKT cell over-activation and cell death, which lead to further hepatic NKT cell deficiency in the development of NAFLD.

Pro-inflammatory activated Kupffer cells by lipids induce hepatic NKT cells deficiency through activation-induced cell death.

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Tang, Tongfang; Sui, Yongheng; Lian, Min; Li, Zhiping; Hua, Jing

2013-01-01

Dietary lipids play an important role in the progression of non-alcoholic fatty liver disease (NAFLD) through alternation of liver innate immune response. The present study was to investigate the effect of lipid on Kupffer cells phenotype and function in vivo and in vitro. And further to investigate the impact of lipid on ability of Kupffer cell lipid antigen presentation to activate NKT cells. Wild type male C57BL/6 mice were fed either normal or high-fat diet. Hepatic steatosis, Kupffer cell abundance, NKT cell number and cytokine gene expression were evaluated. Antigen presentation assay was performed with Kupffer cells treated with certain fatty acids in vitro and co-cultured with NKT cells. High-fat diet induced hepatosteatosis, significantly increased Kupffer cells and decreased hepatic NKT cells. Lipid treatment in vivo or in vitro induced increase of pro-inflammatory cytokines gene expression and toll-like receptor 4 (TLR4) expression in Kupffer cells. Kupffer cells expressed high levels of CD1d on cell surface and only presented exogenous lipid antigen to activate NKT cells. Ability of Kupffer cells to present antigen and activate NKT cells was enhanced after lipid treatment. In addition, pro-inflammatory activated Kupffer cells by lipid treatment induced hepatic NKT cells activation-induced apoptosis and necrosis. High-fat diet increase Kupffer cells number and induce their pro-inflammatory status. Pro-inflammatory activated Kupfffer cells by lipid promote hepatic NKT cell over-activation and cell death, which lead to further hepatic NKT cell deficiency in the development of NAFLD.

E2F1-Mediated Induction of NFYB Attenuates Apoptosis via Joint Regulation of a Pro-Survival Transcriptional Program.

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Xiaolei Jiang

Full Text Available The E2F1 transcription factor regulates cell proliferation and apoptosis through the control of a considerable variety of target genes. Previous work has detailed the role of other transcription factors in mediating the specificity of E2F function. Here we identify the NF-YB transcription factor as a novel direct E2F1 target. Genome-wide expression analysis of the effects of NFYB knockdown on E2F1-mediated transcription identified a large group of genes that are co-regulated by E2F1 and NFYB. We also provide evidence that knockdown of NFYB enhances E2F1-induced apoptosis, suggesting a pro-survival function of the NFYB/E2F1 joint transcriptional program. Bioinformatic analysis suggests that deregulation of these NFY-dependent E2F1 target genes might play a role in sarcomagenesis as well as drug resistance.

Epigenetic silencing of host cell defense genes enhances intracellular survival of the rickettsial pathogen Anaplasma phagocytophilum.

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Jose C Garcia-Garcia

2009-06-01

Full Text Available Intracellular bacteria have evolved mechanisms that promote survival within hostile host environments, often resulting in functional dysregulation and disease. Using the Anaplasma phagocytophilum-infected granulocyte model, we establish a link between host chromatin modifications, defense gene transcription and intracellular bacterial infection. Infection of THP-1 cells with A. phagocytophilum led to silencing of host defense gene expression. Histone deacetylase 1 (HDAC1 expression, activity and binding to the defense gene promoters significantly increased during infection, which resulted in decreased histone H3 acetylation in infected cells. HDAC1 overexpression enhanced infection, whereas pharmacologic and siRNA HDAC1 inhibition significantly decreased bacterial load. HDAC2 does not seem to be involved, since HDAC2 silencing by siRNA had no effect on A. phagocytophilum intracellular propagation. These data indicate that HDAC up-regulation and epigenetic silencing of host cell defense genes is required for A. phagocytophilum infection. Bacterial epigenetic regulation of host cell gene transcription could be a general mechanism that enhances intracellular pathogen survival while altering cell function and promoting disease.

A gene expression signature associated with survival in metastatic melanoma

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Mandruzzato, Susanna; Callegaro, Andrea; Turcatel, Gianluca; Francescato, Samuela; Montesco, Maria C; Chiarion-Sileni, Vanna; Mocellin, Simone; Rossi, Carlo R; Bicciato, Silvio; Wang, Ena; Marincola, Francesco M; Zanovello, Paola

2006-01-01

Background Current clinical and histopathological criteria used to define the prognosis of melanoma patients are inadequate for accurate prediction of clinical outcome. We investigated whether genome screening by means of high-throughput gene microarray might provide clinically useful information on patient survival. Methods Forty-three tumor tissues from 38 patients with stage III and stage IV melanoma were profiled with a 17,500 element cDNA microarray. Expression data were analyzed using significance analysis of microarrays (SAM) to identify genes associated with patient survival, and supervised principal components (SPC) to determine survival prediction. Results SAM analysis revealed a set of 80 probes, corresponding to 70 genes, associated with survival, i.e. 45 probes characterizing longer and 35 shorter survival times, respectively. These transcripts were included in a survival prediction model designed using SPC and cross-validation which allowed identifying 30 predicting probes out of the 80 associated with survival. Conclusion The longer-survival group of genes included those expressed in immune cells, both innate and acquired, confirming the interplay between immunological mechanisms and the natural history of melanoma. Genes linked to immune cells were totally lacking in the poor-survival group, which was instead associated with a number of genes related to highly proliferative and invasive tumor cells. PMID:17129373

A gene expression signature associated with survival in metastatic melanoma

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Rossi Carlo R

2006-11-01

Full Text Available Abstract Background Current clinical and histopathological criteria used to define the prognosis of melanoma patients are inadequate for accurate prediction of clinical outcome. We investigated whether genome screening by means of high-throughput gene microarray might provide clinically useful information on patient survival. Methods Forty-three tumor tissues from 38 patients with stage III and stage IV melanoma were profiled with a 17,500 element cDNA microarray. Expression data were analyzed using significance analysis of microarrays (SAM to identify genes associated with patient survival, and supervised principal components (SPC to determine survival prediction. Results SAM analysis revealed a set of 80 probes, corresponding to 70 genes, associated with survival, i.e. 45 probes characterizing longer and 35 shorter survival times, respectively. These transcripts were included in a survival prediction model designed using SPC and cross-validation which allowed identifying 30 predicting probes out of the 80 associated with survival. Conclusion The longer-survival group of genes included those expressed in immune cells, both innate and acquired, confirming the interplay between immunological mechanisms and the natural history of melanoma. Genes linked to immune cells were totally lacking in the poor-survival group, which was instead associated with a number of genes related to highly proliferative and invasive tumor cells.

Cisplatin-induced apoptosis inhibits autophagy, which acts as a pro-survival mechanism in human melanoma cells.

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Del Bello, Barbara; Toscano, Marzia; Moretti, Daniele; Maellaro, Emilia

2013-01-01

The interplay between a non-lethal autophagic response and apoptotic cell death is still a matter of debate in cancer cell biology. In the present study performed on human melanoma cells, we investigate the role of basal or stimulated autophagy in cisplatin-induced cytotoxicity, as well as the contribution of cisplatin-induced activation of caspases 3/7 and conventional calpains. The results show that, while down-regulating Beclin-1, Atg14 and LC3-II, cisplatin treatment inhibits the basal autophagic response, impairing a physiological pro-survival response. Consistently, exogenously stimulated autophagy, obtained with trehalose or calpains inhibitors (MDL-28170 and calpeptin), protects from cisplatin-induced apoptosis, and such a protection is reverted by inhibiting autophagy with 3-methyladenine or ATG5 silencing. In addition, during trehalose-stimulated autophagy, the cisplatin-induced activation of calpains is abrogated, suggesting the existence of a feedback loop between the autophagic process and calpains. On the whole, our results demonstrate that in human melanoma cells autophagy may function as a beneficial stress response, hindered by cisplatin-induced death mechanisms. In a therapeutic perspective, these findings suggest that the efficacy of cisplatin-based polychemotherapies for melanoma could be potentiated by inhibitors of autophagy.

Effect of suicidal gene combined with irradiation on esophageal carcinoma cell line

International Nuclear Information System (INIS)

Pan Jianji; Wang Jiezhong; Zheng Tianrong; Zheng Qiuhong

2005-01-01

Objective: As generally known that non-cytotoxic pro-drag can be transformed into cytotoxic drug by suicide gene, this work is to investigate the effect of Coli cytosine deaminase/5-fluorocytosine suicide gene (CD/5-FC) used alone or combined with irradiation in esophageal carcinoma cell line(EC). Methods: CD gene was amplified from Coli DNA genome library with PCR technique, with the eukaryotic vector pcDNA3.1-CD then constructed. ECl09 cells were transfected with pcDNA3.1-CD by liposome method. The cytotoxic effect, bystander effect and radiosensitization effect of CD/5-FC in ECl09 was analyzed. Results: The transfection of CD gene into ECl09 and its transcription was confirmed by RT-PCR method. In vitro, 5-FC showed significantly cytotoxic effect on the EPC cell transfected with CD gene. After adding 5-FC , the survival rate of cultured cell containing 5 % transfect CD gene cell was 41.8 % ± 14.2% while that in the control group was 94.6 ± 4.3 %, (t=3.14, P < 0.05). The survival rate of cultured cell containing 10% transfected CD gene cell was 37.8 ± 4.4% compared to 95.6% ± 5.4% in the control group, (t=9.75, P<0.01). CD/5-FC showed significant radiosen-sitization effect, the survival fraction of CD transfected cell was much lower in 5-FC combined with irradiation, when compared with 5-FC alone and radiotherapy alone group together, (F=11.50, P < 0.01 ). When it was compared with 5-FC alone group and irradiation alone group separately, the difference was also significant( F=4.11, P < 0.05 and F10.53, P < 0.01, respectively). Conclusions: Suicide gene CD/5-FC shows conspicuous by-stander effect and radiosensitization effect. (authors)

MicroRNA-184 inhibits neuroblastoma cell survival through targeting the serine/threonine kinase AKT2

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Murphy Derek M

2010-04-01

Full Text Available Abstract Background Neuroblastoma is a paediatric cancer of the sympathetic nervous system. The single most important genetic indicator of poor clinical outcome is amplification of the MYCN transcription factor. One of many down-stream MYCN targets is miR-184, which is either directly or indirectly repressed by this transcription factor, possibly due to its pro-apoptotic effects when ectopically over-expressed in neuroblastoma cells. The purpose of this study was to elucidate the molecular mechanism by which miR-184 conveys pro-apoptotic effects. Results We demonstrate that the knock-down of endogenous miR-184 has the opposite effect of ectopic up-regulation, leading to enhanced neuroblastoma cell numbers. As a mechanism of how miR-184 causes apoptosis when over-expressed, and increased cell numbers when inhibited, we demonstrate direct targeting and degradation of AKT2, a major downstream effector of the phosphatidylinositol 3-kinase (PI3K pathway, one of the most potent pro-survival pathways in cancer. The pro-apoptotic effects of miR-184 ectopic over-expression in neuroblastoma cell lines is reproduced by siRNA inhibition of AKT2, while a positive effect on cell numbers similar to that obtained by the knock-down of endogenous miR-184 can be achieved by ectopic up-regulation of AKT2. Moreover, co-transfection of miR-184 with an AKT2 expression vector lacking the miR-184 target site in the 3'UTR rescues cells from the pro-apoptotic effects of miR-184. Conclusions MYCN contributes to tumorigenesis, in part, by repressing miR-184, leading to increased levels of AKT2, a direct target of miR-184. Thus, two important genes with positive effects on cell growth and survival, MYCN and AKT2, can be linked into a common genetic pathway through the actions of miR-184. As an inhibitor of AKT2, miR-184 could be of potential benefit in miRNA mediated therapeutics of MYCN amplified neuroblastoma and other forms of cancer.

3'UTR Shortening Potentiates MicroRNA-Based Repression of Pro-differentiation Genes in Proliferating Human Cells.

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Yonit Hoffman

2016-02-01

Full Text Available Most mammalian genes often feature alternative polyadenylation (APA sites and hence diverse 3'UTR lengths. Proliferating cells were reported to favor APA sites that result in shorter 3'UTRs. One consequence of such shortening is escape of mRNAs from targeting by microRNAs (miRNAs whose binding sites are eliminated. Such a mechanism might provide proliferation-related genes with an expression gain during normal or cancerous proliferation. Notably, miRNA sites tend to be more active when located near both ends of the 3'UTR compared to those located more centrally. Accordingly, miRNA sites located near the center of the full 3'UTR might become more active upon 3'UTR shortening. To address this conjecture we performed 3' sequencing to determine the 3' ends of all human UTRs in several cell lines. Remarkably, we found that conserved miRNA binding sites are preferentially enriched immediately upstream to APA sites, and this enrichment is more prominent in pro-differentiation/anti-proliferative genes. Binding sites of the miR17-92 cluster, upregulated in rapidly proliferating cells, are particularly enriched just upstream to APA sites, presumably conferring stronger inhibitory activity upon shortening. Thus 3'UTR shortening appears not only to enable escape from inhibition of growth promoting genes but also to potentiate repression of anti-proliferative genes.

PRAP1 is a novel executor of p53-dependent mechanisms in cell survival after DNA damage.

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Huang, B H; Zhuo, J L; Leung, C H W; Lu, G D; Liu, J J; Yap, C T; Hooi, S C

2012-12-13

p53 has a crucial role in governing cellular mechanisms in response to a broad range of genotoxic stresses. During DNA damage, p53 can either promote cell survival by activating senescence or cell-cycle arrest and DNA repair to maintain genomic integrity for cell survival or direct cells to undergo apoptosis to eliminate extensively damaged cells. The ability of p53 to execute these two opposing cell fates depends on distinct signaling pathways downstream of p53. In this study, we showed that under DNA damage conditions induced by chemotherapeutic drugs, gamma irradiation and hydrogen peroxide, p53 upregulates a novel protein, proline-rich acidic protein 1 (PRAP1). We identified functional p53-response elements within intron 1 of PRAP1 gene and showed that these regions interact directly with p53 using ChIP assays, indicating that PRAP1 is a novel p53 target gene. The induction of PRAP1 expression by p53 may promote resistance of cancer cells to chemotherapeutic drugs such as 5-fluorouracil (5-FU), as knockdown of PRAP1 increases apoptosis in cancer cells after 5-FU treatment. PRAP1 appears to protect cells from apoptosis by inducing cell-cycle arrest, suggesting that the induction of PRAP1 expression by p53 in response to DNA-damaging agents contributes to cancer cell survival. Our findings provide a greater insight into the mechanisms underlying the pro-survival role of p53 in response to cytotoxic treatments.

No evidence that genetic variation in the myeloid-derived suppressor cell pathway influences ovarian cancer survival

DEFF Research Database (Denmark)

Sucheston-Campbell, Lara E; Cannioto, Rikki; Clay, Alyssa I

2017-01-01

BACKGROUND: The precise mechanism by which the immune system is adversely affected in cancer patients remains poorly understood, but the accumulation of immune suppressive/pro-tumorigenic myeloid-derived suppressor cells (MDSCs) is thought to be one prominent mechanism contributing to immunologic...... tolerance of malignant cells in epithelial ovarian cancer (EOC). To this end, we hypothesized genetic variation in MDSC pathway genes would be associated with survival after EOC diagnoses. METHODS: We measured the hazard of death due to EOC within 10 years of diagnosis, overall and by invasive subtype...

Isthmin exerts pro-survival and death-promoting effect on endothelial cells through alphavbeta5 integrin depending on its physical state.

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Zhang, Y; Chen, M; Venugopal, S; Zhou, Y; Xiang, W; Li, Y-H; Lin, Q; Kini, R M; Chong, Y-S; Ge, R

2011-05-05

Isthmin (ISM) is a 60 kDa secreted-angiogenesis inhibitor that suppresses tumor growth in mouse and disrupts vessel patterning in zebrafish embryos. It selectively binds to alphavbeta5 (αvβ5) integrin on the surface of endothelial cells (ECs), but the mechanism of its antiangiogenic action remains unknown. In this work, we establish that soluble ISM suppresses in vitro angiogenesis and induces EC apoptosis by interacting with its cell surface receptor αvβ5 integrin through a novel 'RKD' motif localized within its adhesion-associated domain in MUC4 and other proteins domain. ISM induces EC apoptosis through integrin-mediated death (IMD) by direct recruitment and activation of caspase-8 without causing anoikis. On the other hand, immobilized ISM loses its antiangiogenic function and instead promotes EC adhesion, survival and migration through αvβ5 integrin by activating focal adhesion kinase (FAK). ISM unexpectedly has both a pro-survival and death-promoting effect on ECs depending on its physical state. This dual function of a single antiangiogenic protein may impact its antiangiogenic efficacy in vivo.

Promotion of Metastasis-associated Gene Expression in Survived PANC-1 Cells Following Trichostatin A Treatment.

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Chen, Zongjing; Yang, Yunxiu; Liu, Biao; Wang, Benquan; Sun, Meng; Zhang, Ling; Chen, Bicheng; You, Heyi; Zhou, Mengtao

2015-01-01

Histone deacetylase inhibitors represent a promising class of potential anticancer agents for the treatment of human malignancies. In this study, the effects of trichostatin A (TSA) on apoptosis, metastasis-associated gene expression, and activation of the Notch pathway in human pancreatic cancer cell lines were investigated. After treatment with TSA, cell viability and apoptosis were evaluated using the MTT [3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide] assay, Hoechst 33258 staining, and flow cytometry. Moreover, RT-PCR and western blot analyses were performed to measure the expression levels of apoptosis-associated genes (Bcl-2, Bax, and caspase-3), metastasis-associated genes (E-cadherin, vimentin, and matrix metalloproteinases), and Notch pathway activation (Notch intracellular domain, NICD). The levels of matrix metalloproteinase 2 and NICD were also semi-quantified by immunoassay. Following treatment with TSA for 24 h, PANC-1, SW1990, and MIATACA-2 cells exhibited cell death. The MTT assay revealed that TSA significantly decreased cell viability in a dose-dependent manner in PANC-1 cells. The Hoechst 33258 staining and flow cytometry results evidenced a significant increase in PANC-1 cell apoptosis following TSA treatment. The expression levels of Bax and caspase-3 were increased significantly, whereas Bcl-2 was down-regulated after TSA treatment. In the PANC-1 cells that survived after TSA treatment, the expression levels of vimentin, E-cadherin, and MMP genes were altered by the promotion of potential metastasis and increased expression of NICD. TSA can induce apoptosis of pancreatic cancer cells. In addition, the up-regulation of metastasis-related genes and the activation of the Notch pathway in the survived PANC-1 cells may be associated with a too-low level of TSA or resistance to TSA.

In vitro non-viral murine pro-neurotrophin 3 gene transfer into rat bone marrow stromal cells.

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Darabi, Shahram; Tiraihi, Taki; Delshad, AliReza; Sadeghizadeh, Majid; Khalil, Wisam; Taheri, Taher

2017-04-15

Neurotrophin 3 (NT-3) is an important factor for promoting prenatal neural development, as well as regeneration, axogenesis and plasticity in postnatal life. Therapy with NT-3 was reported to improve the condition of patients suffering from degenerative diseases and traumatic injuries, however, the disadvantage of NT-3 protein delivery is its short half-life, thus our alternative approach is the use of NT-3 gene therapy. In this study, the bone marrow stromal cells (BMSCs) were isolated from adult rats, cultured for 4 passages and transfected with either pEGFP-N1 or a constructed vector containing murine proNT-3 (pSecTag2/HygroB-murine proNT-3) using Lipofectamine 2000 followed by Hygromycin B (200mg/kg). The transfection efficiency of the transiently transfected BMSCs was evaluated using the green fluorescence protein containing vector (pEGFP-N1). A quantitative evaluation of the NT-3 expression of mRNA using real time qRT-PCR shows that there was double fold increase in NT-3 gene expression compared with non-transfected BMSCs, also, the culture supernatant yielded double fold increase in NT-3 using ELISA technique, the data were supported by immunoblotting technique. This suggests that the use of this transfection technique can be useful for gene therapy in different neurological disorders with neurodegenerative or traumatic origins. Copyright © 2017 Elsevier B.V. All rights reserved.

The co-repressor SMRT delays DNA damage-induced caspase activation by repressing pro-apoptotic genes and modulating the dynamics of checkpoint kinase 2 activation.

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Claudio Scafoglio

Full Text Available Checkpoint kinase 2 (Chk2 is a major regulator of DNA damage response and can induce alternative cellular responses: cell cycle arrest and DNA repair or programmed cell death. Here, we report the identification of a new role of Chk2 in transcriptional regulation that also contributes to modulating the balance between survival and apoptosis following DNA damage. We found that Chk2 interacts with members of the NCoR/SMRT transcriptional co-regulator complexes and serves as a functional component of the repressor complex, being required for recruitment of SMRT on the promoter of pro-apoptotic genes upon DNA damage. Thus, the co-repressor SMRT exerts a critical protective action against genotoxic stress-induced caspase activation, repressing a functionally important cohort of pro-apoptotic genes. Amongst them, SMRT is responsible for basal repression of Wip1, a phosphatase that de-phosphorylates and inactivates Chk2, thus affecting a feedback loop responsible for licensing the correct timing of Chk2 activation and the proper execution of the DNA repair process.

Cell cycle and aging, morphogenesis, and response to stimuli genes are individualized biomarkers of glioblastoma progression and survival

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Southey Bruce R

2011-06-01

. Biological processes associated glioblastoma survival included morphogenesis, cell cycle, aging, response to stimuli, and programmed cell death. Conclusions Known biomarkers of glioblastoma survival were confirmed, and new general and clinical-dependent gene profiles were uncovered. The comparison of biomarkers across glioblastoma phases and functional analyses offered insights into the role of genes. These findings support the development of more accurate and personalized prognostic tools and gene-based therapies that improve the survival and quality of life of individuals afflicted by glioblastoma multiforme.

3’UTR Shortening Potentiates MicroRNA-Based Repression of Pro-differentiation Genes in Proliferating Human Cells

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Hoffman, Yonit; Bublik, Debora Rosa; P. Ugalde, Alejandro; Elkon, Ran; Biniashvili, Tammy; Agami, Reuven; Oren, Moshe; Pilpel, Yitzhak

2016-01-01

Most mammalian genes often feature alternative polyadenylation (APA) sites and hence diverse 3’UTR lengths. Proliferating cells were reported to favor APA sites that result in shorter 3’UTRs. One consequence of such shortening is escape of mRNAs from targeting by microRNAs (miRNAs) whose binding sites are eliminated. Such a mechanism might provide proliferation-related genes with an expression gain during normal or cancerous proliferation. Notably, miRNA sites tend to be more active when located near both ends of the 3’UTR compared to those located more centrally. Accordingly, miRNA sites located near the center of the full 3’UTR might become more active upon 3'UTR shortening. To address this conjecture we performed 3' sequencing to determine the 3' ends of all human UTRs in several cell lines. Remarkably, we found that conserved miRNA binding sites are preferentially enriched immediately upstream to APA sites, and this enrichment is more prominent in pro-differentiation/anti-proliferative genes. Binding sites of the miR17-92 cluster, upregulated in rapidly proliferating cells, are particularly enriched just upstream to APA sites, presumably conferring stronger inhibitory activity upon shortening. Thus 3’UTR shortening appears not only to enable escape from inhibition of growth promoting genes but also to potentiate repression of anti-proliferative genes. PMID:26908102

Bim and Mcl-1 exert key roles in regulating JAK2V617F cell survival

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Rubert, Joëlle; Qian, Zhiyan; Andraos, Rita; Guthy, Daniel A; Radimerski, Thomas

2011-01-01

The JAK2 V617F mutation plays a major role in the pathogenesis of myeloproliferative neoplasms and is found in the vast majority of patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia or from primary myelofibrosis. The V617F mutation is thought to provide hematopoietic stem cells and myeloid progenitors with a survival and proliferation advantage. It has previously been shown that activated JAK2 promotes cell survival by upregulating the anti-apoptotic STAT5 target gene Bcl-xL. In this study, we have investigated the role of additional apoptotic players, the pro-apoptotic protein Bim as well as the anti-apoptotic protein Mcl-1. Pharmacological inhibition of JAK2/STAT5 signaling in JAK2 V617F mutant SET-2 and MB-02 cells was used to study effects on signaling, cell proliferation and apoptosis by Western blot analysis, WST-1 proliferation assays and flow cytometry. Cells were transfected with siRNA oligos to deplete candidate pro- and anti-apoptotic proteins. Co-immunoprecipitation assays were performed to assess the impact of JAK2 inhibition on complexes of pro- and anti-apoptotic proteins. Treatment of JAK2 V617F mutant cell lines with a JAK2 inhibitor was found to trigger Bim activation. Furthermore, Bim depletion by RNAi suppressed JAK2 inhibitor-induced cell death. Bim activation following JAK2 inhibition led to enhanced sequestration of Mcl-1, besides Bcl-xL. Importantly, Mcl-1 depletion by RNAi was sufficient to compromise JAK2 V617F mutant cell viability and sensitized the cells to JAK2 inhibition. We conclude that Bim and Mcl-1 have key opposing roles in regulating JAK2 V617F cell survival and propose that inactivation of aberrant JAK2 signaling leads to changes in Bim complexes that trigger cell death. Thus, further preclinical evaluation of combinations of JAK2 inhibitors with Bcl-2 family antagonists that also tackle Mcl-1, besides Bcl-xL, is warranted to assess the therapeutic potential

The Role of Vitamin D in the Immune System as a Pro-survival Molecule.

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Chirumbolo, Salvatore; Bjørklund, Geir; Sboarina, Andrea; Vella, Antonio

2017-05-01

Vitamin D is a fascinating and attractive molecule that has gained particular attention in medicine in recent years. Its immunomodulatory and anti-inflammatory potential might resemble the activity of many nature-derived molecules (eg, flavonoids), but its role in biology was selected during a long evolutionary pathway to dampen the damaging effect of cell stress response and of the immune reaction. In this sense, this molecule can be considered an ancient hormone that serves, in its primary role, as a pro-survival agent. The goal of this review was to elucidate this topic. The article reviews current literature on the field, focusing on issues regarding the role of vitamin D in immunity. Vitamin D participates in the survival machinery used by the cell, and in particular it plays a major role in synchronizing calcium oscillatory signaling to allow cell autophagy or apoptosis during a stress response. Vitamin D should be better highlighted in its molecular action and vitamin D receptor genomics to conceive a more suited therapeutic supplementation protocol in clinics. Copyright © 2017 Elsevier HS Journals, Inc. All rights reserved.

RNA-seq methods for identifying differentially expressed gene in human pancreatic islet cells treated with pro-inflammatory cytokines.

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Li, Bo; Bi, Chang Long; Lang, Ning; Li, Yu Ze; Xu, Chao; Zhang, Ying Qi; Zhai, Ai Xia; Cheng, Zhi Feng

2014-01-01

Type 1 diabetes is a chronic autoimmune disease in which pancreatic beta cells are killed by the infiltrating immune cells as well as the cytokines released by these cells. Many studies indicate that inflammatory mediators have an essential role in this disease. In the present study, we profiled the transcriptome in human islets of langerhans under control conditions or following exposure to the pro-inflammatory cytokines based on the RNA sequencing dataset downloaded from SRA database. After filtered the low-quality ones, the RNA readers was aligned to human genome hg19 by TopHat and then assembled by Cufflinks. The expression value of each transcript was calculated and consequently differentially expressed genes were screened out. Finally, a total of 63 differentially expressed genes were identified including 60 up-regulated and three down-regulated genes. GBP5 and CXCL9 stood out as the top two most up-regulated genes in cytokines treated samples with the log2 fold change of 12.208 and 10.901, respectively. Meanwhile, PTF1A and REG3G were identified as the top two most down-regulated genes with the log2 fold change of -3.759 and -3.606, respectively. Of note, we also found 262 lncRNAs (long non-coding RNA), 177 of which were inferred as novel lncRNAs. Further in-depth follow-up analysis of the transcriptional regulation reported in this study may shed light on the specific function of these lncRNA.

Gene mutations, chromosome aberrations and survival after X-ray irradiation of cultured Chinese hamster cells at cysteamine protection

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Elisova, I.V.; Feoktistova, I.P.

1983-01-01

The culture of Chinese hamster cells (clone 431) has been used to study cysteamine action on mutagenous effect of X-rays, determined by the induction of resistance of gene mutations to 6-thioguanine and chromosomal abberations, as well as on the reproductive form of death of irradiated cells. Dose--- effect curves are obtained under conditions of irradiation with and without protector. The factor of dose alteration is 2.0 for chromosomal aberrations and cell survival, and 2.8 for gene mutations. It is sUpposed that cysteamine affects the general mechanisms, which take part in the realis zation of injuries that bring about gene mutations, chromosomal aberrations and cell lethality

HC-Pro silencing suppressor significantly alters the gene expression profile in tobacco leaves and flowers

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Lehto Kirsi

2011-04-01

Full Text Available Abstract Background RNA silencing is used in plants as a major defence mechanism against invasive nucleic acids, such as viruses. Accordingly, plant viruses have evolved to produce counter defensive RNA-silencing suppressors (RSSs. These factors interfere in various ways with the RNA silencing machinery in cells, and thereby disturb the microRNA (miRNA mediated endogene regulation and induce developmental and morphological changes in plants. In this study we have explored these effects using previously characterized transgenic tobacco plants which constitutively express (under CaMV 35S promoter the helper component-proteinase (HC-Pro derived from a potyviral genome. The transcript levels of leaves and flowers of these plants were analysed using microarray techniques (Tobacco 4 × 44 k, Agilent. Results Over expression of HC-Pro RSS induced clear phenotypic changes both in growth rate and in leaf and flower morphology of the tobacco plants. The expression of 748 and 332 genes was significantly changed in the leaves and flowers, respectively, in the HC-Pro expressing transgenic plants. Interestingly, these transcriptome alterations in the HC-Pro expressing tobacco plants were similar as those previously detected in plants infected with ssRNA-viruses. Particularly, many defense-related and hormone-responsive genes (e.g. ethylene responsive transcription factor 1, ERF1 were differentially regulated in these plants. Also the expression of several stress-related genes, and genes related to cell wall modifications, protein processing, transcriptional regulation and photosynthesis were strongly altered. Moreover, genes regulating circadian cycle and flowering time were significantly altered, which may have induced a late flowering phenotype in HC-Pro expressing plants. The results also suggest that photosynthetic oxygen evolution, sugar metabolism and energy levels were significantly changed in these transgenic plants. Transcript levels of S

Loss of a Single Mcl-1 Allele Inhibits MYC-Driven Lymphomagenesis by Sensitizing Pro-B Cells to Apoptosis

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Stephanie Grabow

2016-03-01

Full Text Available MCL-1 is critical for progenitor cell survival during emergency hematopoiesis, but its role in sustaining cells undergoing transformation and in lymphomagenesis is only poorly understood. We investigated the importance of MCL-1 in the survival of B lymphoid progenitors undergoing MYC-driven transformation and its functional interactions with pro-apoptotic BIM and PUMA and the tumor suppressor p53 in lymphoma development. Loss of one Mcl-1 allele almost abrogated MYC-driven-lymphoma development owing to a reduction in lymphoma initiating pre-B cells. Although loss of the p53 target PUMA had minor impact, loss of one p53 allele substantially accelerated lymphoma development when MCL-1 was limiting, most likely because p53 loss also causes defects in non-apoptotic tumor suppressive processes. Remarkably, loss of BIM restored the survival of lymphoma initiating cells and rate of tumor development. Thus, MCL-1 has a major role in lymphoma initiating pro-B cells to oppose BIM, which is upregulated in response to oncogenic stress.

Predicting survival in patients with metastatic kidney cancer by gene-expression profiling in the primary tumor.

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Vasselli, James R; Shih, Joanna H; Iyengar, Shuba R; Maranchie, Jodi; Riss, Joseph; Worrell, Robert; Torres-Cabala, Carlos; Tabios, Ray; Mariotti, Andra; Stearman, Robert; Merino, Maria; Walther, McClellan M; Simon, Richard; Klausner, Richard D; Linehan, W Marston

2003-06-10

To identify potential molecular determinants of tumor biology and possible clinical outcomes, global gene-expression patterns were analyzed in the primary tumors of patients with metastatic renal cell cancer by using cDNA microarrays. We used grossly dissected tumor masses that included tumor, blood vessels, connective tissue, and infiltrating immune cells to obtain a gene-expression "profile" from each primary tumor. Two patterns of gene expression were found within this uniformly staged patient population, which correlated with a significant difference in overall survival between the two patient groups. Subsets of genes most significantly associated with survival were defined, and vascular cell adhesion molecule-1 (VCAM-1) was the gene most predictive for survival. Therefore, despite the complex biological nature of metastatic cancer, basic clinical behavior as defined by survival may be determined by the gene-expression patterns expressed within the compilation of primary gross tumor cells. We conclude that survival in patients with metastatic renal cell cancer can be correlated with the expression of various genes based solely on the expression profile in the primary kidney tumor.

Perturbed microRNA Expression by Mycobacterium tuberculosis Promotes Macrophage Polarization Leading to Pro-survival Foam Cell.

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Ahluwalia, Pankaj Kumar; Pandey, Rajan Kumar; Sehajpal, Prabodh Kumar; Prajapati, Vijay Kumar

2017-01-01

Tuberculosis (TB) is one of the prevalent causes of death worldwide, with 95% of these deaths occurring in developing countries, like India. The causative agent, Mycobacterium tuberculosis (MTb) has the tenacious ability to circumvent the host's immune system for its own advantage. Macrophages are one of the phagocytic cells that are central to immunity against MTb. These are highly plastic cells dependent on the milieu and can showcase M1/M2 polarization. M1 macrophages are bactericidal in action, but M2 macrophages are anti-inflammatory in their immune response. This computational study is an effort to elucidate the role of miRNAs that influences the survival of MTb in the macrophage. To identify the miRNAs against critical transcription factors, we selected only conserved hits from TargetScan database. Further, validation of these miRNAs was achieved using four databases viz . DIANA-microT, miRDB, miRanda-mirSVR, and miRNAMap. All miRNAs were identified through a conserved seed sequence against the 3'-UTR of transcription factors. This bioinformatics study found that miR-27a and miR-27b has a putative binding site at 3'-UTR of IRF4, and miR-302c against IRF5. miR-155, miR-132, and miR-455-5p are predicted microRNAs against suppressor of cytokine signaling transcription factors. Several other microRNAs, which have an affinity for critical transcription factors, are also predicted in this study. This MTb-associated modulation of microRNAs to modify the expression of the target gene(s) plays a critical role in TB pathogenesis. Other than M1/M2 plasticity, MTb has the ability to convert macrophage into foam cells that are rich in lipids and cholesterol. We have highlighted few microRNAs which overlap between M2/foam cell continuums. miR-155, miR-33, miR-27a, and miR-27b plays a dual role in deciding macrophage polarity and its conversion to foam cells. This study shows a glimpse of microRNAs which can be modulated by MTb not only to prevent its elimination but also

Astrocytes protect glioma cells from chemotherapy and upregulate survival genes via gap junctional communication.

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Lin, Qingtang; Liu, Zhao; Ling, Feng; Xu, Geng

2016-02-01

Gliomas are the most common type of primary brain tumor. Using current standard treatment regimens, the prognosis of patients with gliomas remains poor, which is predominantly due to the resistance of glioma cells to chemotherapy. The organ microenvironment has been implicated in the pathogenesis and survival of tumor cells. Thus, the aim of the present study was to test the hypothesis that astrocytes (the housekeeping cells of the brain microenvironment) may protect glioma cells from chemotherapy and to investigate the underlying mechanism. Immunofluorescent and scanning electron microscopy demonstrated that glioma cells were surrounded and infiltrated by activated astrocytes. In vitro co-culture of glioma cells with astrocytes significantly reduced the cytotoxic effects on glioma cells caused by various chemotherapeutic agents, as demonstrated by fluorescein isothiocyanate-propidium iodide flow cytometry. Transwell experiments indicated that this protective effect was dependent on physical contact and the gap junctional communication (GJC) between astrocytes and glioma cells. Microarray expression profiling further revealed that astrocytes upregulated the expression levels of various critical survival genes in the glioma cells via GJC. The results of the present study indicated that the organ microenvironment may affect the biological behavior of tumor cells and suggest a novel mechanism of resistance in glioma cells, which may be of therapeutic relevance clinically.

Cadmium induces Wnt signaling to upregulate proliferation and survival genes in sub-confluent kidney proximal tubule cells

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Wolff Natascha A

2010-05-01

Full Text Available Abstract Background The class 1 carcinogen cadmium (Cd2+ disrupts the E-cadherin/β-catenin complex of epithelial adherens junctions (AJs and causes renal cancer. Deregulation of E-cadherin adhesion and changes in Wnt/β-catenin signaling are known to contribute to carcinogenesis. Results We investigated Wnt signaling after Cd2+-induced E-cadherin disruption in sub-confluent cultured kidney proximal tubule cells (PTC. Cd2+ (25 μM, 3-9 h caused nuclear translocation of β-catenin and triggered a Wnt response measured by TOPflash reporter assays. Cd2+ reduced the interaction of β-catenin with AJ components (E-cadherin, α-catenin and increased binding to the transcription factor TCF4 of the Wnt pathway, which was upregulated and translocated to the nucleus. While Wnt target genes (c-Myc, cyclin D1 and ABCB1 were up-regulated by Cd2+, electromobility shift assays showed increased TCF4 binding to cyclin D1 and ABCB1 promoter sequences with Cd2+. Overexpression of wild-type and mutant TCF4 confirmed Cd2+-induced Wnt signaling. Wnt signaling elicited by Cd2+ was not observed in confluent non-proliferating cells, which showed increased E-cadherin expression. Overexpression of E-cadherin reduced Wnt signaling, PTC proliferation and Cd2+ toxicity. Cd2+ also induced reactive oxygen species dependent expression of the pro-apoptotic ER stress marker and Wnt suppressor CHOP/GADD153 which, however, did not abolish Wnt response and cell viability. Conclusions Cd2+ induces Wnt signaling in PTC. Hence, Cd2+ may facilitate carcinogenesis of PTC by promoting Wnt pathway-mediated proliferation and survival of pre-neoplastic cells.

Pro-angiogenic cell-based therapy for the treatment of ischemic cardiovascular diseases.

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Silvestre, Jean-Sébastien

2012-10-01

Pro-angiogenic cell therapy has emerged as a promising option to treat patients with acute myocardial infarction or with critical limb ischemia. Exciting pre-clinical studies have prompted the initiation of numerous clinical trials based on administration of stem/progenitor cells with pro-angiogenic potential. Most of the clinical studies performed so far have used bone marrow-derived or peripheral blood-derived mononuclear cells and showed, overall, a modest but significant benefit on tissue remodeling and function in patients with ischemic diseases. These mixed results pave the way for the development of strategies to overcome the limitation of autologous cell therapy and to propose more efficient approaches. Such strategies include pretreatment of cells with activators to augment cell recruitment and survival in the ischemic target area and/or the improvement of cell functions such as their paracrine ability to release proangiogenic factors and vasoactive molecules. In addition, efforts should be directed towards stimulation of both angiogenesis and vessel maturation, the development of a composite product consisting of stem/progenitor cells encapsulated in a biomaterial and the use of additional sources of regenerative cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

Inherited Inflammatory Response Genes Are Associated with B-Cell Non-Hodgkin's Lymphoma Risk and Survival.

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Kaspar René Nielsen

Full Text Available Malignant B-cell clones are affected by both acquired genetic alterations and by inherited genetic variations changing the inflammatory tumour microenvironment.We investigated 50 inflammatory response gene polymorphisms in 355 B-cell non-Hodgkin's lymphoma (B-NHL samples encompassing 216 diffuse large B cell lymphoma (DLBCL and 139 follicular lymphoma (FL and 307 controls. The effect of single genes and haplotypes were investigated and gene-expression analysis was applied for selected genes. Since interaction between risk genes can have a large impact on phenotype, two-way gene-gene interaction analysis was included.We found inherited SNPs in genes critical for inflammatory pathways; TLR9, IL4, TAP2, IL2RA, FCGR2A, TNFA, IL10RB, GALNT12, IL12A and IL1B were significantly associated with disease risk and SELE, IL1RN, TNFA, TAP2, MBL2, IL5, CX3CR1, CHI3L1 and IL12A were, associated with overall survival (OS in specific diagnostic entities of B-NHL. We discovered noteworthy interactions between DLBCL risk alleles on IL10 and IL4RA and FL risk alleles on IL4RA and IL4. In relation to OS, a highly significant interaction was observed in DLBCL for IL4RA (rs1805010 * IL10 (rs1800890 (HR = 0.11 (0.02-0.50. Finally, we explored the expression of risk genes from the gene-gene interaction analysis in normal B-cell subtypes showing a different expression of IL4RA, IL10, IL10RB genes supporting a pathogenetic effect of these interactions in the germinal center.The present findings support the importance of inflammatory genes in B-cell lymphomas. We found association between polymorphic sites in inflammatory response genes and risk as well as outcome in B-NHL and suggest an effect of gene-gene interactions during the stepwise oncogenesis.

Recombinant EPF/chaperonin 10 promotes the survival of O4-positive pro-oligodendrocytes prepared from neonatal rat brain.

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McCombe, P A

2008-12-01

Chaperonin 10 (cpn 10) is a small heat-shock protein that is usually intracellular. Early pregnancy factor (EPF), a biologically active protein that was first described in the serum of pregnant mammals, is homologous to cpn 10. EPF/cpn 10 has been reported to have effects on immunomodulation and cell survival and to inhibit activation of toll-like receptors by lipopolysaccharide. We found that recombinant EPF/cpn 10 was able to suppress experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, which is a disease causing inflammation and demyelination of the brain and spinal cord. This beneficial effect could be due to anti-inflammatory and/or cell survival properties of EPF/cpn 10. We aimed to assess the effects of cpn 10 on cells of the oligodendrocyte lineage because oligodendrocytes are the brain cells that produce myelin and that are depleted in multiple sclerosis. Two forms of recombinant EPF/cpn 10 were prepared in the pGEX expression system and in the baculovirus expression system. Purified O4(+) pro-oligodendrocytes were prepared from the brains of day-old Wistar rats and isolated by cell sorting with flow cytometry. Single cells were dispensed into micro-well plates and tested for survival in the presence of a range of concentrations of the two forms of cpn 10. We also studied the effects of bFGF, PDGF, IGF-1 and insulin as controls. With cpn 10 present, there was enhanced survival of O4(+) cells.

Reduced Erg Dosage Impairs Survival of Hematopoietic Stem and Progenitor Cells.

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Xie, Ying; Koch, Mia Lee; Zhang, Xin; Hamblen, Melanie J; Godinho, Frank J; Fujiwara, Yuko; Xie, Huafeng; Klusmann, Jan-Henning; Orkin, Stuart H; Li, Zhe

2017-07-01

ERG, an ETS family transcription factor frequently overexpressed in human leukemia, has been implicated as a key regulator of hematopoietic stem cells. However, how ERG controls normal hematopoiesis, particularly at the stem and progenitor cell level, and how it contributes to leukemogenesis remain incompletely understood. Using homologous recombination, we generated an Erg knockdown allele (Erg kd ) in which Erg expression can be conditionally restored by Cre recombinase. Erg kd/kd animals die at E10.5-E11.5 due to defects in endothelial and hematopoietic cells, but can be completely rescued by Tie2-Cre-mediated restoration of Erg in these cells. In Erg kd/+ mice, ∼40% reduction in Erg dosage perturbs both fetal liver and bone marrow hematopoiesis by reducing the numbers of Lin - Sca-1 + c-Kit + (LSK) hematopoietic stem and progenitor cells (HSPCs) and megakaryocytic progenitors. By genetic mosaic analysis, we find that Erg-restored HSPCs outcompete Erg kd/+ HSPCs for contribution to adult hematopoiesis in vivo. This defect is in part due to increased apoptosis of HSPCs with reduced Erg dosage, a phenotype that becomes more drastic during 5-FU-induced stress hematopoiesis. Expression analysis reveals that reduced Erg expression leads to changes in expression of a subset of ERG target genes involved in regulating survival of HSPCs, including increased expression of a pro-apoptotic regulator Bcl2l11 (Bim) and reduced expression of Jun. Collectively, our data demonstrate that ERG controls survival of HSPCs, a property that may be used by leukemic cells. Stem Cells 2017;35:1773-1785. © 2017 AlphaMed Press.

Selenium is critical for cancer-signaling gene expression but not cell proliferation in human colon Caco-2 cells.

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Zeng, Huawei; Botnen, James H

2007-01-01

Selenium (Se) is a potential anticarcinogenic nutrient, and the essential role of Se in cell growth is well recognized but certain cancer cells appear to have acquired a survival advantage under conditions of Se-deficiency. To understand the molecular basis of Se-anticancer effects at nutritional doses (nmol/L) for cultured cells, we generated Se-deficient colon Caco-2 cells by gradually reducing serum in media because serum contains a trace amount of Se. The glutathione peroxidase (GPx) activity of Se-deficient Caco-2 cells was 10.8 mU/mg protein compared to 133.6 approximately 146.3 mU/mg protein in Caco-2 cells supplemented with 500 nmol/L selenite, SeMSC or SeMet (three tested Se-chemical forms) after 7-d culture in serum free media. Interestingly, there were no detectable differences in cell growth, cell cycle progression between Se-deficient cells and cells supplemented with 500 nmol/L Se. To examine differential cancer signaling-gene expression between Se-deficient and Se-supplemented cells, we employed a cancer signal pathway-specific array assay coupled with the real time PCR analysis. Our data demonstrate that although Caco-2 cells are resistant to Se deprivation, Se may exert its anticancer property through increasing the expression of humoral defense gene (A2M) and tumor suppressor-related genes (IGFBP3, HHIP) while decreasing pro-inflammatory gene (CXC L9, HSPB2) expression.

Inhibition of HSP27 alone or in combination with pAKT inhibition as therapeutic approaches to target SPARC-induced glioma cell survival

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Schultz Chad R

2012-04-01

Full Text Available Abstract Background The current treatment regimen for glioma patients is surgery, followed by radiation therapy plus temozolomide (TMZ, followed by 6 months of adjuvant TMZ. Despite this aggressive treatment regimen, the overall survival of all surgically treated GBM patients remains dismal, and additional or different therapies are required. Depending on the cancer type, SPARC has been proposed both as a therapeutic target and as a therapeutic agent. In glioma, SPARC promotes invasion via upregulation of the p38 MAPK/MAPKAPK2/HSP27 signaling pathway, and promotes tumor cell survival by upregulating pAKT. As HSP27 and AKT interact to regulate the activity of each other, we determined whether inhibition of HSP27 was better than targeting SPARC as a therapeutic approach to inhibit both SPARC-induced glioma cell invasion and survival. Results Our studies found the following. 1 SPARC increases the expression of tumor cell pro-survival and pro-death protein signaling in balance, and, as a net result, tumor cell survival remains unchanged. 2 Suppressing SPARC increases tumor cell survival, indicating it is not a good therapeutic target. 3 Suppressing HSP27 decreases tumor cell survival in all gliomas, but is more effective in SPARC-expressing tumor cells due to the removal of HSP27 inhibition of SPARC-induced pro-apoptotic signaling. 4 Suppressing total AKT1/2 paradoxically enhanced tumor cell survival, indicating that AKT1 or 2 are poor therapeutic targets. 5 However, inhibiting pAKT suppresses tumor cell survival. 6 Inhibiting both HSP27 and pAKT synergistically decreases tumor cell survival. 7 There appears to be a complex feedback system between SPARC, HSP27, and AKT. 8 This interaction is likely influenced by PTEN status. With respect to chemosensitization, we found the following. 1 SPARC enhances pro-apoptotic signaling in cells exposed to TMZ. 2 Despite this enhanced signaling, SPARC protects cells against TMZ. 3 This protection can be reduced

Gene array analysis of PD-1H overexpressing monocytes reveals a pro-inflammatory profile

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Preeti Bharaj

2018-02-01

Full Text Available We have previously reported that overexpression of Programmed Death -1 Homolog (PD-1H in human monocytes leads to activation and spontaneous secretion of multiple pro inflammatory cytokines. Here we evaluate changes in monocytes gene expression after enforced PD-1H expression by gene array. The results show that there are significant alterations in 51 potential candidate genes that relate to immune response, cell adhesion and metabolism. Genes corresponding to pro-inflammatory cytokines showed the highest upregulation, 7, 3.2, 3.0, 5.8, 4.4 and 3.1 fold upregulation of TNF-α, IL-1 β, IFN-α, γ, λ and IL-27 relative to vector control. The data are in agreement with cytometric bead array analysis showing induction of proinflammatory cytokines, IL-6, IL-1β and TNF-α by PD-1H. Other genes related to inflammation, include transglutaminase 2 (TG2, NF-κB (p65 and p50 and toll like receptors (TLR 3 and 4 were upregulated 5, 4.5 and 2.5 fold, respectively. Gene set enrichment analysis (GSEA also revealed that signaling pathways related to inflammatory response, such as NFκB, AT1R, PYK2, MAPK, RELA, TNFR1, MTOR and proteasomal degradation, were significantly upregulated in response to PD-1H overexpression. We validated the results utilizing a standard inflammatory sepsis model in humanized BLT mice, finding that PD-1H expression was highly correlated with proinflammatory cytokine production. We therefore conclude that PD-1H functions to enhance monocyte activation and the induction of a pro-inflammatory gene expression profile.

Connection between cell phone use, p53 gene expression in different zones of glioblastoma multiforme and survival prognoses

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Reza Akhavan-Sigari

2014-08-01

Full Text Available The aim of this paper is to investigate p53 gene expression in the central and peripheral zones of glioblastoma multiforme using a real-time reverse transcription polymerase chain reaction (RT-PCR technique in patients who use cell phones ≥3 hours a day and determine its relationship to clinicopathological findings and overall survival. Sixty-three patients (38 males and 25 females, diagnosed with glioblastoma multiforme (GBM, underwent tumor resection between 2008 and 2011. Patient ages ranged from 25 to 88 years, with a mean age of 55. The levels of expression of p53 in the central and peripheral zone of the GBM were quantified by RT-PCR. Data on p53 gene expression from the central and peripheral zone, the related malignancy and the clinicopatholagical findings (age, gender, tumor location and size, as well as overall survival, were analyzed. Forty-one out of 63 patients (65% with the highest level of cell phone use (≥3 hours/day had higher mutant type p53 expression in the peripheral zone of the glioblastoma; the difference was statistically significant (P=0.034. Results from the present study on the use of mobile phones for ≥3 hours a day show a consistent pattern of increased risk for the mutant type of p53 gene expression in the peripheral zone of the glioblastoma, and that this increase was significantly correlated with shorter overall survival time. The risk was not higher for ipsilateral exposure. We found that the mutant type of p53 gene expression in the peripheral zone of the glioblastoma was increased in 65% of patients using cell phones ≥3 hours a day.

Depression of p53-independent Akt survival signals in human oral cancer cells bearing mutated p53 gene after exposure to high-LET radiation

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Nakagawa, Yosuke [Department of Oral and Maxillofacial Surgery, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Takahashi, Akihisa [Advanced Scientific Research Leader Development Unit, Gunma University, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511 (Japan); Kajihara, Atsuhisa; Yamakawa, Nobuhiro; Imai, Yuichiro [Department of Oral and Maxillofacial Surgery, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Ota, Ichiro; Okamoto, Noritomo [Department of Otorhinolaryngology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Mori, Eiichiro [Department of Radiation Oncology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Noda, Taichi [Department of Dermatology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Furusawa, Yoshiya [Heavy-ion Radiobiology Research Group, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Kirita, Tadaaki [Department of Oral and Maxillofacial Surgery, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Ohnishi, Takeo, E-mail: tohnishi@naramed-u.ac.jp [Department of Radiation Oncology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan)

2012-07-13

Highlights: Black-Right-Pointing-Pointer High-LET radiation induces efficiently apoptosis regardless of p53 gene status. Black-Right-Pointing-Pointer We examined whether high-LET radiation depresses the Akt-survival signals. Black-Right-Pointing-Pointer High-LET radiation depresses of survival signals even in the mp53 cancer cells. Black-Right-Pointing-Pointer High-LET radiation activates Caspase-9 through depression of survival signals. Black-Right-Pointing-Pointer High-LET radiation suppresses cell growth through depression of survival signals. -- Abstract: Although mutations and deletions in the p53 tumor suppressor gene lead to resistance to low linear energy transfer (LET) radiation, high-LET radiation efficiently induces cell lethality and apoptosis regardless of the p53 gene status in cancer cells. Recently, it has been suggested that the induction of p53-independent apoptosis takes place through the activation of Caspase-9 which results in the cleavage of Caspase-3 and poly (ADP-ribose) polymerase (PARP). This study was designed to examine if high-LET radiation depresses serine/threonine protein kinase B (PKB, also known as Akt) and Akt-related proteins. Human gingival cancer cells (Ca9-22 cells) harboring a mutated p53 (mp53) gene were irradiated with 2 Gy of X-rays or Fe-ion beams. The cellular contents of Akt-related proteins participating in cell survival signaling were analyzed with Western Blotting 1, 2, 3 and 6 h after irradiation. Cell cycle distributions after irradiation were assayed with flow cytometric analysis. Akt-related protein levels decreased when cells were irradiated with high-LET radiation. High-LET radiation increased G{sub 2}/M phase arrests and suppressed the progression of the cell cycle much more efficiently when compared to low-LET radiation. These results suggest that high-LET radiation enhances apoptosis through the activation of Caspase-3 and Caspase-9, and suppresses cell growth by suppressing Akt-related signaling, even in mp

The cytotoxic type 3 secretion system 1 of Vibrio rewires host gene expression to subvert cell death and activate cell survival pathways.

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De Nisco, Nicole J; Kanchwala, Mohammed; Li, Peng; Fernandez, Jessie; Xing, Chao; Orth, Kim

2017-05-16

Bacterial effectors potently manipulate host signaling pathways. The marine bacterium Vibrio parahaemolyticus ( V. para ) delivers effectors into host cells through two type 3 secretion systems (T3SSs). T3SS1 is vital for V. para survival in the environment, whereas T3SS2 causes acute gastroenteritis in human hosts. Although the natural host is undefined, T3SS1 effectors attack highly conserved cellular processes and pathways to orchestrate nonapoptotic cell death. To understand how the concerted action of T3SS1 effectors globally affects host cell signaling, we compared gene expression changes over time in primary fibroblasts infected with V. para that have a functional T3SS1 (T3SS1 + ) to those in cells infected with V. para lacking T3SS1 (T3SS1 - ). Overall, the host transcriptional response to both T3SS1 + and T3SS1 - V. para was rapid, robust, and temporally dynamic. T3SS1 rewired host gene expression by specifically altering the expression of 398 genes. Although T3SS1 effectors targeted host cells at the posttranslational level to cause cytotoxicity, V. para T3SS1 also precipitated a host transcriptional response that initially activated cell survival and repressed cell death networks. The increased expression of several key prosurvival transcripts mediated by T3SS1 depended on a host signaling pathway that is silenced posttranslationally later in infection. Together, our analysis reveals a complex interplay between the roles of T3SS1 as both a transcriptional and posttranslational manipulator of host cell signaling. Copyright © 2017, American Association for the Advancement of Science.

VEGF improves survival of mesenchymal stem cells in infarcted hearts

International Nuclear Information System (INIS)

Pons, Jennifer; Huang Yu; Arakawa-Hoyt, Janice; Washko, Daniel; Takagawa, Junya; Ye, Jianqin; Grossman, William; Su Hua

2008-01-01

Bone marrow-derived mesenchymal stem cells (MSC) are a promising source for cell-based treatment of myocardial infarction (MI), but existing strategies are restricted by low cell survival and engraftment. We examined whether vascular endothelial growth factor (VEGF) improve MSC viability in infracted hearts. We found long-term culture increased MSC-cellular stress: expressing more cell cycle inhibitors, p16 INK , p21 and p19 ARF . VEGF treatment reduced cellular stress, increased pro-survival factors, phosphorylated-Akt and Bcl-xL expression and cell proliferation. Co-injection of MSCs with VEGF to MI hearts increased cell engraftment and resulted in better improvement of cardiac function than that injected with MSCs or VEGF alone. In conclusion, VEGF protects MSCs from culture-induce cellular stress and improves their viability in ischemic myocardium, which results in improvements of their therapeutic effect for the treatment of MI

Glycosylphosphatidylinositol Anchor Modification Machinery Deficiency Is Responsible for the Formation of Pro-Prion Protein (PrP) in BxPC-3 Protein and Increases Cancer Cell Motility*

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Yang, Liheng; Gao, Zhenxing; Hu, Lipeng; Wu, Guiru; Yang, Xiaowen; Zhang, Lihua; Zhu, Ying; Wong, Boon-Seng; Xin, Wei; Sy, Man-Sun; Li, Chaoyang

2016-01-01

The normal cellular prion protein (PrP) is a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein. However, in pancreatic ductal adenocarcinoma cell lines, such as BxPC-3, PrP exists as a pro-PrP retaining its glycosylphosphatidylinositol (GPI) peptide signaling sequence. Here, we report the identification of another pancreatic ductal adenocarcinoma cell line, AsPC-1, which expresses a mature GPI-anchored PrP. Comparison of the 24 genes involved in the GPI anchor modification pathway between AsPC-1 and BxPC-3 revealed 15 of the 24 genes, including PGAP1 and PIG-F, were down-regulated in the latter cells. We also identified six missense mutations in DPM2, PIG-C, PIG-N, and PIG-P alongside eight silent mutations. When BxPC-3 cells were fused with Chinese hamster ovary (CHO) cells, which lack endogenous PrP, pro-PrP was successfully converted into mature GPI-anchored PrP. Expression of the individual gene, such as PGAP1, PIG-F, or PIG-C, into BxPC-3 cells does not result in phosphoinositide-specific phospholipase C sensitivity of PrP. However, when PIG-F but not PIG-P is expressed in PGAP1-expressing BxPC-3 cells, PrP on the surface of the cells becomes phosphoinositide-specific phospholipase C-sensitive. Thus, low expression of PIG-F and PGAP1 is the major factor contributing to the accumulation of pro-PrP. More importantly, BxPC-3 cells expressing GPI-anchored PrP migrate much slower than BxPC-3 cells bearing pro-PrP. In addition, GPI-anchored PrP-bearing AsPC-1 cells also migrate slower than pro-PrP bearing BxPC-3 cells, although both cells express filamin A. “Knocking out” PRNP in BxPC-3 cell drastically reduces its migration. Collectively, these results show that multiple gene irregularity in BxPC-3 cells is responsible for the formation of pro-PrP, and binding of pro-PrP to filamin A contributes to enhanced tumor cell motility. PMID:26683373

The truncated splice variant of peroxisome proliferator-activated receptor alpha, PPARα-tr, autonomously regulates proliferative and pro-inflammatory genes

International Nuclear Information System (INIS)

Thomas, Maria; Bayha, Christine; Klein, Kathrin; Müller, Simon; Weiss, Thomas S.; Schwab, Matthias; Zanger, Ulrich M.

2015-01-01

The peroxisome proliferator-activated receptor alpha (PPARα) controls lipid/energy homeostasis and inflammatory responses. The truncated splice variant PPARα-tr was suggested to exert a dominant negative function despite being unable to bind consensus PPARα DNA response elements. The distribution and variability factor of each PPARα variant were assessed in the well-characterized cohort of human liver samples (N = 150) on the mRNA and protein levels. Specific siRNA-mediated downregulation of each transcript as well as specific overexpression with subsequent qRT-PCR analysis of downstream genes was used for investigation of specific functional roles of PPARα-wt and PPARα-tr forms in primary human hepatocytes. Bioinformatic analyses of genome-wide liver expression profiling data suggested a possible role of PPARα-tr in downregulating proliferative and pro-inflammatory genes. Specific gene silencing of both forms in primary human hepatocytes showed that induction of metabolic PPARα-target genes by agonist WY14,643 was prevented by PPARα-wt knock-down but neither prevented nor augmented by PPARα-tr knock-down. WY14,643 treatment did not induce proliferative genes including MYC, CDK1, and PCNA, and knock-down of PPARα-wt had no effect, while PPARα-tr knock-down caused up to 3-fold induction of these genes. Similarly, induction of pro-inflammatory genes IL1B, PTGS2, and CCL2 by IL-6 was augmented by knock-down of PPARα-tr but not of PPARα-wt. In contrast to human proliferative genes, orthologous mouse genes were readily inducible by WY14,643 in PPARα-tr non-expressing AML12 mouse hepatocytes. Induction was augmented by overexpression of PPARα-wt and attenuated by overexpression of PPARα-tr. Pro-inflammatory genes including IL-1β, CCL2 and TNFα were induced by WY14,643 in mouse and human cells and both PPARα forms attenuated induction. As potential mechanism of PPARα-tr inhibitory action we suggest crosstalk with WNT/β-catenin pathway. Finally

Knockout Serum Replacement Promotes Cell Survival by Preventing BIM from Inducing Mitochondrial Cytochrome C Release.

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Yuki Ishii

Full Text Available Knockout serum replacement (KOSR is a nutrient supplement commonly used to replace serum for culturing stem cells. We show here that KOSR has pro-survival activity in chronic myelogenous leukemia (CML cells transformed by the BCR-ABL oncogene. Inhibitors of BCR-ABL tyrosine kinase kill CML cells by stimulating pro-apoptotic BIM and inhibiting anti-apoptotic BCL2, BCLxL and MCL1. We found that KOSR protects CML cells from killing by BCR-ABL inhibitors--imatinib, dasatinib and nilotinib. The protective effect of KOSR is reversible and not due to the selective outgrowth of drug-resistant clones. In KOSR-protected CML cells, imatinib still inhibited the BCR-ABL tyrosine kinase, reduced the phosphorylation of STAT, ERK and AKT, down-regulated BCL2, BCLxL, MCL1 and up-regulated BIM. However, these pro-apoptotic alterations failed to cause cytochrome c release from the mitochondria. With mitochondria isolated from KOSR-cultured CML cells, we showed that addition of recombinant BIM protein also failed to cause cytochrome c release. Besides the kinase inhibitors, KOSR could protect cells from menadione, an inducer of oxidative stress, but it did not protect cells from DNA damaging agents. Switching from serum to KOSR caused a transient increase in reactive oxygen species and AKT phosphorylation in CML cells that were protected by KOSR but not in those that were not protected by this nutrient supplement. Treatment of KOSR-cultured cells with the PH-domain inhibitor MK2206 blocked AKT phosphorylation, abrogated the formation of BIM-resistant mitochondria and stimulated cell death. These results show that KOSR has cell-context dependent pro-survival activity that is linked to AKT activation and the inhibition of BIM-induced cytochrome c release from the mitochondria.

Selective targeting of pro-inflammatory Th1 cells by microRNA-148a-specific antagomirs in vivo.

Science.gov (United States)

Maschmeyer, Patrick; Petkau, Georg; Siracusa, Francesco; Zimmermann, Jakob; Zügel, Franziska; Kühl, Anja Andrea; Lehmann, Katrin; Schimmelpfennig, Sarah; Weber, Melanie; Haftmann, Claudia; Riedel, René; Bardua, Markus; Heinz, Gitta Anne; Tran, Cam Loan; Hoyer, Bimba Franziska; Hiepe, Falk; Herzog, Sebastian; Wittmann, Jürgen; Rajewsky, Nikolaus; Melchers, Fritz Georg; Chang, Hyun-Dong; Radbruch, Andreas; Mashreghi, Mir-Farzin

2018-05-01

In T lymphocytes, expression of miR-148a is induced by T-bet and Twist1, and is specific for pro-inflammatory Th1 cells. In these cells, miR-148a inhibits the expression of the pro-apoptotic protein Bim and promotes their survival. Here we use sequence-specific cholesterol-modified oligonucleotides against miR-148a (antagomir-148a) for the selective elimination of pro-inflammatory Th1 cells in vivo. In the murine model of transfer colitis, antagomir-148a treatment reduced the number of pro-inflammatory Th1 cells in the colon of colitic mice by 50% and inhibited miR-148a expression by 71% in the remaining Th1 cells. Expression of Bim protein in colonic Th1 cells was increased. Antagomir-148a-mediated reduction of Th1 cells resulted in a significant amelioration of colitis. The effect of antagomir-148a was selective for chronic inflammation. Antigen-specific memory Th cells that were generated by an acute immune reaction to nitrophenylacetyl-coupled chicken gamma globulin (NP-CGG) were not affected by treatment with antagomir-148a, both during the effector and the memory phase. In addition, antibody titers to NP-CGG were not altered. Thus, antagomir-148a might qualify as an effective drug to selectively deplete pro-inflammatory Th1 cells of chronic inflammation without affecting the protective immunological memory. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Transplantation of Gene-Edited Hepatocyte-like Cells Modestly Improves Survival of Arginase-1-Deficient Mice

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Yuan Yan Sin

2018-03-01

Full Text Available Progress in gene editing research has been accelerated by utilizing engineered nucleases in combination with induced pluripotent stem cell (iPSC technology. Here, we report transcription activator-like effector nuclease (TALEN-mediated reincorporation of Arg1 exons 7 and 8 in iPSCs derived from arginase-1-deficient mice possessing Arg1Δ alleles lacking these terminal exons. The edited cells could be induced to differentiate into hepatocyte-like cells (iHLCs in vitro and were subsequently used for transplantation into our previously described (Sin et al., PLoS ONE 2013 tamoxifen-inducible Arg1-Cre arginase-1-deficient mouse model. While successful gene-targeted repair was achieved in iPSCs containing Arg1Δ alleles, only minimal restoration of urea cycle function could be observed in the iHLC-transplanted mice compared to control mice, and survival in this lethal model was extended by up to a week in some mice. The partially rescued phenotype may be due to inadequate regenerative capacity of arginase-1-expressing cells in the correct metabolic zones. Technical hurdles exist and will need to be overcome for gene-edited iPSC to iHLC rescue of arginase-1 deficiency, a rare urea cycle disorder.

Different Effects of BORIS/CTCFL on Stemness Gene Expression, Sphere Formation and Cell Survival in Epithelial Cancer Stem Cells.

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Loredana Alberti

Full Text Available Cancer stem cells are cancer cells characterized by stem cell properties and represent a small population of tumor cells that drives tumor development, progression, metastasis and drug resistance. To date, the molecular mechanisms that generate and regulate cancer stem cells are not well defined. BORIS (Brother of Regulator of Imprinted Sites or CTCFL (CTCF-like is a DNA-binding protein that is expressed in normal tissues only in germ cells and is re-activated in tumors. Recent evidences have highlighted the correlation of BORIS/CTCFL expression with poor overall survival of different cancer patients. We have previously shown an association of BORIS-expressing cells with stemness gene expression in embryonic cancer cells. Here, we studied the role of BORIS in epithelial tumor cells. Using BORIS-molecular beacon that was already validated, we were able to show the presence of BORIS mRNA in cancer stem cell-enriched populations (side population and spheres of cervical, colon and breast tumor cells. BORIS silencing studies showed a decrease of sphere formation capacity in breast and colon tumor cells. Importantly, BORIS-silencing led to down-regulation of hTERT, stem cell (NANOG, OCT4, SOX2 and BMI1 and cancer stem cell markers (ABCG2, CD44 and ALDH1 genes. Conversely, BORIS-induction led to up-regulation of the same genes. These phenotypes were observed in cervical, colon and invasive breast tumor cells. However, a completely different behavior was observed in the non-invasive breast tumor cells (MCF7. Indeed, these cells acquired an epithelial mesenchymal transition phenotype after BORIS silencing. Our results demonstrate that BORIS is associated with cancer stem cell-enriched populations of several epithelial tumor cells and the different phenotypes depend on the origin of tumor cells.

A Hyaluronan-Based Injectable Hydrogel Improves the Survival and Integration of Stem Cell Progeny following Transplantation

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Brian G. Ballios

2015-06-01

Full Text Available The utility of stem cells and their progeny in adult transplantation models has been limited by poor survival and integration. We designed an injectable and bioresorbable hydrogel blend of hyaluronan and methylcellulose (HAMC and tested it with two cell types in two animal models, thereby gaining an understanding of its general applicability for enhanced cell distribution, survival, integration, and functional repair relative to conventional cell delivery in saline. HAMC improves cell survival and integration of retinal stem cell (RSC-derived rods in the retina. The pro-survival mechanism of HAMC is ascribed to the interaction of the CD44 receptor with HA. Transient disruption of the retinal outer limiting membrane, combined with HAMC delivery, results in significantly improved rod survival and visual function. HAMC also improves the distribution, viability, and functional repair of neural stem and progenitor cells (NSCs. The HAMC delivery system improves cell transplantation efficacy in two CNS models, suggesting broad applicability.

Overexpression of pro-gastrin releasing peptide promotes the cell proliferation and progression in small cell lung cancer

International Nuclear Information System (INIS)

Gong, Zhiyun; Lu, Renquan; Xie, Suhong; Jiang, Minglei; Liu, Kai; Xiao, Ran; Shen, Jiabin; Wang, Yanchun; Guo, Lin

2016-01-01

Pro-gastrin releasing peptide (ProGRP) plays the role of oncogene in small cell lung cancer (SCLC). In this study, we aim to explore the biological function of ProGRP in SCLC cells and its potential mechanism. Expression of ProGRP in SCLC tissues and cell lines were detected by immunohistochemistry and western blot analysis, respectively. The transduced cell lines with ProGRP down-regulation were established using RNA interference technology. Cell viability, cologenic, apoptosis-associated assay and the biomarker levels determination for cell supernatant were performed in the transduced cells to elucidate the biological functions and mechanisms of ProGRP in SCLC cells. Our data showed that ProGRP protein was demonstrated a higher level in SCLC tissues and cells compared with the control, and its diagnostic efficiency was better than NSE, further, the higher levels of ProGRP were detected in the patients with extensive disease stage (P < 0.05), were also the unfavorable factor to the prognosis of SCLC patients. Additionally, the concentration of serum ProGRP is a useful biomarker in disease-monitoring of the patients with SCLC. Down-regulation of ProGRP significantly reduced SCLC cell growth, repressed colony formation, but increased cancer cell apoptosis. Additionally, repression of ProGRP also induced change in the cell cycle and output of NSE. Our data indicated that ProGRP serve as the useful biomarker in the management of SCLC and might be a potential therapeutic target. - Highlights: • ProGRP is overexpressed in the tissues and sera of the patients with SCLC. • Down-regulation of ProGRP inhibited cell proliferation. • Inhibition of ProGRP altered cell cycle distribution and triggers the apoptosis of lung cancer cells.

Glycosylphosphatidylinositol Anchor Modification Machinery Deficiency Is Responsible for the Formation of Pro-Prion Protein (PrP) in BxPC-3 Protein and Increases Cancer Cell Motility.

Science.gov (United States)

Yang, Liheng; Gao, Zhenxing; Hu, Lipeng; Wu, Guiru; Yang, Xiaowen; Zhang, Lihua; Zhu, Ying; Wong, Boon-Seng; Xin, Wei; Sy, Man-Sun; Li, Chaoyang

2016-02-19

The normal cellular prion protein (PrP) is a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein. However, in pancreatic ductal adenocarcinoma cell lines, such as BxPC-3, PrP exists as a pro-PrP retaining its glycosylphosphatidylinositol (GPI) peptide signaling sequence. Here, we report the identification of another pancreatic ductal adenocarcinoma cell line, AsPC-1, which expresses a mature GPI-anchored PrP. Comparison of the 24 genes involved in the GPI anchor modification pathway between AsPC-1 and BxPC-3 revealed 15 of the 24 genes, including PGAP1 and PIG-F, were down-regulated in the latter cells. We also identified six missense mutations in DPM2, PIG-C, PIG-N, and PIG-P alongside eight silent mutations. When BxPC-3 cells were fused with Chinese hamster ovary (CHO) cells, which lack endogenous PrP, pro-PrP was successfully converted into mature GPI-anchored PrP. Expression of the individual gene, such as PGAP1, PIG-F, or PIG-C, into BxPC-3 cells does not result in phosphoinositide-specific phospholipase C sensitivity of PrP. However, when PIG-F but not PIG-P is expressed in PGAP1-expressing BxPC-3 cells, PrP on the surface of the cells becomes phosphoinositide-specific phospholipase C-sensitive. Thus, low expression of PIG-F and PGAP1 is the major factor contributing to the accumulation of pro-PrP. More importantly, BxPC-3 cells expressing GPI-anchored PrP migrate much slower than BxPC-3 cells bearing pro-PrP. In addition, GPI-anchored PrP-bearing AsPC-1 cells also migrate slower than pro-PrP bearing BxPC-3 cells, although both cells express filamin A. "Knocking out" PRNP in BxPC-3 cell drastically reduces its migration. Collectively, these results show that multiple gene irregularity in BxPC-3 cells is responsible for the formation of pro-PrP, and binding of pro-PrP to filamin A contributes to enhanced tumor cell motility. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Schistosome tegumental ecto-apyrase (SmATPDase1 degrades exogenous pro-inflammatory and pro-thrombotic nucleotides

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Akram A. Da’dara

2014-03-01

Full Text Available Schistosomes are parasitic worms that can survive in the hostile environment of the human bloodstream where they appear refractory to both immune elimination and thrombus formation. We hypothesize that parasite migration in the bloodstream can stress the vascular endothelium causing this tissue to release chemicals alerting responsive host cells to the stress. Such chemicals are called damage associated molecular patterns (DAMPs and among the most potent is the proinflammatory mediator, adenosine triphosphate (ATP. Furthermore, the ATP derivative ADP is a pro-thrombotic molecule that acts as a strong activator of platelets. Schistosomes are reported to possess at their host interactive tegumental surface a series of enzymes that could, like their homologs in mammals, degrade extracellular ATP and ADP. These are alkaline phosphatase (SmAP, phosphodiesterase (SmNPP-5 and ATP diphosphohydrolase (SmATPDase1. In this work we employ RNAi to knock down expression of the genes encoding these enzymes in the intravascular life stages of the parasite. We then compare the abilities of these parasites to degrade exogenously added ATP and ADP. We find that only SmATPDase1-suppressed parasites are significantly impaired in their ability to degrade these nucleotides. Suppression of SmAP or SmNPP-5 does not appreciably affect the worms’ ability to catabolize ATP or ADP. These findings are confirmed by the functional characterization of the enzymatically active, full-length recombinant SmATPDase1 expressed in CHO-S cells. The enzyme is a true apyrase; SmATPDase1 degrades ATP and ADP in a cation dependent manner. Optimal activity is seen at alkaline pH. The Km of SmATPDase1 for ATP is 0.4 ± 0.02 mM and for ADP, 0.252 ± 0.02 mM. The results confirm the role of tegumental SmATPDase1 in the degradation of the exogenous pro-inflammatory and pro-thrombotic nucleotides ATP and ADP by live intravascular stages of the parasite. By degrading host inflammatory signals

Romidepsin targets multiple survival signaling pathways in malignant T cells

International Nuclear Information System (INIS)

Valdez, B C; Brammer, J E; Li, Y; Murray, D; Liu, Y; Hosing, C; Nieto, Y; Champlin, R E; Andersson, B S

2015-01-01

Romidepsin is a cyclic molecule that inhibits histone deacetylases. It is Food and Drug Administration-approved for treatment of cutaneous and peripheral T-cell lymphoma, but its precise mechanism of action against malignant T cells is unknown. To better understand the biological effects of romidepsin in these cells, we exposed PEER and SUPT1 T-cell lines, and a primary sample from T-cell lymphoma patient (Patient J) to romidepsin. We then examined the consequences in some key oncogenic signaling pathways. Romidepsin displayed IC 50 values of 10.8, 7.9 and 7.0 nm in PEER, SUPT1 and Patient J cells, respectively. Strong inhibition of histone deacetylases and demethylases, increased production of reactive oxygen species and decreased mitochondrial membrane potential were observed, which may contribute to the observed DNA-damage response and apoptosis. The stress-activated protein kinase/c-Jun N-terminal kinase signaling pathway and unfolded protein response in the endoplasmic reticulum were activated, whereas the phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) and β-catenin pro-survival pathways were inhibited. The decreased level of β-catenin correlated with the upregulation of its inhibitor SFRP1 through romidepsin-mediated hypomethylation of its gene promoter. Our results provide new insights into how romidepsin invokes malignant T-cell killing, show evidence of its associated DNA hypomethylating activity and offer a rationale for the development of romidepsin-containing combination therapies

A novel pleiotropic effect of aspirin: Beneficial regulation of pro- and anti-inflammatory mechanisms in microglial cells.

Science.gov (United States)

Kata, Diana; Földesi, Imre; Feher, Liliana Z; Hackler, Laszlo; Puskas, Laszlo G; Gulya, Karoly

2017-06-01

Aspirin, one of the most widely used non-steroidal anti-inflammatory drugs, has extensively studied effects on the cardiovascular system. To reveal further pleiotropic, beneficial effects of aspirin on a number of pro- and anti-inflammatory microglial mechanisms, we performed morphometric and functional studies relating to phagocytosis, pro- and anti-inflammatory cytokine production (IL-1β, tumor necrosis factor-α (TNF-α) and IL-10, respectively) and analyzed the expression of a number of inflammation-related genes, including those related to the above functions, in pure microglial cells. We examined the effects of aspirin (0.1mM and 1mM) in unchallenged (control) and bacterial lipopolysaccharide (LPS)-challenged secondary microglial cultures. Aspirin affected microglial morphology and functions in a dose-dependent manner as it inhibited LPS-elicited microglial activation by promoting ramification and the inhibition of phagocytosis in both concentrations. Remarkably, aspirin strongly reduced the pro-inflammatory IL-1β and TNF-α production, while it increased the anti-inflammatory IL-10 level in LPS-challenged cells. Moreover, aspirin differentially regulated the expression of a number of inflammation-related genes as it downregulated such pro-inflammatory genes as Nos2, Kng1, IL1β, Ptgs2 or Ccr1, while it upregulated some anti-inflammatory genes such as IL10, Csf2, Cxcl1, Ccl5 or Tgfb1. Thus, the use of aspirin could be beneficial for the prophylaxis of certain neurodegenerative disorders as it effectively ameliorates inflammation in the brain. Copyright © 2017 Elsevier Inc. All rights reserved.

Cyclic mechanical stretch down-regulates cathelicidin antimicrobial peptide expression and activates a pro-inflammatory response in human bronchial epithelial cells

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Harpa Karadottir

2015-12-01

Full Text Available Mechanical ventilation (MV of patients can cause damage to bronchoalveolar epithelium, leading to a sterile inflammatory response, infection and in severe cases sepsis. Limited knowledge is available on the effects of MV on the innate immune defense system in the human lung. In this study, we demonstrate that cyclic stretch of the human bronchial epithelial cell lines VA10 and BCi NS 1.1 leads to down-regulation of cathelicidin antimicrobial peptide (CAMP gene expression. We show that treatment of VA10 cells with vitamin D3 and/or 4-phenyl butyric acid counteracted cyclic stretch mediated down-regulation of CAMP mRNA and protein expression (LL-37. Further, we observed an increase in pro-inflammatory responses in the VA10 cell line subjected to cyclic stretch. The mRNA expression of the genes encoding pro-inflammatory cytokines IL-8 and IL-1β was increased after cyclic stretching, where as a decrease in gene expression of chemokines IP-10 and RANTES was observed. Cyclic stretch enhanced oxidative stress in the VA10 cells. The mRNA expression of toll-like receptor (TLR 3, TLR5 and TLR8 was reduced, while the gene expression of TLR2 was increased in VA10 cells after cyclic stretch. In conclusion, our in vitro results indicate that cyclic stretch may differentially modulate innate immunity by down-regulation of antimicrobial peptide expression and increase in pro-inflammatory responses.

Pretreating mesenchymal stem cells with electrical stimulation causes sustained long-lasting pro-osteogenic effects

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Maria Eischen-Loges

2018-06-01

Full Text Available Background Electrical stimulation (ES has a long history of successful use in the clinical treatment of refractory, non-healing bone fractures and has recently been proposed as an adjunct to bone tissue-engineering treatments to optimize their therapeutic potential. This idea emerged from ES’s demonstrated positive effects on stem cell migration, proliferation, differentiation and adherence to scaffolds, all cell behaviors recognized to be advantageous in Bone Tissue Engineering (BTE. In previous in vitro experiments we demonstrated that direct current ES, administered daily, accelerates Mesenchymal Stem Cell (MSC osteogenic differentiation. In the present study, we sought to define the optimal ES regimen for maximizing this pro-osteogenic effect. Methods Rat bone marrow-derived MSC were exposed to 100 mV/mm, 1 hr/day for three, seven, and 14 days, then osteogenic differentiation was assessed at Day 14 of culture by measuring collagen production, calcium deposition, alkaline phosphatase activity and osteogenic marker gene expression. Results We found that exposing MSC to ES for three days had minimal effect, while seven and 14 days resulted in increased osteogenic differentiation, as indicated by significant increases in collagen and calcium deposits, and expression of osteogenic marker genes Col1a1, Osteopontin, Osterix and Calmodulin. We also found that cells treated with ES for seven days, maintained this pro-osteogenic activity long (for at least seven days after discontinuing ES exposure. Discussion This study showed that while three days of ES is insufficient to solicit pro-osteogenic effects, seven and 14 days significantly increases osteogenic differentiation. Importantly, we found that cells treated with ES for only seven days, maintained this pro-osteogenic activity long after discontinuing ES exposure. This sustained positive osteogenic effect is likely due to the enhanced expression of RunX2 and Calmodulin we observed. This

Identifying activated T cells in reconstituted RAG deficient mice using retrovirally transduced Pax5 deficient pro-B cells.

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Nadesan Gajendran

Full Text Available Various methods have been used to identify activated T cells such as binding of MHC tetramers and expression of cell surface markers in addition to cytokine-based assays. In contrast to these published methods, we here describe a strategy to identify T cells that respond to any antigen and track the fate of these activated T cells. We constructed a retroviral double-reporter construct with enhanced green fluorescence protein (EGFP and a far-red fluorescent protein from Heteractis crispa (HcRed. LTR-driven EGFP expression was used to enrich and identify transduced cells, while HcRed expression is driven by the CD40Ligand (CD40L promoter, which is inducible and enables the identification and cell fate tracing of T cells that have responded to infection/inflammation. Pax5 deficient pro-B cells that can give rise to different hematopoietic cells like T cells, were retrovirally transduced with this double-reporter cassette and were used to reconstitute the T cell pool in RAG1 deficient mice that lack T and B cells. By using flow cytometry and histology, we identified activated T cells that had developed from Pax5 deficient pro-B cells and responded to infection with the bacterial pathogen Listeria monocytogenes. Microscopic examination of organ sections allowed visual identification of HcRed-expressing cells. To further characterize the immune response to a given stimuli, this strategy can be easily adapted to identify other cells of the hematopoietic system that respond to infection/inflammation. This can be achieved by using an inducible reporter, choosing the appropriate promoter, and reconstituting mice lacking cells of interest by injecting gene-modified Pax5 deficient pro-B cells.

Patents and innovation in cancer therapeutics: lessons from CellPro.

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Bar-Shalom, Avital; Cook-Deegan, Robert

2002-01-01

This article discusses the interaction between intellectual property and cancer treatment. CellPro developed a stem cell separation technology based on research at the Fred Hutchinson Cancer Center. A patent with broad claims to bone marrow stem cell antibodies had been awarded to Johns Hopkins University and licensed to Baxter Healthcare under the 1980 Bayh-Dole Act to promote commercial use of inventions from federally funded research. CellPro got FDA approval more than two years before Baxter but lost patent infringement litigation. NIH elected not to compel Hopkins to license its patents to CellPro. CellPro went out of business, selling its technology to its competitor. Decisions at both firms and university licensing offices, and policies at the Patent and Trademark Office, NIH, and the courts influenced the outcome.

Identification of human ferritin, heavy polypeptide 1 (FTH1) and yeast RGI1 (YER067W) as pro-survival sequences that counteract the effects of Bax and copper in Saccharomyces cerevisiae

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Eid, Rawan [Department of Chemistry and Chemical Engineering, Royal Military College, Kingston, Ontario (Canada); Department of Biology, Queen' s University, Kingston, Ontario (Canada); Boucher, Eric [Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec (Canada); Gharib, Nada [Department of Chemistry and Chemical Engineering, Royal Military College, Kingston, Ontario (Canada); Khoury, Chamel [Department of Chemistry and Chemical Engineering, Royal Military College, Kingston, Ontario (Canada); Department of Biology, Queen' s University, Kingston, Ontario (Canada); Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec (Canada); Arab, Nagla T.T. [Department of Chemistry and Chemical Engineering, Royal Military College, Kingston, Ontario (Canada); Department of Biology, Queen' s University, Kingston, Ontario (Canada); Murray, Alistair [Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec (Canada); Young, Paul G. [Department of Biology, Queen' s University, Kingston, Ontario (Canada); Mandato, Craig A. [Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec (Canada); Greenwood, Michael T., E-mail: michael.greenwood@rmc.ca [Department of Chemistry and Chemical Engineering, Royal Military College, Kingston, Ontario (Canada)

2016-03-01

Ferritin is a sub-family of iron binding proteins that form multi-subunit nanotype iron storage structures and prevent oxidative stress induced apoptosis. Here we describe the identification and characterization of human ferritin, heavy polypeptide 1 (FTH1) as a suppressor of the pro-apoptotic murine Bax sequence in yeast. In addition we demonstrate that FTH1 is a general pro-survival sequence since it also prevents the cell death inducing effects of copper when heterologously expressed in yeast. Although ferritins are phylogenetically widely distributed and are present in most species of Bacteria, Archaea and Eukarya, ferritin is conspicuously absent in most fungal species including Saccharomyces cerevisiae. An in silico analysis of the yeast proteome lead to the identification of the 161 residue RGI1 (YER067W) encoded protein as a candidate for being a yeast ferritin. In addition to sharing 20% sequence identity with the 183 residue FTH1, RGI1 also has similar pro-survival properties as ferritin when overexpressed in yeast. Analysis of recombinant protein by SDS-PAGE and by electron microscopy revealed the expected formation of higher-order structures for FTH1 that was not observed with Rgi1p. Further analysis revealed that cells overexpressing RGI1 do not show increased resistance to iron toxicity and do not have enhanced capacity to store iron. In contrast, cells lacking RGI1 were found to be hypersensitive to the toxic effects of iron. Overall, our results suggest that Rgi1p is a novel pro-survival protein whose function is not related to ferritin but nevertheless it may have a role in regulating yeast sensitivity to iron stress. - Highlights: • Human ferritin, heavy polypeptide 1 (FTH1) was identified as a suppressor of the pro-apoptotic Bax in yeast. • Based on its similarity to ferritin we examined Rgi1p/YER067W for potential ferritin like functions. • Like human H-ferritin, RGI1 confers increased resistance to apoptotic inducing stresses in yeast. �

Identification of human ferritin, heavy polypeptide 1 (FTH1) and yeast RGI1 (YER067W) as pro-survival sequences that counteract the effects of Bax and copper in Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Eid, Rawan; Boucher, Eric; Gharib, Nada; Khoury, Chamel; Arab, Nagla T.T.; Murray, Alistair; Young, Paul G.; Mandato, Craig A.; Greenwood, Michael T.

2016-01-01

Ferritin is a sub-family of iron binding proteins that form multi-subunit nanotype iron storage structures and prevent oxidative stress induced apoptosis. Here we describe the identification and characterization of human ferritin, heavy polypeptide 1 (FTH1) as a suppressor of the pro-apoptotic murine Bax sequence in yeast. In addition we demonstrate that FTH1 is a general pro-survival sequence since it also prevents the cell death inducing effects of copper when heterologously expressed in yeast. Although ferritins are phylogenetically widely distributed and are present in most species of Bacteria, Archaea and Eukarya, ferritin is conspicuously absent in most fungal species including Saccharomyces cerevisiae. An in silico analysis of the yeast proteome lead to the identification of the 161 residue RGI1 (YER067W) encoded protein as a candidate for being a yeast ferritin. In addition to sharing 20% sequence identity with the 183 residue FTH1, RGI1 also has similar pro-survival properties as ferritin when overexpressed in yeast. Analysis of recombinant protein by SDS-PAGE and by electron microscopy revealed the expected formation of higher-order structures for FTH1 that was not observed with Rgi1p. Further analysis revealed that cells overexpressing RGI1 do not show increased resistance to iron toxicity and do not have enhanced capacity to store iron. In contrast, cells lacking RGI1 were found to be hypersensitive to the toxic effects of iron. Overall, our results suggest that Rgi1p is a novel pro-survival protein whose function is not related to ferritin but nevertheless it may have a role in regulating yeast sensitivity to iron stress. - Highlights: • Human ferritin, heavy polypeptide 1 (FTH1) was identified as a suppressor of the pro-apoptotic Bax in yeast. • Based on its similarity to ferritin we examined Rgi1p/YER067W for potential ferritin like functions. • Like human H-ferritin, RGI1 confers increased resistance to apoptotic inducing stresses in yeast. �

Expression of nm23-H1 gene product in esophageal squamous cell carcinoma and its association with vessel invasion and survival

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Tomita, Masaki; Ayabe, Takanori; Matsuzaki, Yasunori; Edagawa, Masao; Maeda, Masayuki; Shimizu, Tetsuya; Hara, Masaki; Onitsuka, Toshio

2001-01-01

We assessed the nm23-H1 gene product expression and its relationship with lymphatic and blood vessel invasion in patients with esophageal squamous cell carcinoma. Formalin-fixed and paraffin-embedded tissue sections from 45 patients who were treated surgically were used in this study. Pathologists graded lymphatic and blood vessel invasion in each of the tissue samples. Expression of nm23-Hl gene product was determined using a specific monoclonal antibody. Expression of nm23-H1 gene product was present in 17 (37.8%) cases. We found an inverse correlation between nm23-H1 gene product expression and lymphatic vessel invasion, whereas no correlation between nm23-H1 gene product expression and blood vessel invasion. Overall survival rate was not different between nm23-H1 gene product positive and negative patients (p = 0.21). However, reduced expression of nm23-H1 gene product was associated with shorter overall survival in patients with involved lymph nodes (p < 0.05), but not in patients without involved lymph nodes (p = 0.87). In patients with esophageal squamous cell carcinoma, there appears to be an inverse relationship between nm23-H1 gene product expression and lymphatic vessel invasion. Furthermore, nm23-H1 gene product expression might be a prognostic marker in patients with involved lymph nodes. Our data does not demonstrate any correlation between nm23-H1 gene product expression and blood vessel invasion

Molecular and Microenvironmental Determinants of Glioma Stem-Like Cell Survival and Invasion

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Alison Roos

2017-06-01

Full Text Available Glioblastoma multiforme (GBM is the most frequent primary brain tumor in adults with a 5-year survival rate of 5% despite intensive research efforts. The poor prognosis is due, in part, to aggressive invasion into the surrounding brain parenchyma. Invasion is a complex process mediated by cell-intrinsic pathways, extrinsic microenvironmental cues, and biophysical cues from the peritumoral stromal matrix. Recent data have attributed GBM invasion to the glioma stem-like cell (GSC subpopulation. GSCs are slowly dividing, highly invasive, therapy resistant, and are considered to give rise to tumor recurrence. GSCs are localized in a heterogeneous cellular niche, and cross talk between stromal cells and GSCs cultivates a fertile environment that promotes GSC invasion. Pro-migratory soluble factors from endothelial cells, astrocytes, macrophages, microglia, and non-stem-like tumor cells can stimulate peritumoral invasion of GSCs. Therefore, therapeutic efforts designed to target the invasive GSCs may enhance patient survival. In this review, we summarize the current understanding of extrinsic pathways and major stromal and immune players facilitating GSC maintenance and survival.

Increasing pro-survival factors within whole brain tissue of Sprague Dawley rats via intracerebral administration of modified valproic acid

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Ryan C. Bates

2015-08-01

Full Text Available Neural tissue exposure to valproic acid (VPA increases several pro-survival phospho-proteins that can be used as biomarkers for indicating a beneficial drug response (pAktSer473, pGSK3βSer9, pErk1/2Thr202/Tyr204. Unfortunately, targeting VPA to neural tissue is a problem due to severe asymmetrical distribution, wherein the drug tends to remain in peripheral blood rather than localizing within the brain. Intracerebral delivery of an amide-linked VPA–PEG conjugate could address these issues by enhancing retention and promoting cerebro-global increases in pro-survival phospho-proteins. It is necessary to assay for the retained bioactivity of a PEGylated valproic acid molecule, along with locating an intracranial cannula placement that optimizes the increase of a known downstream biomarker for chronic VPA exposure. Here we show an acute injection of VPA–PEG conjugate within brain tissue increased virtually all of the assayed phospho-proteins, including well-known pro-survival factors. In contrast, an acute injection of VPA expectedly decreased signaling throughout the hour. Needle penetration into whole brain tissue is the intentional cause of trauma in this procedure. The trauma to brain tissue was observed to overcome known phospho-protein increases for unmodified VPA in the injected solution, while VPA–PEG conjugate appeared to induce significant increases in pro-survival phospho-proteins, despite the procedural trauma.

Sonic Hedgehog promotes the survival of neural crest cells by limiting apoptosis induced by the dependence receptor CDON during branchial arch development.

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Delloye-Bourgeois, Céline; Rama, Nicolas; Brito, José; Le Douarin, Nicole; Mehlen, Patrick

2014-09-26

Cell-adhesion molecule-related/Downregulated by Oncogenes (CDO or CDON) was identified as a receptor for the classic morphogen Sonic Hedgehog (SHH). It has been shown that, in cell culture, CDO also behaves as a SHH dependence receptor: CDO actively triggers apoptosis in absence of SHH via a proteolytic cleavage in CDO intracellular domain. We present evidence that CDO is also pro-apoptotic in the developing neural tube where SHH is known to act as a survival factor. SHH, produced by the ventral foregut endoderm, was shown to promote survival of facial neural crest cells (NCCs) that colonize the first branchial arch (BA1). We show here that the survival activity of SHH on neural crest cells is due to SHH-mediated inhibition of CDO pro-apoptotic activity. Silencing of CDO rescued NCCs from apoptosis observed upon SHH inhibition in the ventral foregut endoderm. Thus, the pair SHH/dependence receptor CDO may play an important role in neural crest cell survival during the formation of the first branchial arch. Copyright © 2014 Elsevier Inc. All rights reserved.

Hedgehog pathway regulators influence cervical cancer cell proliferation, survival and migration

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Samarzija, Ivana [Ecole Polytechnique Federale Lausanne (EPFL), Department of Life Sciences, Swiss Institute for Experimental Cancer Research (ISREC), 1015 Lausanne (Switzerland); Beard, Peter, E-mail: peter.beard@epfl.ch [Ecole Polytechnique Federale Lausanne (EPFL), Department of Life Sciences, Swiss Institute for Experimental Cancer Research (ISREC), 1015 Lausanne (Switzerland)

2012-08-17

Highlights: Black-Right-Pointing-Pointer Unknown cellular mutations complement papillomavirus-induced carcinogenesis. Black-Right-Pointing-Pointer Hedgehog pathway components are expressed by cervical cancer cells. Black-Right-Pointing-Pointer Hedgehog pathway activators and inhibitors regulate cervical cancer cell biology. Black-Right-Pointing-Pointer Cell immortalization by papillomavirus and activation of Hedgehog are independent. -- Abstract: Human papillomavirus (HPV) infection is considered to be a primary hit that causes cervical cancer. However, infection with this agent, although needed, is not sufficient for a cancer to develop. Additional cellular changes are required to complement the action of HPV, but the precise nature of these changes is not clear. Here, we studied the function of the Hedgehog (Hh) signaling pathway in cervical cancer. The Hh pathway can have a role in a number of cancers, including those of liver, lung and digestive tract. We found that components of the Hh pathway are expressed in several cervical cancer cell lines, indicating that there could exists an autocrine Hh signaling loop in these cells. Inhibition of Hh signaling reduces proliferation and survival of the cervical cancer cells and induces their apoptosis as seen by the up-regulation of the pro-apoptotic protein cleaved caspase 3. Our results indicate that Hh signaling is not induced directly by HPV-encoded proteins but rather that Hh-activating mutations are selected in cells initially immortalized by HPV. Sonic Hedgehog (Shh) ligand induces proliferation and promotes migration of the cervical cancer cells studied. Together, these results indicate pro-survival and protective roles of an activated Hh signaling pathway in cervical cancer-derived cells, and suggest that inhibition of this pathway may be a therapeutic option in fighting cervical cancer.

Hedgehog pathway regulators influence cervical cancer cell proliferation, survival and migration

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Samarzija, Ivana; Beard, Peter

2012-01-01

Highlights: ► Unknown cellular mutations complement papillomavirus-induced carcinogenesis. ► Hedgehog pathway components are expressed by cervical cancer cells. ► Hedgehog pathway activators and inhibitors regulate cervical cancer cell biology. ► Cell immortalization by papillomavirus and activation of Hedgehog are independent. -- Abstract: Human papillomavirus (HPV) infection is considered to be a primary hit that causes cervical cancer. However, infection with this agent, although needed, is not sufficient for a cancer to develop. Additional cellular changes are required to complement the action of HPV, but the precise nature of these changes is not clear. Here, we studied the function of the Hedgehog (Hh) signaling pathway in cervical cancer. The Hh pathway can have a role in a number of cancers, including those of liver, lung and digestive tract. We found that components of the Hh pathway are expressed in several cervical cancer cell lines, indicating that there could exists an autocrine Hh signaling loop in these cells. Inhibition of Hh signaling reduces proliferation and survival of the cervical cancer cells and induces their apoptosis as seen by the up-regulation of the pro-apoptotic protein cleaved caspase 3. Our results indicate that Hh signaling is not induced directly by HPV-encoded proteins but rather that Hh-activating mutations are selected in cells initially immortalized by HPV. Sonic Hedgehog (Shh) ligand induces proliferation and promotes migration of the cervical cancer cells studied. Together, these results indicate pro-survival and protective roles of an activated Hh signaling pathway in cervical cancer-derived cells, and suggest that inhibition of this pathway may be a therapeutic option in fighting cervical cancer.

A PKA survival pathway inhibited by DPT-PKI, a new specific cell permeable PKA inhibitor, is induced by T. annulata in parasitized B-lymphocytes.

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Guergnon, Julien; Dessauge, Frederic; Traincard, François; Cayla, Xavier; Rebollo, Angelita; Bost, Pierre Etienne; Langsley, Gordon; Garcia, Alphonse

2006-08-01

T. annulata, an intracellular pathogenic parasite of the Aplicomplexa protozoan family infects bovine B-lymphocytes and macrophages. Parasitized cells that become transformed survive and proliferate independently of exogenous growth factors. In the present study, we used the isogenic non parasitized BL3 and parasitized TBL3 B cell lines, as a model to evaluate the contribution of two-major PI3-K- and PKA-dependent anti-apoptotic pathways in the survival of T. annulata parasitized B lymphocytes. We found that T. annulata increases PKA activity, induces over-expression of the catalytic subunit and down-regulates the pro-survival phosphorylation state of Akt/PKB. Consistent with a role of PKA activation in survival, two pharmacological inhibitors H89 and KT5720 ablate PKA-dependent survival of parasitized cells. To specifically inhibit PKA pro-survival pathways we linked the DPTsh1 peptide shuttle sequence to PKI(5-24) and we generated DPT-PKI, a cell permeable PKI. DPT-PKI specifically inhibited PKA activity in bovine cell extracts and, as expected, also inhibited the PKA-dependent survival of T. annulata parasitized TBL3 cells. Thus, parasite-dependent constitutive activation of PKA in TBL3 cells generates an anti-apoptotic pathway that can protect T. annulata-infected B cells from apoptosis. These results also indicate that DPT-PKI could be a powerful tool to inhibit PKA pathways in other cell types.

Gene expression profiling of canine osteosarcoma reveals genes associated with short and long survival times

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Rao Nagesha AS

2009-09-01

Full Text Available Abstract Background Gene expression profiling of spontaneous tumors in the dog offers a unique translational opportunity to identify prognostic biomarkers and signaling pathways that are common to both canine and human. Osteosarcoma (OS accounts for approximately 80% of all malignant bone tumors in the dog. Canine OS are highly comparable with their human counterpart with respect to histology, high metastatic rate and poor long-term survival. This study investigates the prognostic gene profile among thirty-two primary canine OS using canine specific cDNA microarrays representing 20,313 genes to identify genes and cellular signaling pathways associated with survival. This, the first report of its kind in dogs with OS, also demonstrates the advantages of cross-species comparison with human OS. Results The 32 tumors were classified into two prognostic groups based on survival time (ST. They were defined as short survivors (dogs with poor prognosis: surviving fewer than 6 months and long survivors (dogs with better prognosis: surviving 6 months or longer. Fifty-one transcripts were found to be differentially expressed, with common upregulation of these genes in the short survivors. The overexpressed genes in short survivors are associated with possible roles in proliferation, drug resistance or metastasis. Several deregulated pathways identified in the present study, including Wnt signaling, Integrin signaling and Chemokine/cytokine signaling are comparable to the pathway analysis conducted on human OS gene profiles, emphasizing the value of the dog as an excellent model for humans. Conclusion A molecular-based method for discrimination of outcome for short and long survivors is useful for future prognostic stratification at initial diagnosis, where genes and pathways associated with cell cycle/proliferation, drug resistance and metastasis could be potential targets for diagnosis and therapy. The similarities between human and canine OS makes the

Globular adiponectin induces a pro-inflammatory response in human astrocytic cells

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Wan, Zhongxiao; Mah, Dorrian; Simtchouk, Svetlana; Klegeris, Andis; Little, Jonathan P.

2014-01-01

Highlights: • Adiponectin receptors are expressed in human astrocytes. • Globular adiponectin induces secretion of IL-6 and MCP-1 from cultured astrocytes. • Adiponectin may play a pro-inflammatory role in astrocytes. - Abstract: Neuroinflammation, mediated in part by activated brain astrocytes, plays a critical role in the development of neurodegenerative disorders, including Alzheimer’s disease (AD). Adiponectin is the most abundant adipokine secreted from adipose tissue and has been reported to exert both anti- and pro-inflammatory effects in peripheral tissues; however, the effects of adiponectin on astrocytes remain unknown. Shifts in peripheral concentrations of adipokines, including adiponectin, could contribute to the observed link between midlife adiposity and increased AD risk. The aim of the present study was to characterize the effects of globular adiponectin (gAd) on pro-inflammatory cytokine mRNA expression and secretion in human U373 MG astrocytic cells and to explore the potential involvement of nuclear factor (NF)-κB, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and phosphatidylinositide 3-kinases (PI3 K) signaling pathways in these processes. We demonstrated expression of adiponectin receptor 1 (adipoR1) and adipoR2 in U373 MG cells and primary human astrocytes. gAd induced secretion of interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1, and gene expression of IL-6, MCP-1, IL-1β and IL-8 in U373 MG cells. Using specific inhibitors, we found that NF-κB, p38MAPK and ERK1/2 pathways are involved in gAd-induced induction of cytokines with ERK1/2 contributing the most. These findings provide evidence that gAd may induce a pro-inflammatory phenotype in human astrocytes

Globular adiponectin induces a pro-inflammatory response in human astrocytic cells

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Wan, Zhongxiao; Mah, Dorrian; Simtchouk, Svetlana [School of Health and Exercise Sciences, University of British Columbia Okanagan, Kelowna, BC (Canada); Klegeris, Andis [Department of Biology, University of British Columbia Okanagan, Kelowna, BC (Canada); Little, Jonathan P., E-mail: jonathan.little@ubc.ca [School of Health and Exercise Sciences, University of British Columbia Okanagan, Kelowna, BC (Canada)

2014-03-28

Highlights: • Adiponectin receptors are expressed in human astrocytes. • Globular adiponectin induces secretion of IL-6 and MCP-1 from cultured astrocytes. • Adiponectin may play a pro-inflammatory role in astrocytes. - Abstract: Neuroinflammation, mediated in part by activated brain astrocytes, plays a critical role in the development of neurodegenerative disorders, including Alzheimer’s disease (AD). Adiponectin is the most abundant adipokine secreted from adipose tissue and has been reported to exert both anti- and pro-inflammatory effects in peripheral tissues; however, the effects of adiponectin on astrocytes remain unknown. Shifts in peripheral concentrations of adipokines, including adiponectin, could contribute to the observed link between midlife adiposity and increased AD risk. The aim of the present study was to characterize the effects of globular adiponectin (gAd) on pro-inflammatory cytokine mRNA expression and secretion in human U373 MG astrocytic cells and to explore the potential involvement of nuclear factor (NF)-κB, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and phosphatidylinositide 3-kinases (PI3 K) signaling pathways in these processes. We demonstrated expression of adiponectin receptor 1 (adipoR1) and adipoR2 in U373 MG cells and primary human astrocytes. gAd induced secretion of interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1, and gene expression of IL-6, MCP-1, IL-1β and IL-8 in U373 MG cells. Using specific inhibitors, we found that NF-κB, p38MAPK and ERK1/2 pathways are involved in gAd-induced induction of cytokines with ERK1/2 contributing the most. These findings provide evidence that gAd may induce a pro-inflammatory phenotype in human astrocytes.

Tumour cell dormancy as a contributor to the reduced survival of GBM patients who received standard therapy.

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Tong, Luqing; Yi, Li; Liu, Peidong; Abeysekera, Iruni Roshanie; Hai, Long; Li, Tao; Tao, Zhennan; Ma, Haiwen; Xie, Yang; Huang, Yubao; Yu, Shengping; Li, Jiabo; Yuan, Feng; Yang, Xuejun

2018-07-01

Glioblastoma multiforme (GBM) is a fatal cancer with varying life expectancy, even for patients undergoing the same standard therapy. Identification of differentially expressed genes in GBM patients with different survival rates may benefit the development of effective therapeutic strategies. In the present study, key pathways and genes correlated with survival in GBM patients were screened with bioinformatic analysis. Included in the study were 136 eligible patients who had undertaken surgical resection of GBM followed by temozolomide (TMZ) chemoradiation and long-term therapy with TMZ. A total of 383 differentially expressed genes (DEGs) related to GBM survival were identified. Gene Ontology and pathway enrichment analysis as well as hub gene screening and module analysis were performed. As expected, angiogenesis and migration of GBM cells were closely correlated with a poor prognosis. Importantly, the results also indicated that cell dormancy was an essential contributor to the reduced survival of GBM patients. Given the lack of specific targeted genes and pathways known to be involved in tumour cell dormancy, we proposed enriched candidate genes related to the negative regulation of cell proliferation, signalling pathways regulating pluripotency of stem cells and neuroactive ligand-receptor interaction, and 3 hub genes (FTH1, GRM1 and DDIT3). Maintaining persistent cell dormancy or preventing tumour cells from entering dormancy during chemoradiation should be a promising therapeutic strategy.

Targeting multiple pro-apoptotic signaling pathways with curcumin in prostate cancer cells.

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Mariela Rivera

Full Text Available Curcumin, an extract from the turmeric rhizome (Curcuma longa, is known to exhibit anti-inflammatory, antioxidant, chemopreventive and antitumoral activities against aggressive and recurrent cancers. Accumulative data indicate that curcumin may induce cancer cell death. However, the detailed mechanism underlying its pro-apoptotic and anti-cancer effects remains to be elucidated. In the present study, we examined the signaling pathways triggered by curcumin, specifically, the exact molecular mechanisms of curcumin-induced apoptosis in highly metastatic human prostate cancer cells. The effect of curcumin was evaluated using for the first time in prostate cancer, a gel-free shotgun quantitative proteomic analysis coupled with Tandem Mass Tag isobaric labeling-based-signaling networks. Results were confirmed at the gene expression level by qRT-PCR and at the protein expression level by western blot and flow cytometry. Our findings revealed that curcumin induced an Endoplasmic Reticulum stress-mediated apoptosis in PC3. The mechanisms by which curcumin promoted cell death in these cells were associated with cell cycle arrest, increased reactive oxygen species, autophagy and the Unfolded Protein Response. Furthermore, the upregulation of ER stress was measured using key indicators of ER stress: Glucose-Regulated Protein 78, Inositol-Requiring Enzyme 1 alpha, Protein Disulfide isomerase and Calreticulin. Chronic ER stress induction was concomitant with the upregulation of pro-apoptotic markers (caspases 3,9,12 and Poly (ADP-ribose polymerase. The downregulated proteins include anti-apoptotic and anti-tumor markers, supporting their curcumin-induced pro-apoptotic role in prostate cancer cells. Taken together, these data suggest that curcumin may serve as a promising anticancer agent by inducing a chronic ER stress mediated cell death and activation of cell cycle arrest, UPR, autophagy and oxidative stress responses.

Targeting multiple pro-apoptotic signaling pathways with curcumin in prostate cancer cells

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Rivera, Mariela; Ramos, Yanilda; Rodríguez-Valentín, Madeline; López-Acevedo, Sheila; Cubano, Luis A.; Zou, Jin; Zhang, Qiang; Wang, Guangdi

2017-01-01

Curcumin, an extract from the turmeric rhizome (Curcuma longa), is known to exhibit anti-inflammatory, antioxidant, chemopreventive and antitumoral activities against aggressive and recurrent cancers. Accumulative data indicate that curcumin may induce cancer cell death. However, the detailed mechanism underlying its pro-apoptotic and anti-cancer effects remains to be elucidated. In the present study, we examined the signaling pathways triggered by curcumin, specifically, the exact molecular mechanisms of curcumin-induced apoptosis in highly metastatic human prostate cancer cells. The effect of curcumin was evaluated using for the first time in prostate cancer, a gel-free shotgun quantitative proteomic analysis coupled with Tandem Mass Tag isobaric labeling-based-signaling networks. Results were confirmed at the gene expression level by qRT-PCR and at the protein expression level by western blot and flow cytometry. Our findings revealed that curcumin induced an Endoplasmic Reticulum stress-mediated apoptosis in PC3. The mechanisms by which curcumin promoted cell death in these cells were associated with cell cycle arrest, increased reactive oxygen species, autophagy and the Unfolded Protein Response. Furthermore, the upregulation of ER stress was measured using key indicators of ER stress: Glucose-Regulated Protein 78, Inositol-Requiring Enzyme 1 alpha, Protein Disulfide isomerase and Calreticulin. Chronic ER stress induction was concomitant with the upregulation of pro-apoptotic markers (caspases 3,9,12) and Poly (ADP-ribose) polymerase. The downregulated proteins include anti-apoptotic and anti-tumor markers, supporting their curcumin-induced pro-apoptotic role in prostate cancer cells. Taken together, these data suggest that curcumin may serve as a promising anticancer agent by inducing a chronic ER stress mediated cell death and activation of cell cycle arrest, UPR, autophagy and oxidative stress responses. PMID:28628644

The SaeR/S gene regulatory system induces a pro-inflammatory cytokine response during Staphylococcus aureus infection.

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Robert L Watkins

Full Text Available Community-associated methicillin-resistant Staphylococcus aureus accounts for a large portion of the increased staphylococcal disease incidence and can cause illness ranging from mild skin infections to rapidly fatal sepsis syndromes. Currently, we have limited understanding of S. aureus-derived mechanisms contributing to bacterial pathogenesis and host inflammation during staphylococcal disease. Herein, we characterize an influential role for the saeR/S two-component gene regulatory system in mediating cytokine induction using mouse models of S. aureus pathogenesis. Invasive S. aureus infection induced the production of localized and systemic pro-inflammatory cytokines, including tumor necrosis factor alpha (TNF-α, interferon gamma (IFN-γ, interleukin (IL-6 and IL-2. In contrast, mice infected with an isogenic saeR/S deletion mutant demonstrated significantly reduced pro-inflammatory cytokine levels. Additionally, secreted factors influenced by saeR/S elicited pro-inflammatory cytokines in human blood ex vivo. Our study further demonstrated robust saeR/S-mediated IFN-γ production during both invasive and subcutaneous skin infections. Results also indicated a critical role for saeR/S in promoting bacterial survival and enhancing host mortality during S. aureus peritonitis. Taken together, this study provides insight into specific mechanisms used by S. aureus during staphylococcal disease and characterizes a relationship between a bacterial global regulator of virulence and the production of pro-inflammatory mediators.

Gene trapping identifies a putative tumor suppressor and a new inducer of cell migration

International Nuclear Information System (INIS)

Guardiola-Serrano, Francisca; Haendeler, Judith; Lukosz, Margarete; Sturm, Karsten; Melchner, Harald von; Altschmied, Joachim

2008-01-01

Tumor necrosis factor alpha (TNFα) is a pleiotropic cytokine involved in apoptotic cell death, cellular proliferation, differentiation, inflammation, and tumorigenesis. In tumors it is secreted by tumor associated macrophages and can have both pro- and anti-tumorigenic effects. To identify genes regulated by TNFα, we performed a gene trap screen in the mammary carcinoma cell line MCF-7 and recovered 64 unique, TNFα-induced gene trap integration sites. Among these were the genes coding for the zinc finger protein ZC3H10 and for the transcription factor grainyhead-like 3 (GRHL3). In line with the dual effects of TNFα on tumorigenesis, we found that ZC3H10 inhibits anchorage independent growth in soft agar suggesting a tumor suppressor function, whereas GRHL3 strongly stimulated the migration of endothelial cells which is consistent with an angiogenic, pro-tumorigenic function

A transcriptomic computational analysis of mastic oil-treated Lewis lung carcinomas reveals molecular mechanisms targeting tumor cell growth and survival

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Roussos Charis

2009-12-01

Full Text Available Abstract Background Mastic oil from Pistacia lentiscus variation chia, a blend of bioactive terpenes with recognized medicinal properties, has been recently shown to exert anti-tumor growth activity through inhibition of cancer cell proliferation, survival, angiogenesis and inflammatory response. However, no studies have addressed its mechanisms of action at genome-wide gene expression level. Methods To investigate molecular mechanisms triggered by mastic oil, Lewis Lung Carcinoma cells were treated with mastic oil or DMSO and RNA was collected at five distinct time points (3-48 h. Microarray expression profiling was performed using Illumina mouse-6 v1 beadchips, followed by computational analysis. For a number of selected genes, RT-PCR validation was performed in LLC cells as well as in three human cancer cell lines of different origin (A549, HCT116, K562. PTEN specific inhibition by a bisperovanadium compound was applied to validate its contribution to mastic oil-mediated anti-tumor growth effects. Results In this work we demonstrated that exposure of Lewis lung carcinomas to mastic oil caused a time-dependent alteration in the expression of 925 genes. GO analysis associated expression profiles with several biological processes and functions. Among them, modifications on cell cycle/proliferation, survival and NF-κB cascade in conjunction with concomitant regulation of genes encoding for PTEN, E2F7, HMOX1 (up-regulation and NOD1 (down-regulation indicated some important mechanistic links underlying the anti-proliferative, pro-apoptotic and anti-inflammatory effects of mastic oil. The expression profiles of Hmox1, Pten and E2f7 genes were similarly altered by mastic oil in the majority of test cancer cell lines. Inhibition of PTEN partially reversed mastic oil effects on tumor cell growth, indicating a multi-target mechanism of action. Finally, k-means clustering, organized the significant gene list in eight clusters demonstrating a similar

Alternative splicing, a new target to block cellular gene expression by poliovirus 2A protease

International Nuclear Information System (INIS)

Alvarez, Enrique; Castello, Alfredo; Carrasco, Luis; Izquierdo, Jose M.

2011-01-01

Highlights: → Novel role for poliovirus 2A protease as splicing modulator. → Poliovirus 2A protease inhibits the alternative splicing of pre-mRNAs. → Poliovirus 2A protease blocks the second catalytic step of splicing. -- Abstract: Viruses have developed multiple strategies to interfere with the gene expression of host cells at different stages to ensure their own survival. Here we report a new role for poliovirus 2A pro modulating the alternative splicing of pre-mRNAs. Expression of 2A pro potently inhibits splicing of reporter genes in HeLa cells. Low amounts of 2A pro abrogate Fas exon 6 skipping, whereas higher levels of protease fully abolish Fas and FGFR2 splicing. In vitro splicing of MINX mRNA using nuclear extracts is also strongly inhibited by 2A pro , leading to accumulation of the first exon and the lariat product containing the unspliced second exon. These findings reveal that the mechanism of action of 2A pro on splicing is to selectively block the second catalytic step.

Alternative splicing, a new target to block cellular gene expression by poliovirus 2A protease

Energy Technology Data Exchange (ETDEWEB)

Alvarez, Enrique, E-mail: ealvarez@cbm.uam.es [Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Nicolas Cabrera, 1 Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); Castello, Alfredo; Carrasco, Luis; Izquierdo, Jose M. [Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Nicolas Cabrera, 1 Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain)

2011-10-14

Highlights: {yields} Novel role for poliovirus 2A protease as splicing modulator. {yields} Poliovirus 2A protease inhibits the alternative splicing of pre-mRNAs. {yields} Poliovirus 2A protease blocks the second catalytic step of splicing. -- Abstract: Viruses have developed multiple strategies to interfere with the gene expression of host cells at different stages to ensure their own survival. Here we report a new role for poliovirus 2A{sup pro} modulating the alternative splicing of pre-mRNAs. Expression of 2A{sup pro} potently inhibits splicing of reporter genes in HeLa cells. Low amounts of 2A{sup pro} abrogate Fas exon 6 skipping, whereas higher levels of protease fully abolish Fas and FGFR2 splicing. In vitro splicing of MINX mRNA using nuclear extracts is also strongly inhibited by 2A{sup pro}, leading to accumulation of the first exon and the lariat product containing the unspliced second exon. These findings reveal that the mechanism of action of 2A{sup pro} on splicing is to selectively block the second catalytic step.

Pro-survival role for Parkinson's associated gene DJ-1 revealed in trophically impaired dopaminergic neurons.

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Liviu Aron

2010-04-01

Full Text Available The mechanisms underlying the selective death of substantia nigra (SN neurons in Parkinson disease (PD remain elusive. While inactivation of DJ-1, an oxidative stress suppressor, causes PD, animal models lacking DJ-1 show no overt dopaminergic (DA neuron degeneration in the SN. Here, we show that aging mice lacking DJ-1 and the GDNF-receptor Ret in the DA system display an accelerated loss of SN cell bodies, but not axons, compared to mice that only lack Ret signaling. The survival requirement for DJ-1 is specific for the GIRK2-positive subpopulation in the SN which projects exclusively to the striatum and is more vulnerable in PD. Using Drosophila genetics, we show that constitutively active Ret and associated Ras/ERK, but not PI3K/Akt, signaling components interact genetically with DJ-1. Double loss-of-function experiments indicate that DJ-1 interacts with ERK signaling to control eye and wing development. Our study uncovers a conserved interaction between DJ-1 and Ret-mediated signaling and a novel cell survival role for DJ-1 in the mouse. A better understanding of the molecular connections between trophic signaling, cellular stress and aging could uncover new targets for drug development in PD.

p63 promotes cell survival through fatty acid synthase.

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Venkata Sabbisetti

2009-06-01

Full Text Available There is increasing evidence that p63, and specifically DeltaNp63, plays a central role in both development and tumorigenesis by promoting epithelial cell survival. However, few studies have addressed the molecular mechanisms through which such important function is exerted. Fatty acid synthase (FASN, a key enzyme that synthesizes long-chain fatty acids and is involved in both embryogenesis and cancer, has been recently proposed as a direct target of p53 family members, including p63 and p73. Here we show that knockdown of either total or DeltaN-specific p63 isoforms in squamous cell carcinoma (SCC9 or immortalized prostate epithelial (iPrEC cells caused a decrease in cell viability by inducing apoptosis without affecting the cell cycle. p63 silencing significantly reduced both the expression and the activity of FASN. Importantly, stable overexpression of either FASN or myristoylated AKT (myr-AKT was able to partially rescue cells from cell death induced by p63 silencing. FASN induced AKT phosphorylation and a significant reduction in cell viability was observed when FASN-overexpressing SCC9 cells were treated with an AKT inhibitor after p63 knockdown, indicating that AKT plays a major role in FASN-mediated survival. Activated AKT did not cause any alteration in the FASN protein levels but induced its activity, suggesting that the rescue from apoptosis documented in the p63-silenced cells expressing myr-AKT cells may be partially mediated by FASN. Finally, we demonstrated that p63 and FASN expression are positively associated in clinical squamous cell carcinoma samples as well as in the developing prostate. Taken together, our findings demonstrate that FASN is a functionally relevant target of p63 and is required for mediating its pro-survival effects.

Pancreatic β-cells activate a JunB/ATF3-dependent survival pathway during inflammation

DEFF Research Database (Denmark)

Gurzov, E N; Barthson, J; Marhfour, I

2012-01-01

Destruction of insulin-producing pancreatic β-cells by local autoimmune inflammation is a hallmark of type 1 diabetes. Histochemical analysis of pancreases from non-obese diabetic mice indicated activation of the transcription factor JunB/AP-1 (activator protein-1) after autoimmune infiltration......-cells and human islet cells against pro-inflammatory mediators. These results were confirmed in genetically modified islets derived from Ubi-JunB transgenic mice. Our findings identify ATF3 as a novel downstream target of JunB in the survival mechanism of β-cells under inflammatory stress....

Deletion of Notch1 converts pro-T cells to dendritic cells and promotes thymic B cells by cell-extrinsic and cell-intrinsic mechanisms.

Science.gov (United States)

Feyerabend, Thorsten B; Terszowski, Grzegorz; Tietz, Annette; Blum, Carmen; Luche, Hervé; Gossler, Achim; Gale, Nicholas W; Radtke, Freddy; Fehling, Hans Jörg; Rodewald, Hans-Reimer

2009-01-16

Notch1 signaling is required for T cell development and has been implicated in fate decisions in the thymus. We showed that Notch1 deletion in progenitor T cells (pro-T cells) revealed their latent developmental potential toward becoming conventional and plasmacytoid dendritic cells. In addition, Notch1 deletion in pro-T cells resulted in large numbers of thymic B cells, previously explained by T-to-B cell fate conversion. Single-cell genotyping showed, however, that the majority of these thymic B cells arose from Notch1-sufficient cells by a cell-extrinsic pathway. Fate switching nevertheless exists for a subset of thymic B cells originating from Notch1-deleted pro-T cells. Chimeric mice lacking the Notch ligand delta-like 4 (Dll4) in thymus epithelium revealed an essential role for Dll4 in T cell development. Thus, Notch1-Dll4 signaling fortifies T cell commitment by suppressing non-T cell lineage potential in pro-T cells, and normal Notch1-driven T cell development repels excessive B cells in the thymus.

GCN5 regulates the activation of PI3K/Akt survival pathway in B cells exposed to oxidative stress via controlling gene expressions of Syk and Btk.

Science.gov (United States)

Kikuchi, Hidehiko; Kuribayashi, Futoshi; Takami, Yasunari; Imajoh-Ohmi, Shinobu; Nakayama, Tatsuo

2011-02-25

Histone acetyltransferase(s) (HATs) are involved in the acetylation of core histones, which is an important event for transcription regulation through alterations in the chromatin structure in eukaryotes. General control non-depressible 5 (GCN5) was first identified as a global coactivator and transcription-related HAT. Here we report that GCN5 regulates the activation of phosphatidylinositol 3-kinase (PI3K)/acutely transforming retrovirus AKT8 in rodent T cell lymphoma (Akt) survival pathway in B cells exposed to oxidative stress via controlling gene expressions of spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (Btk). The GCN5-deficiency remarkably caused apoptotic cell death by treatment with exogenous hydrogen peroxide (H(2)O(2)) in chicken DT40 cells. In GCN5-deficient DT40 cells, gene expressions of Syk and Btk, which are involved in activation of PI3K/Akt survival pathway in DT40 cells exposed to exogenous H(2)O(2), were remarkably decreased compared with those in wild type DT40 cells. In addition, phosphorylation of Akt in H(2)O(2)-treated GCN5-deficient cells was remarkably suppressed as compared to that of DT40. Chromatin immunoprecipitation assay revealed that GCN5 binds to proximal 5'-upstream regions of Syk and Btk genes in vivo. These results suggest that GCN5 takes part in transcriptional regulations of the Syk and Btk genes, and plays a key role in epigenetic regulation of PI3K/Akt survival pathway in B cells exposed to reactive oxygen species such as H(2)O(2). Copyright © 2011 Elsevier Inc. All rights reserved.

The Growing Complexity of Cancer Cell Response to DNA-Damaging Agents: Caspase 3 Mediates Cell Death or Survival?

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Razmik Mirzayans

2016-05-01

Full Text Available It is widely stated that wild-type p53 either mediates the activation of cell cycle checkpoints to facilitate DNA repair and promote cell survival, or orchestrates apoptotic cell death following exposure to cancer therapeutic agents. This reigning paradigm has been challenged by numerous discoveries with different human cell types, including solid tumor-derived cell lines. Thus, activation of the p53 signaling pathway by ionizing radiation and other DNA-damaging agents hinders apoptosis and triggers growth arrest (e.g., through premature senescence in some genetic backgrounds; such growth arrested cells remain viable, secrete growth-promoting factors, and give rise to progeny with stem cell-like properties. In addition, caspase 3, which is best known for its role in the execution phase of apoptosis, has been recently reported to facilitate (rather than suppress DNA damage-induced genomic instability and carcinogenesis. This observation is consistent with an earlier report demonstrating that caspase 3 mediates secretion of the pro-survival factor prostaglandin E2, which in turn promotes enrichment of tumor repopulating cells. In this article, we review these and related discoveries and point out novel cancer therapeutic strategies. One of our objectives is to demonstrate the growing complexity of the DNA damage response beyond the conventional “repair and survive, or die” hypothesis.

Gender-Dependent Survival of Allogeneic Trophoblast Stem Cells in Liver

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Epple-Farmer, Jessica; Debeb, Bisrat G.; Smithies, Oliver; Binas, Bert

2012-01-01

In view of the well-known phenomenon of trophoblast immune privilege, trophoblast stem cells (TSCs) might be expected to be immune privileged, which could be of interest for cell or gene therapies. Yet in the ectopic sites tested so far, TSC transplants fail to show noticeable immune privilege and seem to lack physiological support. However, we show here that after portal venous injection, green fluorescent protein (GFP)-labeled TSCs survive for several months in the livers of allogeneic female but not male mice. Gonadectomy experiments revealed that this survival does not require the presence of ovarian hormones but does require the absence of testicular factors. By contrast, GFP-labeled allogeneic embryonic stem cells (ESCs) are reliably rejected; however, these same ESCs survive when mixed with unlabeled TSCs. The protective effect does not require immunological compatibility between ESCs and TSCs. Tumors were not observed in animals with either successfully engrafted TSCs or coinjected ESCs. We conclude that in a suitable hormonal context and location, ectopic TSCs can exhibit and confer immune privilege. These findings suggest applications in cell and gene therapy as well as a new model for studying trophoblast immunology and physiology. PMID:19523327

System-level modeling and simulation of the cell culture microfluidic biochip ProCell

DEFF Research Database (Denmark)

Minhass, Wajid Hassan; Pop, Paul; Madsen, Jan

2010-01-01

Microfluidic biochips offer a promising alternative to a conventional biochemical laboratory. There are two technologies for the microfluidic biochips: droplet-based and flow-based. In this paper we are interested in flow-based microfluidic biochips, where the liquid flows continuously through pre......-defined micro-channels using valves and pumps. We present an approach to the system-level modeling and simulation of a cell culture microfluidic biochip called ProCell, Programmable Cell Culture Chip. ProCell contains a cell culture chamber, which is envisioned to run 256 simultaneous experiments (viewed...

Functional Consequences of Intracellular Proline Levels Manipulation Affecting PRODH/POX-Dependent Pro-Apoptotic Pathways in a Novel in Vitro Cell Culture Model.

Science.gov (United States)

Zareba, Ilona; Surazynski, Arkadiusz; Chrusciel, Marcin; Miltyk, Wojciech; Doroszko, Milena; Rahman, Nafis; Palka, Jerzy

2017-01-01

The effect of impaired intracellular proline availability for proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis was studied. We generated a constitutively knocked-down PRODH/POX MCF-7 breast cancer cell line (MCF-7shPRODH/POX) as a model to analyze the functional consequences of impaired intracellular proline levels. We have used inhibitor of proline utilization in collagen biosynthesis, 2-metoxyestradiol (MOE), inhibitor of prolidase that generate proline, rapamycin (Rap) and glycyl-proline (GlyPro), substrate for prolidase. Collagen and DNA biosynthesis were evaluated by radiometric assays. Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. PRODH/POX knockdown decreased DNA and collagen biosynthesis, whereas increased prolidase activity and intracellular proline level in MCF-7shPRODH/POX cells. All studied compounds decreased cell viability in MCF-7 and MCF-7shPRODH/POX cells. DNA biosynthesis was similarly inhibited by Rap and MOE in both cell lines, but GlyPro inhibited the process only in MCF-7shPRODH/POX and MOE+GlyPro only in MCF-7 cells. All the compounds inhibited collagen biosynthesis, increased prolidase activity and cytoplasmic proline level in MCF-7shPRODH/POX cells and contributed to the induction of pro-survival mode only in MCF-7shPRODH/POX cells. In contrast, all studied compounds upregulated expression of pro-apoptotic protein only in MCF-7 cells. PRODH/POX was confirmed as a driver of apoptosis and proved the eligibility of MCF-7shPRODH/POX cell line as a highly effective model to elucidate the different mechanisms underlying proline utilization or generation in PRODH/POX-dependent pro-apoptotic pathways. © 2017 The Author(s). Published by S. Karger AG, Basel.

Mast cells exert pro-inflammatory effects of relevance to the pathophyisology of tendinopathy.

Science.gov (United States)

Behzad, Hayedeh; Sharma, Aishwariya; Mousavizadeh, Rouhollah; Lu, Alex; Scott, Alex

2013-01-01

We have previously found an increased mast cell density in tendon biopsies from patients with patellar tendinopathy compared to controls. This study examined the influence of mast cells on basic tenocyte functions, including production of the inflammatory mediator prostaglandin E2 (PGE2), extracellular matrix remodeling and matrix metalloproteinase (MMP) gene transcription, and collagen synthesis. Primary human tenocytes were stimulated with an established human mast cell line (HMC-1). Extracellular matrix remodeling was studied by culturing tenocytes in a three-dimensional collagen lattice. Survival/proliferation was assessed with the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay. Levels of mRNA for COX-2, COL1A1, MMP1, and MMP7 were determined by quantitative real-time polymerase chain reaction (qPCR). Cox-2 protein level was assessed by Western blot analysis and type I procollagen was detected by immunofluorescent staining. PGE2 levels were determined using an enzyme-linked immunosorbent assay (ELISA). Mast cells stimulated tenocytes to produce increased levels of COX-2 and the pro-inflammatory mediator PGE2, which in turn decreased COL1A1 mRNA expression. Additionally, mast cells reduced the type I procollagen protein levels produced by tenocytes. Transforming growth factor beta 1 (TGF-β1) was responsible for the induction of Cox-2 and PGE2 by tenocytes. Mast cells increased MMP1 and MMP7 transcription and increased the contraction of a three-dimensional collagen lattice by tenocytes, a phenomenon which was blocked by a pan-MMP inhibitor (Batimastat). Our data demonstrate that mast cell-derived PGE2 reduces collagen synthesis and enhances expression and activities of MMPs in human tenocytes.

Mechanisms of Sensorineural Cell Damage, Death and Survival in the Cochlea

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Allen Frederic Ryan

2015-04-01

Full Text Available The majority of acquired hearing loss, including presbycusis, is caused by irreversible damage to the sensorineural tissues of the cochlea. This article reviews the intracellular mechanisms that contribute to sensorineural damage in the cochlea, as well as the survival signaling pathways that can provide endogenous protection and tissue rescue. These data have primarily been generated in hearing loss not directly related to age. However, there is evidence that similar mechanisms operate in presbycusis. Moreover, accumulation of damage from other causes can contribute to age-related hearing loss. Potential therapeutic interventions to balance opposing but interconnected cell damage and survival pathways, such as antioxidants, anti-apoptotics, and pro-inflammatory cytokine inhibitors, are also discussed.

File list: ALL.Bld.20.AllAg.Pro-B_cells [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available ALL.Bld.20.AllAg.Pro-B_cells mm9 All antigens Blood Pro-B cells SRX1553109,SRX15531...3,SRX1143907 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Bld.20.AllAg.Pro-B_cells.bed ...

Survival advantages conferred to colon cancer cells by E-selectin-induced activation of the PI3K-NFκB survival axis downstream of Death receptor-3

International Nuclear Information System (INIS)

Porquet, Nicolas; Huot, Jacques; Poirier, Andrée; Houle, François; Pin, Anne-Laure; Gout, Stéphanie; Tremblay, Pierre-Luc; Paquet, Éric R; Klinck, Roscoe; Auger, François A

2011-01-01

Extravasation of circulating cancer cells is a key event of metastatic dissemination that is initiated by the adhesion of cancer cells to endothelial cells. It requires interactions between adhesion receptors on endothelial cells and their counter-receptors on cancer cells. Notably, E-selectin, a major endothelial adhesion receptor, interacts with Death receptor-3 present on metastatic colon carcinoma cells. This interaction confers metastatic properties to colon cancer cells by promoting the adhesion of cancer cells to endothelial cells and triggering the activation of the pro-migratory p38 and pro-survival ERK pathways in the cancer cells. In the present study, we investigated further the mechanisms by which the E-selectin-activated pathways downstream of DR3 confer a survival advantage to colon cancer cells. Cell survival has been ascertained by using the WST-1 assay and by evaluating the activation of the PI3 kinase/NFκB survival axis. Apoptosis has been assayed by determining DNA fragmentation by Hoechst staining and by measuring cleavage of caspases-8 and -3. DR3 isoforms have been identified by PCR. For more precise quantification, targeted PCR reactions were carried out, and the amplified products were analyzed by automated chip-based microcapillary electrophoresis on an Agilent 2100 Bioanalyzer instrument. Interaction between DR3-expressing HT29 colon carcinoma cells and E-selectin induces the activation of the PI3K/Akt pathway. Moreover, p65/RelA, the anti-apoptotic subunit of NFκB, is rapidly translocated to the nucleus in response to E-selectin. This translocation is impaired by the PI3K inhibitor LY294002. Furthermore, inhibition of the PI3K/Akt pathway increases the cleavage of caspase 8 in colon cancer cells treated with E-selectin and this effect is still further increased when both ERK and PI3K pathways are concomitantly inhibited. Intriguingly, metastatic colon cancer cell lines such as HT29 and SW620 express higher levels of a splice variant of

Survival advantages conferred to colon cancer cells by E-selectin-induced activation of the PI3K-NFκB survival axis downstream of Death receptor-3

Directory of Open Access Journals (Sweden)

Paquet Éric R

2011-07-01

Full Text Available Abstract Background Extravasation of circulating cancer cells is a key event of metastatic dissemination that is initiated by the adhesion of cancer cells to endothelial cells. It requires interactions between adhesion receptors on endothelial cells and their counter-receptors on cancer cells. Notably, E-selectin, a major endothelial adhesion receptor, interacts with Death receptor-3 present on metastatic colon carcinoma cells. This interaction confers metastatic properties to colon cancer cells by promoting the adhesion of cancer cells to endothelial cells and triggering the activation of the pro-migratory p38 and pro-survival ERK pathways in the cancer cells. In the present study, we investigated further the mechanisms by which the E-selectin-activated pathways downstream of DR3 confer a survival advantage to colon cancer cells. Methods Cell survival has been ascertained by using the WST-1 assay and by evaluating the activation of the PI3 kinase/NFκB survival axis. Apoptosis has been assayed by determining DNA fragmentation by Hoechst staining and by measuring cleavage of caspases-8 and -3. DR3 isoforms have been identified by PCR. For more precise quantification, targeted PCR reactions were carried out, and the amplified products were analyzed by automated chip-based microcapillary electrophoresis on an Agilent 2100 Bioanalyzer instrument. Results Interaction between DR3-expressing HT29 colon carcinoma cells and E-selectin induces the activation of the PI3K/Akt pathway. Moreover, p65/RelA, the anti-apoptotic subunit of NFκB, is rapidly translocated to the nucleus in response to E-selectin. This translocation is impaired by the PI3K inhibitor LY294002. Furthermore, inhibition of the PI3K/Akt pathway increases the cleavage of caspase 8 in colon cancer cells treated with E-selectin and this effect is still further increased when both ERK and PI3K pathways are concomitantly inhibited. Intriguingly, metastatic colon cancer cell lines such as HT

Foxp1 controls mature B cell survival and the development of follicular and B-1 B cells

Science.gov (United States)

Patzelt, Thomas; Keppler, Selina J.; Gorka, Oliver; Thoene, Silvia; Wartewig, Tim; Reth, Michael; Förster, Irmgard; Lang, Roland; Buchner, Maike; Ruland, Jürgen

2018-01-01

The transcription factor Foxp1 is critical for early B cell development. Despite frequent deregulation of Foxp1 in B cell lymphoma, the physiological functions of Foxp1 in mature B cells remain unknown. Here, we used conditional gene targeting in the B cell lineage and report that Foxp1 disruption in developing and mature B cells results in reduced numbers and frequencies of follicular and B-1 B cells and in impaired antibody production upon T cell-independent immunization in vivo. Moreover, Foxp1-deficient B cells are impaired in survival even though they exhibit an increased capacity to proliferate. Transcriptional analysis identified defective expression of the prosurvival Bcl-2 family gene Bcl2l1 encoding Bcl-xl in Foxp1-deficient B cells, and we identified Foxp1 binding in the regulatory region of Bcl2l1. Transgenic overexpression of Bcl2 rescued the survival defect in Foxp1-deficient mature B cells in vivo and restored peripheral B cell numbers. Thus, our results identify Foxp1 as a physiological regulator of mature B cell survival mediated in part via the control of Bcl-xl expression and imply that this pathway might contribute to the pathogenic function of aberrant Foxp1 expression in lymphoma. PMID:29507226

Tumour-Derived Interleukin-1 Beta Induces Pro-inflammatory Response in Human Mesenchymal Stem Cells

DEFF Research Database (Denmark)

Alajez, Nehad M; Al-toub, Mashael; Almusa, Abdulaziz

’ secreted factors as represented by a panel of human cancer cell lines (breast (MCF7 and MDA-MB-231); prostate (PC-3); lung (NCI-H522); colon (HT-29) and head & neck (FaDu)) on the biological characteristics of MSCs. Background Over the past several years, significant amount of research has emerged......, the goal of this study was to assess the cellular and molecular changes in MSCs in response to secreted factors present in conditioned media (CM) from a panel of human tumor cell lines covering a spectrum of human cancers (Breast, Prostate, Lung, colon, and head and neck). Research Morphological changes...... with bipolar processes. In association with phenotypic changes, genome-wide gene expression and bioinformatics analysis revealed an enhanced pro-inflammatory response of those MSCs. Pharmacological inhibitions of FAK and MAPKK severely impaired the pro-inflammatory response of MSCs to tumor CM (~80-99%, and 55...

File list: His.Bld.50.AllAg.Pro-B_cells [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available His.Bld.50.AllAg.Pro-B_cells mm9 Histone Blood Pro-B cells SRX668836,SRX1184113,SRX...09,SRX759800,SRX1143916,SRX1143902 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Bld.50.AllAg.Pro-B_cells.bed ...

File list: His.Bld.20.AllAg.Pro-B_cells [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available His.Bld.20.AllAg.Pro-B_cells mm9 Histone Blood Pro-B cells SRX668836,SRX1184113,SRX...9,SRX1143910,SRX1143916,SRX1143902 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Bld.20.AllAg.Pro-B_cells.bed ...

RACK1 downregulates levels of the pro-apoptotic protein Fem1b in apoptosis-resistant colon cancer cells.

Science.gov (United States)

Subauste, M Cecilia; Ventura-Holman, Tereza; Du, Liqin; Subauste, Jose S; Chan, Shing-Leng; Yu, Victor C; Maher, Joseph F

2009-12-01

Evasion of apoptosis plays an important role in colon cancer progression. Following loss of the Apc tumor suppressor gene in mice, the gene encoding Fem1b is upregulated early in neoplastic intestinal epithelium. Fem1b is a pro-apoptotic protein that interacts with Fas, TNFR1 and Apaf-1, and increased expression of Fem1b induces apoptosis of cancer cells. Fem1b is a homolog of FEM-1, a protein in Caenorhabditis elegans that is negatively regulated by ubiquitination and proteasomal degradation. To study Fem1b regulation in colon cancer progression, we used apoptotis-sensitive SW480 cells, derived from a primary colon cancer, and their isogenic, apoptosis-resistant counterparts SW620 cells, derived from a subsequent metastatic lesion in the same patient. Treatment with proteasome inhibitor increased Fem1b protein levels in SW620 cells, but not in SW480 cells. In SW620 cells we found that endogenous Fem1b co-immunoprecipitates in complexes with RACK1, a protein known to mediate ubiquitination and proteasomal degradation of other pro-apoptotic proteins and to be upregulated in colon cancer. Full-length Fem1b, or the N-terminal region of Fem1b, associated with RACK1 when co-expressed in HEK293T cells, and RACK1 stimulated ubiquitination of Fem1b. RACK1 overexpression in SW620 cells led to downregulation of Fem1b protein levels. Conversely, downregulation of RACK1 led to upregulation of Fem1b protein levels, associated with induction of apoptosis, and this apoptosis was inhibited by blocking Fem1b protein upregulation. In conclusion, RACK1 downregulates levels of the pro-apoptotic protein Fem1b in metastatic, apoptosis-resistant colon cancer cells, which may promote apoptosis-resistance during progression of colon cancer.

[Merkel cell carcinoma: cutaneous manifestation of a highly malignant pre-/pro-B cell neoplasia? : Novel concept about the cellular origin of Merkel cell carcinoma].

Science.gov (United States)

Sauer, C M; Chteinberg, E; Rennspiess, D; Kurz, A K; Zur Hausen, A

2017-03-01

Merkel cell carcinoma (MCC) is a relatively rare but highly malignant non-melanoma skin cancer of the elderly and immunosuppressed patients. The discovery of the Merkel cell polyomavirus (MCPyV) in 2008 significantly impacted the understanding of the etiopathogenesis of MCC. MCPyV is clonally integrated into the MCC genome and approximately 80% of MCC are MCPyV-positive. Recent results of clinical trials using blockade of the PD-1 immune modulatory pathway are promising for the future treatment of MCC. Despite this major progress of the past few years, the cellular origin of MCC still remains obscure. Based on histomorphology, gene expression profiling, and molecular analyses, we have recently hypothesized that MCC originates from pre‑/pro-B cells. Here we review putative cells of MCC, including Merkel cells, (epi‑)dermal stem cells, and pro‑/pre-B cells. In the present work, the focus is on the concept of pre‑/pro-B cells as the cellular origin of MCC, which might also impact the understanding of other human small cell malignancies of unknown cellular origin, such as small cell carcinomas of the lung and other anatomical locations. In addition, this concept might pave the way for novel treatment options, especially for advanced MCC.

Metagenes Associated with Survival in Non-Small Cell Lung Cancer

Science.gov (United States)

Urgard, Egon; Vooder, Tõnu; Võsa, Urmo; Välk, Kristjan; Liu, Mingming; Luo, Cheng; Hoti, Fabian; Roosipuu, Retlav; Annilo, Tarmo; Laine, Jukka; Frenz, Christopher M.; Zhang, Liqing; Metspalu, Andres

2011-01-01

NSCLC (non-small cell lung cancer) comprises about 80% of all lung cancer cases worldwide. Surgery is most effective treatment for patients with early-stage disease. However, 30%–55% of these patients develop recurrence within 5 years. Therefore, markers that can be used to accurately classify early-stage NSCLC patients into different prognostic groups may be helpful in selecting patients who should receive specific therapies. A previously published dataset was used to evaluate gene expression profiles of different NSCLC subtypes. A moderated two-sample t-test was used to identify differentially expressed genes between all tumor samples and cancer-free control tissue, between SCC samples and AC/BC samples and between stage I tumor samples and all other tumor samples. Gene expression microarray measurements were validated using qRT-PCR. Bayesian regression analysis and Kaplan-Meier survival analysis were performed to determine metagenes associated with survival. We identified 599 genes which were down-regulated and 402 genes which were up-regulated in NSCLC compared to the normal lung tissue and 112 genes which were up-regulated and 101 genes which were down-regulated in AC/BC compared to the SCC. Further, for stage Ib patients the metagenes potentially associated with survival were identified. Genes that expressed differently between normal lung tissue and cancer showed enrichment in gene ontology terms which were associated with mitosis and proliferation. Bayesian regression and Kaplan-Meier analysis showed that gene-expression patterns and metagene profiles can be applied to predict the probability of different survival outcomes in NSCLC patients. PMID:21695068

Functional microarray analysis suggests repressed cell-cell signaling and cell survival-related modules inhibit progression of head and neck squamous cell carcinoma

Directory of Open Access Journals (Sweden)

Soares Fernando A

2011-04-01

Full Text Available Abstract Background Cancer shows a great diversity in its clinical behavior which cannot be easily predicted using the currently available clinical or pathological markers. The identification of pathways associated with lymph node metastasis (N+ and recurrent head and neck squamous cell carcinoma (HNSCC may increase our understanding of the complex biology of this disease. Methods Tumor samples were obtained from untreated HNSCC patients undergoing surgery. Patients were classified according to pathologic lymph node status (positive or negative or tumor recurrence (recurrent or non-recurrent tumor after treatment (surgery with neck dissection followed by radiotherapy. Using microarray gene expression, we screened tumor samples according to modules comprised by genes in the same pathway or functional category. Results The most frequent alterations were the repression of modules in negative lymph node (N0 and in non-recurrent tumors rather than induction of modules in N+ or in recurrent tumors. N0 tumors showed repression of modules that contain cell survival genes and in non-recurrent tumors cell-cell signaling and extracellular region modules were repressed. Conclusions The repression of modules that contain cell survival genes in N0 tumors reinforces the important role that apoptosis plays in the regulation of metastasis. In addition, because tumor samples used here were not microdissected, tumor gene expression data are represented together with the stroma, which may reveal signaling between the microenvironment and tumor cells. For instance, in non-recurrent tumors, extracellular region module was repressed, indicating that the stroma and tumor cells may have fewer interactions, which disable metastasis development. Finally, the genes highlighted in our analysis can be implicated in more than one pathway or characteristic, suggesting that therapeutic approaches to prevent tumor progression should target more than one gene or pathway

Osteocalcin protects pancreatic beta cell function and survival under high glucose conditions

Energy Technology Data Exchange (ETDEWEB)

Kover, Karen, E-mail: kkover@cmh.edu [Division of Endocrine/Diabetes, Children' s Mercy Hospital & Clinics, Kansas City, MO 64108 (United States); University of Missouri-Kansas City School of Medicine, Kansas City, MO 64108 (United States); Yan, Yun; Tong, Pei Ying; Watkins, Dara; Li, Xiaoyu [Division of Endocrine/Diabetes, Children' s Mercy Hospital & Clinics, Kansas City, MO 64108 (United States); University of Missouri-Kansas City School of Medicine, Kansas City, MO 64108 (United States); Tasch, James; Hager, Melissa [Kansas City University Medical Biosciences, Kansas City, MO (United States); Clements, Mark; Moore, Wayne V. [Division of Endocrine/Diabetes, Children' s Mercy Hospital & Clinics, Kansas City, MO 64108 (United States); University of Missouri-Kansas City School of Medicine, Kansas City, MO 64108 (United States)

2015-06-19

Diabetes is characterized by progressive beta cell dysfunction and loss due in part to oxidative stress that occurs from gluco/lipotoxicity. Treatments that directly protect beta cell function and survival in the diabetic milieu are of particular interest. A growing body of evidence suggests that osteocalcin, an abundant non-collagenous protein of bone, supports beta cell function and proliferation. Based on previous gene expression data by microarray, we hypothesized that osteocalcin protects beta cells from glucose-induced oxidative stress. To test our hypothesis we cultured isolated rat islets and INS-1E cells in the presence of normal, high, or high glucose ± osteocalcin for up to 72 h. Oxidative stress and viability/mitochondrial function were measured by H{sub 2}O{sub 2} assay and Alamar Blue assay, respectively. Caspase 3/7 activity was also measured as a marker of apoptosis. A functional test, glucose stimulated insulin release, was conducted and expression of genes/protein was measured by qRT-PCR/western blot/ELISA. Osteocalcin treatment significantly reduced high glucose-induced H{sub 2}O{sub 2} levels while maintaining viability/mitochondrial function. Osteocalcin also significantly improved glucose stimulated insulin secretion and insulin content in rat islets after 48 h of high glucose exposure compared to untreated islets. As expected sustained high glucose down-regulated gene/protein expression of INS1 and BCL2 while increasing TXNIP expression. Interestingly, osteocalcin treatment reversed the effects of high glucose on gene/protein expression. We conclude that osteocalcin can protect beta cells from the negative effects of glucose-induced oxidative stress, in part, by reducing TXNIP expression, thereby preserving beta cell function and survival. - Highlights: • Osteocalcin reduces glucose-induced oxidative stress in beta cells. • Osteocalcin preserves beta cell function and survival under stress conditions. • Osteocalcin reduces glucose

Osteocalcin protects pancreatic beta cell function and survival under high glucose conditions

International Nuclear Information System (INIS)

Kover, Karen; Yan, Yun; Tong, Pei Ying; Watkins, Dara; Li, Xiaoyu; Tasch, James; Hager, Melissa; Clements, Mark; Moore, Wayne V.

2015-01-01

Diabetes is characterized by progressive beta cell dysfunction and loss due in part to oxidative stress that occurs from gluco/lipotoxicity. Treatments that directly protect beta cell function and survival in the diabetic milieu are of particular interest. A growing body of evidence suggests that osteocalcin, an abundant non-collagenous protein of bone, supports beta cell function and proliferation. Based on previous gene expression data by microarray, we hypothesized that osteocalcin protects beta cells from glucose-induced oxidative stress. To test our hypothesis we cultured isolated rat islets and INS-1E cells in the presence of normal, high, or high glucose ± osteocalcin for up to 72 h. Oxidative stress and viability/mitochondrial function were measured by H 2 O 2 assay and Alamar Blue assay, respectively. Caspase 3/7 activity was also measured as a marker of apoptosis. A functional test, glucose stimulated insulin release, was conducted and expression of genes/protein was measured by qRT-PCR/western blot/ELISA. Osteocalcin treatment significantly reduced high glucose-induced H 2 O 2 levels while maintaining viability/mitochondrial function. Osteocalcin also significantly improved glucose stimulated insulin secretion and insulin content in rat islets after 48 h of high glucose exposure compared to untreated islets. As expected sustained high glucose down-regulated gene/protein expression of INS1 and BCL2 while increasing TXNIP expression. Interestingly, osteocalcin treatment reversed the effects of high glucose on gene/protein expression. We conclude that osteocalcin can protect beta cells from the negative effects of glucose-induced oxidative stress, in part, by reducing TXNIP expression, thereby preserving beta cell function and survival. - Highlights: • Osteocalcin reduces glucose-induced oxidative stress in beta cells. • Osteocalcin preserves beta cell function and survival under stress conditions. • Osteocalcin reduces glucose-induced TXNIP

Pro-inflammatory stimulation of meniscus cells increases production of matrix metalloproteinases and additional catabolic factors involved in osteoarthritis pathogenesis

Science.gov (United States)

Stone, Austin V.; Loeser, Richard F.; Vanderman, Kadie S.; Long, David L.; Clark, Stephanie C.; Ferguson, Cristin M.

2014-01-01

Objective Meniscus injury increases the risk of osteoarthritis; however, the biologic mechanism remains unknown. We hypothesized that pro-inflammatory stimulation of meniscus would increase production of matrix-degrading enzymes, cytokines and chemokines which cause joint tissue destruction and could contribute to osteoarthritis development. Design Meniscus and cartilage tissue from healthy tissue donors and total knee arthroplasties was cultured. Primary cell cultures were stimulated with pro-inflammatory factors [IL-1β, IL-6, or fibronectin fragments (FnF)] and cellular responses were analyzed by real-time PCR, protein arrays and immunoblots. To determine if NF-κB was required for MMP production, meniscus cultures were treated with inflammatory factors with and without the NF-κB inhibitor, hypoestoxide. Results Normal and osteoarthritic meniscus cells increased their MMP secretion in response to stimulation, but specific patterns emerged that were unique to each stimulus with the greatest number of MMPs expressed in response to FnF. Meniscus collagen and connective tissue growth factor gene expression was reduced. Expression of cytokines (IL-1α, IL-1β, IL-6), chemokines (IL-8, CXCL1, CXCL2, CSF1) and components of the NF-κB and tumor necrosis factor (TNF) family were significantly increased. Cytokine and chemokine protein production was also increased by stimulation. When primary cell cultures were treated with hypoestoxide in conjunction with pro-inflammatory stimulation, p65 activation was reduced as were MMP-1 and MMP-3 production. Conclusions Pro-inflammatory stimulation of meniscus cells increased matrix metalloproteinase production and catabolic gene expression. The meniscus could have an active biologic role in osteoarthritis development following joint injury through increased production of cytokines, chemokines, and matrix-degrading enzymes. PMID:24315792

Chronic occupational exposure to arsenic induces carcinogenic gene signaling networks and neoplastic transformation in human lung epithelial cells

International Nuclear Information System (INIS)

Stueckle, Todd A.; Lu, Yongju; Davis, Mary E.; Wang, Liying; Jiang, Bing-Hua; Holaskova, Ida; Schafer, Rosana; Barnett, John B.; Rojanasakul, Yon

2012-01-01

Chronic arsenic exposure remains a human health risk; however a clear mode of action to understand gene signaling-driven arsenic carcinogenesis is currently lacking. This study chronically exposed human lung epithelial BEAS-2B cells to low-dose arsenic trioxide to elucidate cancer promoting gene signaling networks associated with arsenic-transformed (B-As) cells. Following a 6 month exposure, exposed cells were assessed for enhanced cell proliferation, colony formation, invasion ability and in vivo tumor formation compared to control cell lines. Collected mRNA was subjected to whole genome expression microarray profiling followed by in silico Ingenuity Pathway Analysis (IPA) to identify lung carcinogenesis modes of action. B-As cells displayed significant increases in proliferation, colony formation and invasion ability compared to BEAS-2B cells. B-As injections into nude mice resulted in development of primary and secondary metastatic tumors. Arsenic exposure resulted in widespread up-regulation of genes associated with mitochondrial metabolism and increased reactive oxygen species protection suggesting mitochondrial dysfunction. Carcinogenic initiation via reactive oxygen species and epigenetic mechanisms was further supported by altered DNA repair, histone, and ROS-sensitive signaling. NF-κB, MAPK and NCOR1 signaling disrupted PPARα/δ-mediated lipid homeostasis. A ‘pro-cancer’ gene signaling network identified increased survival, proliferation, inflammation, metabolism, anti-apoptosis and mobility signaling. IPA-ranked signaling networks identified altered p21, EF1α, Akt, MAPK, and NF-κB signaling networks promoting genetic disorder, altered cell cycle, cancer and changes in nucleic acid and energy metabolism. In conclusion, transformed B-As cells with their whole genome expression profile provide an in vitro arsenic model for future lung cancer signaling research and data for chronic arsenic exposure risk assessment. Highlights: ► Chronic As 2 O 3

Chronic occupational exposure to arsenic induces carcinogenic gene signaling networks and neoplastic transformation in human lung epithelial cells

Energy Technology Data Exchange (ETDEWEB)

Stueckle, Todd A., E-mail: tstueckle@hsc.wvu.edu [Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown, WV 26506 (United States); Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WV 26505 (United States); Lu, Yongju, E-mail: yongju6@hotmail.com [Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown, WV 26506 (United States); Davis, Mary E., E-mail: mdavis@wvu.edu [Department of Physiology, West Virginia University, Morgantown, WV 26506 (United States); Wang, Liying, E-mail: lmw6@cdc.gov [Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WV 26505 (United States); Jiang, Bing-Hua, E-mail: bhjiang@jefferson.edu [Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107 (United States); Holaskova, Ida, E-mail: iholaskova@hsc.wvu.edu [Department of Microbiology, Immunology and Cell Biology, West Virginia University, Morgantown, WV 26506 (United States); Schafer, Rosana, E-mail: rschafer@hsc.wvu.edu [Department of Microbiology, Immunology and Cell Biology, West Virginia University, Morgantown, WV 26506 (United States); Barnett, John B., E-mail: jbarnett@hsc.wvu.edu [Department of Microbiology, Immunology and Cell Biology, West Virginia University, Morgantown, WV 26506 (United States); Rojanasakul, Yon, E-mail: yrojan@hsc.wvu.edu [Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown, WV 26506 (United States)

2012-06-01

Chronic arsenic exposure remains a human health risk; however a clear mode of action to understand gene signaling-driven arsenic carcinogenesis is currently lacking. This study chronically exposed human lung epithelial BEAS-2B cells to low-dose arsenic trioxide to elucidate cancer promoting gene signaling networks associated with arsenic-transformed (B-As) cells. Following a 6 month exposure, exposed cells were assessed for enhanced cell proliferation, colony formation, invasion ability and in vivo tumor formation compared to control cell lines. Collected mRNA was subjected to whole genome expression microarray profiling followed by in silico Ingenuity Pathway Analysis (IPA) to identify lung carcinogenesis modes of action. B-As cells displayed significant increases in proliferation, colony formation and invasion ability compared to BEAS-2B cells. B-As injections into nude mice resulted in development of primary and secondary metastatic tumors. Arsenic exposure resulted in widespread up-regulation of genes associated with mitochondrial metabolism and increased reactive oxygen species protection suggesting mitochondrial dysfunction. Carcinogenic initiation via reactive oxygen species and epigenetic mechanisms was further supported by altered DNA repair, histone, and ROS-sensitive signaling. NF-κB, MAPK and NCOR1 signaling disrupted PPARα/δ-mediated lipid homeostasis. A ‘pro-cancer’ gene signaling network identified increased survival, proliferation, inflammation, metabolism, anti-apoptosis and mobility signaling. IPA-ranked signaling networks identified altered p21, EF1α, Akt, MAPK, and NF-κB signaling networks promoting genetic disorder, altered cell cycle, cancer and changes in nucleic acid and energy metabolism. In conclusion, transformed B-As cells with their whole genome expression profile provide an in vitro arsenic model for future lung cancer signaling research and data for chronic arsenic exposure risk assessment. Highlights: ► Chronic As{sub 2}O

Comparative analysis of the survival and gene expression of pathogenic strains Vibrio harveyi after starvation.

Science.gov (United States)

Sun, Jingjing; Gao, Xiaojian; Qun, Jiang; Du, Xuedi; Bi, Keran; Zhang, Xiaojun; Lin, Li

2016-11-01

This study aimed to evaluate the survival and gene expression of Vibrio harveyi under starvation conditions. The microcosms V. harveyi were incubated in sterilized seawater for 4 weeks at room temperature. Overall, the cell numeration declined rapidly about 10 3 CFU/ml during starvation, with a tiny rebound at day 21. Scanning electron microscopy revealed that rod-shaped cells became sphere with a rippled cell surface. By polymerase chain reaction (PCR) assay, nine genes, named luxR, toxR, vhhB, flaA, topA, fur, rpoS, mreB and ftsZ, were detected in the non-starved cells. In the starved cells, the expression levels of the detected genes declined substantially ranging from 0.005-fold to 0.028-fold compared to the non-starved cells performed by reverse transcription quantitative real-time PCR with 16S rRNA as the internal control. In the recovering cells, the expression levels of the detected genes, except luxR and mreB, were upregulated dramatically compared to the wild, especially topA (23.720-fold), fur (39.400-fold) and toxR (9.837-fold), validating that the expressions of both the metabolism and virulence genes were important for growth and survival of V. harveyi. The results may shed a new light on understanding of stress adaptation in bacteria. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Collagen Promotes Higher Adhesion, Survival and Proliferation of Mesenchymal Stem Cells.

Directory of Open Access Journals (Sweden)

Chinnapaka Somaiah

Full Text Available Mesenchymal stem cells (MSC can differentiate into several cell types and are desirable candidates for cell therapy and tissue engineering. However, due to poor cell survival, proliferation and differentiation in the patient, the therapy outcomes have not been satisfactory. Although several studies have been done to understand the conditions that promote proliferation, differentiation and migration of MSC in vitro and in vivo, still there is no clear understanding on the effect of non-cellular bio molecules. Of the many factors that influence the cell behavior, the immediate cell microenvironment plays a major role. In this context, we studied the effect of extracellular matrix (ECM proteins in controlling cell survival, proliferation, migration and directed MSC differentiation. We found that collagen promoted cell proliferation, cell survival under stress and promoted high cell adhesion to the cell culture surface. Increased osteogenic differentiation accompanied by high active RHOA (Ras homology gene family member A levels was exhibited by MSC cultured on collagen. In conclusion, our study shows that collagen will be a suitable matrix for large scale production of MSC with high survival rate and to obtain high osteogenic differentiation for therapy.

Resveratrol post-transcriptionally regulates pro-inflammatory gene expression via regulation of KSRP RNA binding activity

Science.gov (United States)

Bollmann, Franziska; Art, Julia; Henke, Jenny; Schrick, Katharina; Besche, Verena; Bros, Matthias; Li, Huige; Siuda, Daniel; Handler, Norbert; Bauer, Florian; Erker, Thomas; Behnke, Felix; Mönch, Bettina; Härdle, Lorena; Hoffmann, Markus; Chen, Ching-Yi; Förstermann, Ulrich; Dirsch, Verena M.; Werz, Oliver; Kleinert, Hartmut; Pautz, Andrea

2014-01-01

Resveratrol shows beneficial effects in inflammation-based diseases like cancer, cardiovascular and chronic inflammatory diseases. Therefore, the molecular mechanisms of the anti-inflammatory resveratrol effects deserve more attention. In human epithelial DLD-1 and monocytic Mono Mac 6 cells resveratrol decreased the expression of iNOS, IL-8 and TNF-α by reducing mRNA stability without inhibition of the promoter activity. Shown by pharmacological and siRNA-mediated inhibition, the observed effects are SIRT1-independent. Target-fishing and drug responsive target stability experiments showed selective binding of resveratrol to the RNA-binding protein KSRP, a central post-transcriptional regulator of pro-inflammatory gene expression. Knockdown of KSRP expression prevented resveratrol-induced mRNA destabilization in human and murine cells. Resveratrol did not change KSRP expression, but immunoprecipitation experiments indicated that resveratrol reduces the p38 MAPK-related inhibitory KSRP threonine phosphorylation, without blocking p38 MAPK activation or activity. Mutation of the p38 MAPK target site in KSRP blocked the resveratrol effect on pro-inflammatory gene expression. In addition, resveratrol incubation enhanced KSRP-exosome interaction, which is important for mRNA degradation. Finally, resveratrol incubation enhanced its intra-cellular binding to the IL-8, iNOS and TNF-α mRNA. Therefore, modulation of KSRP mRNA binding activity and, thereby, enhancement of mRNA degradation seems to be the common denominator of many anti-inflammatory effects of resveratrol. PMID:25352548

Association of mRNA expression of TP53 and the TP53 codon 72 Arg/Pro gene polymorphism with colorectal cancer risk in Asian population: a bioinformatics analysis and meta-analysis.

Science.gov (United States)

Dong, Zhiyong; Zheng, Longzhi; Liu, Weimin; Wang, Cunchuan

2018-01-01

The relationship between TP53 codon 72 Pro/Arg gene polymorphism and colorectal cancer risk in Asians is still controversial, and this bioinformatics analysis and meta-analysis was performed to assess the associations. The association studies were identified from PubMed, and eligible reports were included. RevMan 5.3.1 software, Oncolnc, cBioPortal, and Oncomine online tools were used for statistical analysis. A random/fixed effects model was used in meta-analysis. The data were reported as risk ratios or mean differences with corresponding 95% CI. We confirmed that TP53 was associated with colorectal cancer, the alteration frequency of TP53 was 53% mutation and 7% deep deletion, and TP53 mRNA expression was different in different types of colorectal cancer based on The Cancer Genome Atlas database. Then, 18 studies were included that examine the association of TP53 codon 72 gene polymorphism with colorectal cancer risk in Asians. The meta-analysis indicated that TP53 Pro allele and Pro/Pro genotype were associated with colorectal cancer risk in Asian population, but Arg/Arg genotype was not (Pro allele: odds ratios [OR]=1.20, 95% CI: 1.06 to 1.35, P =0.003; Pro/Pro genotype: OR=1.39, 95% CI: 1.15 to 1.69, P =0.0007; Arg/Arg genotype: OR=0.86, 95% CI: 0.74 to 1.00, P =0.05). Interestingly, in the meta-analysis of the controls from the population-based studies, we found that TP53 codon 72 Pro/Arg gene polymorphism was associated with colorectal cancer risk (Pro allele: OR=1.33, 95% CI: 1.15 to 1.55, P =0.0002; Pro/Pro genotype: OR=1.61, 95% CI: 1.28 to 2.02, P colorectal cancer, but the different value levels of mRNA expression were not associated with survival rate of colon and rectal cancer. TP53 Pro allele and Pro/Pro genotype were associated with colorectal cancer risk in Asians.

The temporal dynamics of differential gene expression in Aspergillus fumigatus interacting with human immature dendritic cells in vitro.

LENUS (Irish Health Repository)

Morton, Charles O

2011-01-01

Dendritic cells (DC) are the most important antigen presenting cells and play a pivotal role in host immunity to infectious agents by acting as a bridge between the innate and adaptive immune systems. Monocyte-derived immature DCs (iDC) were infected with viable resting conidia of Aspergillus fumigatus (Af293) for 12 hours at an MOI of 5; cells were sampled every three hours. RNA was extracted from both organisms at each time point and hybridised to microarrays. iDC cell death increased at 6 h in the presence of A. fumigatus which coincided with fungal germ tube emergence; >80% of conidia were associated with iDC. Over the time course A. fumigatus differentially regulated 210 genes, FunCat analysis indicated significant up-regulation of genes involved in fermentation, drug transport, pathogenesis and response to oxidative stress. Genes related to cytotoxicity were differentially regulated but the gliotoxin biosynthesis genes were down regulated over the time course, while Aspf1 was up-regulated at 9 h and 12 h. There was an up-regulation of genes in the subtelomeric regions of the genome as the interaction progressed. The genes up-regulated by iDC in the presence of A. fumigatus indicated that they were producing a pro-inflammatory response which was consistent with previous transcriptome studies of iDC interacting with A. fumigatus germ tubes. This study shows that A. fumigatus adapts to phagocytosis by iDCs by utilising genes that allow it to survive the interaction rather than just up-regulation of specific virulence genes.

The temporal dynamics of differential gene expression in Aspergillus fumigatus interacting with human immature dendritic cells in vitro.

Directory of Open Access Journals (Sweden)

Charles O Morton

2011-01-01

Full Text Available Dendritic cells (DC are the most important antigen presenting cells and play a pivotal role in host immunity to infectious agents by acting as a bridge between the innate and adaptive immune systems. Monocyte-derived immature DCs (iDC were infected with viable resting conidia of Aspergillus fumigatus (Af293 for 12 hours at an MOI of 5; cells were sampled every three hours. RNA was extracted from both organisms at each time point and hybridised to microarrays. iDC cell death increased at 6 h in the presence of A. fumigatus which coincided with fungal germ tube emergence; >80% of conidia were associated with iDC. Over the time course A. fumigatus differentially regulated 210 genes, FunCat analysis indicated significant up-regulation of genes involved in fermentation, drug transport, pathogenesis and response to oxidative stress. Genes related to cytotoxicity were differentially regulated but the gliotoxin biosynthesis genes were down regulated over the time course, while Aspf1 was up-regulated at 9 h and 12 h. There was an up-regulation of genes in the subtelomeric regions of the genome as the interaction progressed. The genes up-regulated by iDC in the presence of A. fumigatus indicated that they were producing a pro-inflammatory response which was consistent with previous transcriptome studies of iDC interacting with A. fumigatus germ tubes. This study shows that A. fumigatus adapts to phagocytosis by iDCs by utilising genes that allow it to survive the interaction rather than just up-regulation of specific virulence genes.

Radiation Gene-expression Signatures in Primary Breast Cancer Cells.

Science.gov (United States)

Minafra, Luigi; Bravatà, Valentina; Cammarata, Francesco P; Russo, Giorgio; Gilardi, Maria C; Forte, Giusi I

2018-05-01

In breast cancer (BC) care, radiation therapy (RT) is an efficient treatment to control localized tumor. Radiobiological research is needed to understand molecular differences that affect radiosensitivity of different tumor subtypes and the response variability. The aim of this study was to analyze gene expression profiling (GEP) in primary BC cells following irradiation with doses of 9 Gy and 23 Gy delivered by intraoperative electron radiation therapy (IOERT) in order to define gene signatures of response to high doses of ionizing radiation. We performed GEP by cDNA microarrays and evaluated cell survival after IOERT treatment in primary BC cell cultures. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to validate candidate genes. We showed, for the first time, a 4-gene and a 6-gene signature, as new molecular biomarkers, in two primary BC cell cultures after exposure at 9 Gy and 23 Gy respectively, for which we observed a significantly high survival rate. Gene signatures activated by different doses of ionizing radiation may predict response to RT and contribute to defining a personalized biological-driven treatment plan. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Differential gene expression in porcine SK6 cells infected with wild-type and SAP domain-mutant foot-and-mouth disease virus.

Science.gov (United States)

Ni, Zixin; Yang, Fan; Cao, Weijun; Zhang, Xiangle; Jin, Ye; Mao, Ruoqing; Du, Xiaoli; Li, Weiwei; Guo, Jianhong; Liu, Xiangtao; Zhu, Zixiang; Zheng, Haixue

2016-06-01

Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious disease in livestock. The viral proteinase L(pro) of FMDV is involved in pathogenicity, and mutation of the L(pro) SAP domain reduces FMDV pathogenicity in pigs. To determine the gene expression profiles associated with decreased pathogenicity in porcine cells, we performed transcriptome analysis using next-generation sequencing technology and compared differentially expressed genes in SK6 cells infected with FMDV containing L(pro) with either a wild-type or mutated version of the SAP domain. This analysis yielded 1,853 genes that exhibited a ≥ 2-fold change in expression and was validated by real-time quantitative PCR detection of several differentially expressed genes. Many of the differentially expressed genes correlated with antiviral responses corresponded to genes associated with transcription factors, immune regulation, cytokine production, inflammatory response, and apoptosis. Alterations in gene expression profiles may be responsible for the variations in pathogenicity observed between the two FMDV variants. Our results provided genes of interest for the further study of antiviral pathways and pathogenic mechanisms related to FMDV L(pro).

Intermittent Hypoxia Alters Gene Expression in Peripheral Blood Mononuclear Cells of Healthy Volunteers.

Science.gov (United States)

Polotsky, Vsevolod Y; Bevans-Fonti, Shannon; Grigoryev, Dmitry N; Punjabi, Naresh M

2015-01-01

Obstructive sleep apnea is associated with high cardiovascular morbidity and mortality. Intermittent hypoxia of obstructive sleep apnea is implicated in the development and progression of insulin resistance and atherosclerosis, which have been attributed to systemic inflammation. Intermittent hypoxia leads to pro-inflammatory gene up-regulation in cell culture, but the effects of intermittent hypoxia on gene expression in humans have not been elucidated. A cross-over study was performed exposing eight healthy men to intermittent hypoxia or control conditions for five hours with peripheral blood mononuclear cell isolation before and after exposures. Total RNA was isolated followed by gene microarrays and confirmatory real time reverse transcriptase PCR. Intermittent hypoxia led to greater than two fold up-regulation of the pro-inflammatory gene toll receptor 2 (TLR2), which was not increased in the control exposure. We hypothesize that up-regulation of TLR2 by intermittent hypoxia may lead to systemic inflammation, insulin resistance and atherosclerosis in patients with obstructive sleep apnea.

[Expression of plant antimicrobial peptide pro-SmAMP2 gene increases resistance of transgenic potato plants to Alternaria and Fusarium pathogens].

Science.gov (United States)

Vetchinkina, E M; Komakhina, V V; Vysotskii, D A; Zaitsev, D V; Smirnov, A N; Babakov, A V; Komakhin, R A

2016-09-01

The chickweed (Stellaria media L.) pro-SmAMP2 gene encodes the hevein-like peptides that have in vitro antimicrobial activity against certain harmful microorganisms. These peptides play an important role in protecting the chickweed plants from infection, and the pro-SmAMP2 gene was previously used to protect transgenic tobacco and Arabidopsis plants from phytopathogens. In this study, the pro-SmAMP2 gene under control of viral CaMV35S promoter or under control of its own pro-SmAMP2 promoter was transformed into cultivated potato plants of two cultivars, differing in the resistance to Alternaria: Yubiley Zhukova (resistant) and Skoroplodny (susceptible). With the help of quantitative real-time PCR, it was demonstrated that transgenic potato plants expressed the pro-SmAMP2 gene under control of both promoters at the level comparable to or exceeding the level of the potato actin gene. Assessment of the immune status of the transformants demonstrated that expression of antimicrobial peptide pro-SmAMP2 gene was able to increase the resistance to a complex of Alternaria sp. and Fusarium sp. phytopathogens only in potato plants of the Yubiley Zhukova cultivar. The possible role of the pro-SmAMP2 products in protecting potatoes from Alternaria sp. and Fusarium sp. is discussed.

SIRT1 regulates MAPK pathways in vitiligo skin: insight into the molecular pathways of cell survival

Science.gov (United States)

Becatti, Matteo; Fiorillo, Claudia; Barygina, Victoria; Cecchi, Cristina; Lotti, Torello; Prignano, Francesca; Silvestro, Agrippino; Nassi, Paolo; Taddei, Niccolò

2014-01-01

Vitiligo is an acquired and progressive hypomelanotic disease that manifests as circumscribed depigmented patches on the skin. The aetiology of vitiligo remains unclear, but recent experimental data underline the interactions between melanocytes and other typical skin cells, particularly keratinocytes. Our previous results indicate that keratinocytes from perilesional skin show the features of damaged cells. Sirtuins (silent mating type information regulation 2 homolog) 1, well-known modulators of lifespan in many species, have a role in gene repression, metabolic control, apoptosis and cell survival, DNA repair, development, inflammation, neuroprotection and healthy ageing. In the literature there is no evidence for SIRT1 signalling in vitiligo and its possible involvement in disease progression. Here, biopsies were taken from the perilesional skin of 16 patients suffering from non-segmental vitiligo and SIRT1 signalling was investigated in these cells. For the first time, a new SIRT1/Akt, also known as Protein Kinase B (PKB)/mitogen-activated protein kinase (MAPK) signalling has been revealed in vitiligo. SIRT1 regulates MAPK pathway via Akt-apoptosis signal-regulating kinase-1 and down-regulates pro-apoptotic molecules, leading to decreased oxidative stress and apoptotic cell death in perilesional vitiligo keratinocytes. We therefore propose SIRT1 activation as a novel way of protecting perilesional vitiligo keratinocytes from damage. PMID:24410795

Photoreceptor Differentiation following Transplantation of Allogeneic Retinal Progenitor Cells to the Dystrophic Rhodopsin Pro347Leu Transgenic Pig

DEFF Research Database (Denmark)

Klassen, H; Kiilgaard, Jens Folke; Warfvinge, K

2012-01-01

Purpose. Transplantation of stem, progenitor, or precursor cells has resulted in photoreceptor replacement and evidence of functional efficacy in rodent models of retinal degeneration. Ongoing work has been directed toward the replication of these results in a large animal model, namely, the pig....... Methods. Retinal progenitor cells were derived from the neural retina of GFP-transgenic pigs and transplanted to the subretinal space of rhodopsin Pro347Leu-transgenic allorecipients, in the early stage of the degeneration and the absence of immune suppression. Results. Results confirm the survival...

Integrative analysis of survival-associated gene sets in breast cancer.

Science.gov (United States)

Varn, Frederick S; Ung, Matthew H; Lou, Shao Ke; Cheng, Chao

2015-03-12

Patient gene expression information has recently become a clinical feature used to evaluate breast cancer prognosis. The emergence of prognostic gene sets that take advantage of these data has led to a rich library of information that can be used to characterize the molecular nature of a patient's cancer. Identifying robust gene sets that are consistently predictive of a patient's clinical outcome has become one of the main challenges in the field. We inputted our previously established BASE algorithm with patient gene expression data and gene sets from MSigDB to develop the gene set activity score (GSAS), a metric that quantitatively assesses a gene set's activity level in a given patient. We utilized this metric, along with patient time-to-event data, to perform survival analyses to identify the gene sets that were significantly correlated with patient survival. We then performed cross-dataset analyses to identify robust prognostic gene sets and to classify patients by metastasis status. Additionally, we created a gene set network based on component gene overlap to explore the relationship between gene sets derived from MSigDB. We developed a novel gene set based on this network's topology and applied the GSAS metric to characterize its role in patient survival. Using the GSAS metric, we identified 120 gene sets that were significantly associated with patient survival in all datasets tested. The gene overlap network analysis yielded a novel gene set enriched in genes shared by the robustly predictive gene sets. This gene set was highly correlated to patient survival when used alone. Most interestingly, removal of the genes in this gene set from the gene pool on MSigDB resulted in a large reduction in the number of predictive gene sets, suggesting a prominent role for these genes in breast cancer progression. The GSAS metric provided a useful medium by which we systematically investigated how gene sets from MSigDB relate to breast cancer patient survival. We used

A Large-Scale RNAi Screen Identifies SGK1 as a Key Survival Kinase for GBM Stem Cells.

Science.gov (United States)

Kulkarni, Shreya; Goel-Bhattacharya, Surbhi; Sengupta, Sejuti; Cochran, Brent H

2018-01-01

Glioblastoma multiforme (GBM) is the most common type of primary malignant brain cancer and has a very poor prognosis. A subpopulation of cells known as GBM stem-like cells (GBM-SC) have the capacity to initiate and sustain tumor growth and possess molecular characteristics similar to the parental tumor. GBM-SCs are known to be enriched in hypoxic niches and may contribute to therapeutic resistance. Therefore, to identify genetic determinants important for the proliferation and survival of GBM stem cells, an unbiased pooled shRNA screen of 10,000 genes was conducted under normoxic as well as hypoxic conditions. A number of essential genes were identified that are required for GBM-SC growth, under either or both oxygen conditions, in two different GBM-SC lines. Interestingly, only about a third of the essential genes were common to both cell lines. The oxygen environment significantly impacts the cellular genetic dependencies as 30% of the genes required under hypoxia were not required under normoxic conditions. In addition to identifying essential genes already implicated in GBM such as CDK4, KIF11 , and RAN , the screen also identified new genes that have not been previously implicated in GBM stem cell biology. The importance of the serum and glucocorticoid-regulated kinase 1 (SGK1) for cellular survival was validated in multiple patient-derived GBM stem cell lines using shRNA, CRISPR, and pharmacologic inhibitors. However, SGK1 depletion and inhibition has little effect on traditional serum grown glioma lines and on differentiated GBM-SCs indicating its specific importance in GBM stem cell survival. Implications: This study identifies genes required for the growth and survival of GBM stem cells under both normoxic and hypoxic conditions and finds SGK1 as a novel potential drug target for GBM. Mol Cancer Res; 16(1); 103-14. ©2017 AACR . ©2017 American Association for Cancer Research.

p70S6 kinase signals cell survival as well as growth, inactivating the pro-apoptotic molecule BAD

DEFF Research Database (Denmark)

Harada, H; Andersen, Jens S.; Mann, M

2001-01-01

Cytokines often deliver simultaneous, yet distinct, cell growth and cell survival signals. The 70-kDa ribosomal protein S6 kinase (p70S6K) is known to regulate cell growth by inducing protein synthesis components. We purified membrane-based p70S6K as a kinase responsible for site-specific phospho...

File list: NoD.Bld.10.AllAg.Pro-B_cells [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available NoD.Bld.10.AllAg.Pro-B_cells mm9 No description Blood Pro-B cells SRX1553109,SRX155...3108,SRX1553107,SRX1553110 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Bld.10.AllAg.Pro-B_cells.bed ...

File list: NoD.Bld.50.AllAg.Pro-B_cells [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available NoD.Bld.50.AllAg.Pro-B_cells mm9 No description Blood Pro-B cells SRX1553109,SRX155...3108,SRX1553107,SRX1553110 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Bld.50.AllAg.Pro-B_cells.bed ...

Musashi1 modulates cell proliferation genes in the medulloblastoma cell line Daoy

Directory of Open Access Journals (Sweden)

Hung Jaclyn Y

2008-09-01

Full Text Available Abstract Background Musashi1 (Msi1 is an RNA binding protein with a central role during nervous system development and stem cell maintenance. High levels of Msi1 have been reported in several malignancies including brain tumors thereby associating Msi1 and cancer. Methods We used the human medulloblastoma cell line Daoy as model system in this study to knock down the expression of Msi1 and determine the effects upon soft agar growth and neurophere formation. Quantitative RT-PCR was conducted to evaluate the expression of cell proliferation, differentiation and survival genes in Msi1 depleted Daoy cells. Results We observed that MSI1 expression was elevated in Daoy cells cultured as neurospheres compared to those grown as monolayer. These data indicated that Msi1 might be involved in regulating proliferation in cancer cells. Here we show that shRNA mediated Msi1 depletion in Daoy cells notably impaired their ability to form colonies in soft agar and to grow as neurospheres in culture. Moreover, differential expression of a group of Notch, Hedgehog and Wnt pathway related genes including MYCN, FOS, NOTCH2, SMO, CDKN1A, CCND2, CCND1, and DKK1, was also found in the Msi1 knockdown, demonstrating that Msi1 modulated the expression of a subset of cell proliferation, differentiation and survival genes in Daoy. Conclusion Our data suggested that Msi1 may promote cancer cell proliferation and survival as its loss seems to have a detrimental effect in the maintenance of medulloblastoma cancer cells. In this regard, Msi1 might be a positive regulator of tumor progression and a potential target for therapy.

Cell model for the study of receptor and regulatory functions of human proHB-EGF

Directory of Open Access Journals (Sweden)

N. V. Korotkevych

2014-08-01

Full Text Available Developing of new models and approaches, particularly with fluorescent techniques, for investigation of intracellular transport of proHB-EGF and its ligand-receptor complexes is strongly required. In order to create a model for studying proHB-EGF functions the genetic construction pEGFP-N1-proHB-EGF, encoding proHB-EGF-EGFP which is fluorescent-labeled form of proHB-EGF with enhanced green fluorescent protein EGFP in the cytoplasmic terminus of the molecule, was obtained. Eukaryotic cells expressing fusion protein proHB-EGF-EGFP on the cell surface were obtained by transfection with pEGFP-N1-proHB-EGF. Expressed in the Vero cells proHB-EGF-EGFP could bind fluorescent derivative of nontoxic receptor-binding subunit B of diphtheria toxin mCherry-SubB. After stimulation of transfected cells with TPA (12-O-Tetradecanoylphorbol-13-acetate, proHB-EGF-EGFP formed a fluorescentl-labeled C-terminal fragment of the molecule – CTF-EGFP. Thus, the obtained genetic construction pEGFP-N1-proHB-EGF could be helpful in visualization of molecules proHB-EGF and CTF in cells, may open new possibilities for the studying of their functions, such as receptor function of proHB-EGF for diphtheria toxin, intracellular translocation of CTF and provide possibilities for natural proHB-EGF ligands search.

Changes of tumorigenicity and gene expressions of melanoma cells mutated in outer space

International Nuclear Information System (INIS)

Xiang Qing; Xu Mei; Xiao Cheng; Xu Bo; Li Hongyan; Geng Chuanying; Pan Lin; Fang Qing; Guo Yupeng; Tang Jingtian

2006-01-01

Objective: To screen the cell lines with decreased tumorigenicity, and identify those genes related to the development and metastasis of melanoma. Methods: The murine melanoma B16 cells were carried into the outer space by No. 20 retrievable satellite, and the survival cells were cloned after returned to earth. Five monoclonal cell lines(No.1, No.5, No.6, No.7, No.8) were utilized for further study. The cells were injected into C57BL/6J mice subcutaneously and abdominally respectively, then tumor-free time and survival time were recorded, tumor lumps were examined by routine pathological method. Gene chips were used to detect the gene expressions in 2 cell lines(No.1, No.8)with decreased tumorigenicity. Results: Compared with the control group, the tumor-free time was longer for No.1 cell lines (P<0.05). The survival time was significantly increased (P<0.01) and the weights of tumors were significantly decreased (P<0.01) for both No.1 and No.8 cell lines. The lymphocytes were infiltrated into tumors and adjacent tissues in those mice injected with No.1 and No.8 cell lines. Changes in 145 gene expressions were identified in No.1 cell lines, and 124 genes in No.8 cell lines (P<0.05), 9 genes of them were common to both cell lines. Furthermore, prostaglandin D2 synthase gene was markedly upregulated. Conclusion: The study implied that the decrease of tumorigenicity was related to the changes of carcinoma-associated gene expressions. (authors)

Cholesterol and phytosterols differentially regulate the expression of caveolin 1 and a downstream prostate cell growth-suppressor gene

Science.gov (United States)

Ifere, Godwin O.; Equan, Anita; Gordon, Kereen; Nagappan, Peri; Igietseme, Joseph U.; Ananaba, Godwin A.

2010-01-01

Background The purpose of our study was to show the distinction between the apoptotic and anti-proliferative signaling of phytosterols and cholesterol enrichment in prostate cancer cell lines, mediated by the differential transcription of caveolin-1, and N-myc downstream regulated gene1 (NDRG1), a pro-apoptotic androgen-regulated tumor suppressor. Methods PC-3 and DU145 cells were treated with sterols (cholesterol and phytosterols) for 72 h, followed by trypan blue dye exclusion measurement of necrosis and cell growth measured with a Coulter counter. Sterol induction of cell growth-suppressor gene expression was evaluated by mRNA transcription using RT-PCR, while cell cycle analysis was performed by FACS analysis. Altered expression of Ndrg1 protein was confirmed by Western blot analysis. Apoptosis was evaluated by real time RT-PCR amplification of P53, Bcl-2 gene and its related pro- and anti-apoptotic family members. Results Physiological doses (16 µM) of cholesterol and phytosterols were not cytotoxic in these cells. Cholesterol enrichment promoted cell growth (Pphytosterols significantly induced growth-suppression (Pphytosterols decreased mitotic subpopulations. We demonstrated for the first time that cholesterols concertedly attenuated the expression of caveolin-1(cav-1) and NDRG1 genes in both prostate cancer cell lines. Phytosterols had the opposite effect by inducing overexpression of cav-1, a known mediator of androgen-dependent signals that presumably control cell growth or apoptosis. Conclusions Cholesterol and phytosterol treatment differentially regulated the growth of prostate cancer cells and the expression of p53 and cav-1, a gene that regulates androgen-regulated signals. These sterols also differentially regulated cell cycle arrest, downstream pro-apoptotic androgen-regulated tumor-suppressor, NDRG1 suggesting that cav-1 may mediate pro-apoptotic NDRG1 signals. Elucidation of the mechanism for sterol modulation of growth and apoptosis signaling

Progranulin is expressed within motor neurons and promotes neuronal cell survival

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Kay Denis G

2009-10-01

Full Text Available Abstract Background Progranulin is a secreted high molecular weight growth factor bearing seven and one half copies of the cysteine-rich granulin-epithelin motif. While inappropriate over-expression of the progranulin gene has been associated with many cancers, haploinsufficiency leads to atrophy of the frontotemporal lobes and development of a form of dementia (frontotemporal lobar degeneration with ubiquitin positive inclusions, FTLD-U associated with the formation of ubiquitinated inclusions. Recent reports indicate that progranulin has neurotrophic effects, which, if confirmed would make progranulin the only neuroprotective growth factor that has been associated genetically with a neurological disease in humans. Preliminary studies indicated high progranulin gene expression in spinal cord motor neurons. However, it is uncertain what the role of Progranulin is in normal or diseased motor neuron function. We have investigated progranulin gene expression and subcellular localization in cultured mouse embryonic motor neurons and examined the effect of progranulin over-expression and knockdown in the NSC-34 immortalized motor neuron cell line upon proliferation and survival. Results In situ hybridisation and immunohistochemical techniques revealed that the progranulin gene is highly expressed by motor neurons within the mouse spinal cord and in primary cultures of dissociated mouse embryonic spinal cord-dorsal root ganglia. Confocal microscopy coupled to immunocytochemistry together with the use of a progranulin-green fluorescent protein fusion construct revealed progranulin to be located within compartments of the secretory pathway including the Golgi apparatus. Stable transfection of the human progranulin gene into the NSC-34 motor neuron cell line stimulates the appearance of dendritic structures and provides sufficient trophic stimulus to survive serum deprivation for long periods (up to two months. This is mediated at least in part through

Ubiquinol decreases monocytic expression and DNA methylation of the pro-inflammatory chemokine ligand 2 gene in humans

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Fischer Alexandra

2012-10-01

Full Text Available Abstract Background Coenzyme Q10 is an essential cofactor in the respiratory chain and serves in its reduced form, ubiquinol, as a potent antioxidant. Studies in vitro and in vivo provide evidence that ubiquinol reduces inflammatory processes via gene expression. Here we investigate the putative link between expression and DNA methylation of ubiquinol sensitive genes in monocytes obtained from human volunteers supplemented with 150 mg/ day ubiquinol for 14 days. Findings Ubiquinol decreases the expression of the pro-inflammatory chemokine (C-X-C motif ligand 2 gene (CXCL2 more than 10-fold. Bisulfite-/ MALDI-TOF-based analysis of regulatory regions of the CXCL2 gene identified six adjacent CpG islands which showed a 3.4-fold decrease of methylation status after ubiquinol supplementation. This effect seems to be rather gene specific, because ubiquinol reduced the expression of two other pro-inflammatory genes (PMAIP1, MMD without changing the methylation pattern of the respective gene. Conclusion In conclusion, ubiquinol decreases monocytic expression and DNA methylation of the pro-inflammatory CXCL2 gene in humans. Current Controlled Trials ISRCTN26780329.

Clinical prognostic significance and pro-metastatic activity of RANK/RANKL via the AKT pathway in endometrial cancer.

Science.gov (United States)

Wang, Jing; Liu, Yao; Wang, Lihua; Sun, Xiao; Wang, Yudong

2016-02-02

RANK/RANKL plays a key role in metastasis of certain malignant tumors, which makes it a promising target for developing novel therapeutic strategies for cancer. However, the prognostic value and pro-metastatic activity of RANK in endometrial cancer (EC) remain to be determined. Thus, the present study investigated the effect of RANK on the prognosis of EC patients, as well as the pro-metastatic activity of EC cells. The results indicated that those with high expression of RANK showed decreased overall survival and progression-free survival. Statistical analysis revealed the positive correlations between RANK/RANKL expression and metastasis-related factors. Additionally, RANK/RANKL significantly promoted cell migration/invasion via activating AKT/β-catenin/Snail pathway in vitro. However, RANK/RANKL-induced AKT activation could be suppressed after osteoprotegerin (OPG) treatment. Furthermore, the combination of medroxyprogesterone acetate (MPA) and RANKL could in turn attenuate the effect of RANKL alone. Similarly, MPA could partially inhibit the RANK-induced metastasis in an orthotopic mouse model via suppressing AKT/β-catenin/Snail pathway. Therefore, therapeutic inhibition of MPA in RANK/RANKL-induced metastasis was mediated by AKT/β-catenin/Snail pathway both in vitro and in vivo, suggesting a potential target of RANK for gene-based therapy for EC.

Regulation of radiation-induced apoptosis by early growth response-1 gene in solid tumors

International Nuclear Information System (INIS)

Ahmed, M.

2003-01-01

Ionizing radiation exposure is associated with activation of certain immediate-early genes that function as transcription factors. These include members of jun or fos and early growth response (EGR) gene families. In particular, the functional role of EGR-1 in radiation-induced signaling is pivotal since the promoter of EGR-1 contains radiation-inducible CArG DNA sequences. The Egr-1 gene belongs to a family of Egr genes that includes EGR-2, EGR-3, EGR-4, EGR-α and the tumor suppressor, Wilms' tumor gene product, WT1. The Egr-1 gene product, EGR-1, is a nuclear protein that contains three zinc fingers of the C 2 H 2 subtype. The EGR-1 GC-rich consensus target sequence, 5'-GCGT/GGGGCG-3' or 5'-TCCT/ACCTCCTCC-3', has been identified in the promoter regions of transcription factors, growth factors, receptors, cell cycle regulators and pro-apoptotic genes. The gene targets mediated by Egr-1 in response to ionizing radiation include TNF-α , p53, Rb and Bax, all these are effectors of apoptosis. Based on these targets, Egr-1 is a pivotal gene that initiates early signal transduction events in response to ionizing radiation leading to either growth arrest or cell death in tumor cells. There are two potential application of Egr-1 gene in therapy of cancer. First, the Egr-1 promoter contains information for appropriate spatial and temporal expression in-vivo that can be regulated by ionizing radiation to control transcription of genes that have pro-apoptotic and suicidal function. Secondly, EGR-1 protein can eliminate 'induced-radiation resistance' by inhibiting the functions of radiation-induced pro-survival genes (NFκB activity and bcl-2 expression) and activate pro-apoptotic genes (such as bax) to confer a significant radio-sensitizing effect. Together, the reported findings from my laboratory demonstrate clearly that EGR-1 is an early central gene that confers radiation sensitivity and its pro-apoptotic functions are synergized by abrogation of induced radiation

EDAG promotes the expansion and survival of human CD34+ cells.

Directory of Open Access Journals (Sweden)

Ke Zhao

Full Text Available EDAG is multifunctional transcriptional regulator primarily expressed in the linloc-kit+Sca-1+ hematopoietic stem cells (HSC and CD34+ progenitor cells. Previous studies indicate that EDAG is required for maintaining hematopoietic lineage commitment balance. Here using ex vivo culture and HSC transplantation models, we report that EDAG enhances the proliferative potential of human cord blood CD34+ cells, increases survival, prevents cell apoptosis and promotes their repopulating capacity. Moreover, EDAG overexpression induces rapid entry of CD34+ cells into the cell cycle. Gene expression profile analysis indicate that EDAG knockdown leads to down-regulation of various positive cell cycle regulators including cyclin A, B, D, and E. Together these data provides novel insights into EDAG in regulation of expansion and survival of human hematopoietic stem/progenitor cells.

Tumor-adjacent tissue co-expression profile analysis reveals pro-oncogenic ribosomal gene signature for prognosis of resectable hepatocellular carcinoma.

Science.gov (United States)

Grinchuk, Oleg V; Yenamandra, Surya P; Iyer, Ramakrishnan; Singh, Malay; Lee, Hwee Kuan; Lim, Kiat Hon; Chow, Pierce Kah-Hoe; Kuznetsov, Vladamir A

2018-01-01

Currently, molecular markers are not used when determining the prognosis and treatment strategy for patients with hepatocellular carcinoma (HCC). In the present study, we proposed that the identification of common pro-oncogenic pathways in primary tumors (PT) and adjacent non-malignant tissues (AT) typically used to predict HCC patient risks may result in HCC biomarker discovery. We examined the genome-wide mRNA expression profiles of paired PT and AT samples from 321 HCC patients. The workflow integrated differentially expressed gene selection, gene ontology enrichment, computational classification, survival predictions, image analysis and experimental validation methods. We developed a 24-ribosomal gene-based HCC classifier (RGC), which is prognostically significant in both PT and AT. The RGC gene overexpression in PT was associated with a poor prognosis in the training (hazard ratio = 8.2, P = 9.4 × 10 -6 ) and cross-cohort validation (hazard ratio = 2.63, P = 0.004) datasets. The multivariate survival analysis demonstrated the significant and independent prognostic value of the RGC. The RGC displayed a significant prognostic value in AT of the training (hazard ratio = 5.0, P = 0.03) and cross-validation (hazard ratio = 1.9, P = 0.03) HCC groups, confirming the accuracy and robustness of the RGC. Our experimental and bioinformatics analyses suggested a key role for c-MYC in the pro-oncogenic pattern of ribosomal biogenesis co-regulation in PT and AT. Microarray, quantitative RT-PCR and quantitative immunohistochemical studies of the PT showed that DKK1 in PT is the perspective biomarker for poor HCC outcomes. The common co-transcriptional pattern of ribosome biogenesis genes in PT and AT from HCC patients suggests a new scalable prognostic system, as supported by the model of tumor-like metabolic redirection/assimilation in non-malignant AT. The RGC, comprising 24 ribosomal genes, is introduced as a robust and reproducible prognostic model for

File list: InP.Bld.50.AllAg.Pro-B_cells [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available InP.Bld.50.AllAg.Pro-B_cells mm9 Input control Blood Pro-B cells SRX1143898,SRX6688...12,SRX759802,SRX759803,SRX1143899,SRX1143900,SRX1143913,SRX1143907 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Bld.50.AllAg.Pro-B_cells.bed ...

Regulatory dendritic cell infusion prolongs kidney allograft survival in non-human primates

OpenAIRE

Ezzelarab, M.; Zahorchak, A.F.; Lu, L.; Morelli, A.E.; Chalasani, G.; Demetris, A.J.; Lakkis, F.G.; Wijkstrom, M.; Murase, N.; Humar, A.; Shapiro, R.; Cooper, D.K.C.; Thomson, A.W.

2013-01-01

We examined the influence of regulatory dendritic cells (DCreg), generated from cytokine-mobilized donor blood monocytes in vitamin D3 and IL-10, on renal allograft survival in a clinically-relevant rhesus macaque model. DCreg expressed low MHC class II and costimulatory molecules, but comparatively high levels of programmed death ligand-1 (B7-H1), and were resistant to pro-inflammatory cytokine-induced maturation. They were infused intravenously (3.5–10×106/kg), together with the B7-CD28 cos...

A stromal cell free culture system generates mouse pro-T cells that can reconstitute T-cell compartments in vivo.

Science.gov (United States)

Gehre, Nadine; Nusser, Anja; von Muenchow, Lilly; Tussiwand, Roxane; Engdahl, Corinne; Capoferri, Giuseppina; Bosco, Nabil; Ceredig, Rhodri; Rolink, Antonius G

2015-03-01

T-cell lymphopenia following BM transplantation or diseases such as AIDS result in immunodeficiency. Novel approaches to ameliorate this situation are urgently required. Herein, we describe a novel stromal cell free culture system in which Lineage(-) Sca1(+)c-kit(+) BM hematopoietic progenitors very efficiently differentiate into pro-T cells. This culture system consists of plate-bound Delta-like 4 Notch ligand and the cytokines SCF and IL-7. The pro-T cells developing in these cultures express CD25, CD117, and partially CD44; express cytoplasmic CD3ε; and have their TCRβ locus partially D-J rearranged. They could be expanded for over 3 months and used to reconstitute the T-cell compartments of sublethally irradiated T-cell-deficient CD3ε(-/-) mice or lethally irradiated WT mice. Pro-T cells generated in this system could partially correct the T-cell lymphopenia of pre-Tα(-/-) mice. However, reconstituted CD3ε(-/-) mice suffered from a wasting disease that was prevented by co-injection of purified CD4(+) CD25(high) WT Treg cells. In a T-cell-sufficient or T-lymphopenic setting, the development of disease was not observed. Thus, this in vitro culture system represents a powerful tool to generate large numbers of pro-T cells for transplantation and possibly with clinical applications. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Gene expression analysis of cell death induction by Taurolidine in different malignant cell lines

International Nuclear Information System (INIS)

Chromik, Ansgar M; Weyhe, Dirk; Mittelkötter, Ulrich; Uhl, Waldemar; Hahn, Stephan A; Daigeler, Adrien; Flier, Annegret; Bulut, Daniel; May, Christina; Harati, Kamran; Roschinsky, Jan; Sülberg, Dominique

2010-01-01

The anti-infective agent Taurolidine (TRD) has been shown to have cell death inducing properties, but the mechanism of its action is largely unknown. The aim of this study was to identify potential common target genes modulated at the transcriptional level following TRD treatment in tumour cell lines originating from different cancer types. Five different malignant cell lines (HT29, Chang Liver, HT1080, AsPC-1 and BxPC-3) were incubated with TRD (100 μM, 250 μM and 1000 μM). Proliferation after 8 h and cell viability after 24 h were analyzed by BrdU assay and FACS analysis, respectively. Gene expression analyses were carried out using the Agilent -microarray platform to indentify genes which displayed conjoint regulation following the addition of TRD in all cell lines. Candidate genes were subjected to Ingenuity Pathways Analysis and selected genes were validated by qRT-PCR and Western Blot. TRD 250 μM caused a significant inhibition of proliferation as well as apoptotic cell death in all cell lines. Among cell death associated genes with the strongest regulation in gene expression, we identified pro-apoptotic transcription factors (EGR1, ATF3) as well as genes involved in the ER stress response (PPP1R15A), in ubiquitination (TRAF6) and mitochondrial apoptotic pathways (PMAIP1). This is the first conjoint analysis of potential target genes of TRD which was performed simultaneously in different malignant cell lines. The results indicate that TRD might be involved in different signal transduction pathways leading to apoptosis

A hemocyte gene expression signature correlated with predictive capacity of oysters to survive Vibrio infections

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Rosa Rafael

2012-06-01

Full Text Available Abstract Background The complex balance between environmental and host factors is an important determinant of susceptibility to infection. Disturbances of this equilibrium may result in multifactorial diseases as illustrated by the summer mortality syndrome, a worldwide and complex phenomenon that affects the oysters, Crassostrea gigas. The summer mortality syndrome reveals a physiological intolerance making this oyster species susceptible to diseases. Exploration of genetic basis governing the oyster resistance or susceptibility to infections is thus a major goal for understanding field mortality events. In this context, we used high-throughput genomic approaches to identify genetic traits that may characterize inherent survival capacities in C. gigas. Results Using digital gene expression (DGE, we analyzed the transcriptomes of hemocytes (immunocompetent cells of oysters able or not able to survive infections by Vibrio species shown to be involved in summer mortalities. Hemocytes were nonlethally collected from oysters before Vibrio experimental infection, and two DGE libraries were generated from individuals that survived or did not survive. Exploration of DGE data and microfluidic qPCR analyses at individual level showed an extraordinary polymorphism in gene expressions, but also a set of hemocyte-expressed genes whose basal mRNA levels discriminate oyster capacity to survive infections by the pathogenic V. splendidus LGP32. Finally, we identified a signature of 14 genes that predicted oyster survival capacity. Their expressions are likely driven by distinct transcriptional regulation processes associated or not associated to gene copy number variation (CNV. Conclusions We provide here for the first time in oyster a gene expression survival signature that represents a useful tool for understanding mortality events and for assessing genetic traits of interest for disease resistance selection programs.

Xanomeline suppresses excessive pro-inflammatory cytokine responses through neural signal-mediated pathways and improves survival in lethal inflammation

Science.gov (United States)

Rosas-Ballina, Mauricio; Ferrer, Sergio Valdés; Dancho, Meghan; Ochani, Mahendar; Katz, David; Cheng, Kai Fan; Olofsson, Peder S.; Chavan, Sangeeta S.; Al-Abed, Yousef; Tracey, Kevin J.; Pavlov, Valentin A.

2014-01-01

Inflammatory conditions characterized by excessive immune cell activation and cytokine release, are associated with bidirectional immune system-brain communication, underlying sickness behavior and other physiological responses. The vagus nerve has an important role in this communication by conveying sensory information to the brain, and brain-derived immunoregulatory signals that suppress peripheral cytokine levels and inflammation. Brain muscarinic acetylcholine receptor (mAChR)-mediated cholinergic signaling has been implicated in this regulation. However, the possibility of controlling inflammation by peripheral administration of centrally-acting mAChR agonists is unexplored. To provide insight we used the centrally-acting M1 mAChR agonist xanomeline, previously developed in the context of Alzheimer’s disease and schizophrenia. Intraperitoneal administration of xanomeline significantly suppressed serum and splenic TNF levels, alleviated sickness behavior, and increased survival during lethal murine endotoxemia. The anti-inflammatory effects of xanomeline were brain mAChR-mediated and required intact vagus nerve and splenic nerve signaling. The anti-inflammatory efficacy of xanomeline was retained for at least 20h, associated with alterations in splenic lymphocyte, and dendritic cell proportions, and decreased splenocyte responsiveness to endotoxin. These results highlight an important role of the M1 mAChR in a neural circuitry to spleen in which brain cholinergic activation lowers peripheral pro-inflammatory cytokines to levels favoring survival. The therapeutic efficacy of xanomeline was also manifested by significantly improved survival in preclinical settings of severe sepsis. These findings are of interest for strategizing novel therapeutic approaches in inflammatory diseases. PMID:25063706

Disturbance of DKK1 level is partly involved in survival of lung cancer cells via regulation of ROMO1 and γ-radiation sensitivity

Energy Technology Data Exchange (ETDEWEB)

Kim, In Gyu, E-mail: igkim@kaeri.re.kr [Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, P.O. Box 105, Yuseong, Daejeon 305-600 (Korea, Republic of); Department of Radiation Biotechnology and Applied Radioisotope, University of Science and Technology (UST), 989-111 Daedeok-daero, Yuseong-gu, Daejeon 305-353 (Korea, Republic of); Kim, Seo Yoen [Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, P.O. Box 105, Yuseong, Daejeon 305-600 (Korea, Republic of); Biomedical Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of); Kim, Hyun A; Kim, Jeong Yul [Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, P.O. Box 105, Yuseong, Daejeon 305-600 (Korea, Republic of); Lee, Jae Ha; Choi, Soo Im [Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, P.O. Box 105, Yuseong, Daejeon 305-600 (Korea, Republic of); Department of Radiation Biotechnology and Applied Radioisotope, University of Science and Technology (UST), 989-111 Daedeok-daero, Yuseong-gu, Daejeon 305-353 (Korea, Republic of); Han, Jeong Ran; Kim, Kug Chan [Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, P.O. Box 105, Yuseong, Daejeon 305-600 (Korea, Republic of); Cho, Eun Wie [Biomedical Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of)

2014-01-03

Highlights: •DKK1 was expressed differently among non-small-cell lung cancer cell lines. •DKK1 negatively regulated ROMO1 gene expression. •Disturbance of DKK1 level induced the imbalance of cellular ROS. •DKK1/ROMO1-induced ROS imbalance is involved in cell survival in NSCLC. -- Abstract: Dickkopf1 (DKK1), a secreted protein involved in embryonic development, is a potent inhibitor of the Wnt signaling pathway and has been postulated to be a tumor suppressor or tumor promoter depending on the tumor type. In this study, we showed that DKK1 was expressed differently among non-small-cell lung cancer cell lines. The DKK1 expression level was much higher in A549 cells than in H460 cells. We revealed that blockage of DKK1 expression by silencing RNA in A549 cells caused up-regulation of intracellular reactive oxygen species (ROS) modulator (ROMO1) protein, followed by partial cell death, cell growth inhibition, and loss of epithelial–mesenchymal transition property caused by ROS, and it also increased γ-radiation sensitivity. DKK1 overexpression in H460 significantly inhibited cell survival with the decrease of ROMO1 level, which induced the decrease of cellular ROS. Thereafter, exogenous N-acetylcysteine, an antioxidant, or hydrogen peroxide, a pro-oxidant, partially rescued cells from death and growth inhibition. In each cell line, both overexpression and blockage of DKK1 not only elevated p-RB activation, which led to cell growth arrest, but also inactivated AKT/NF-kB, which increased radiation sensitivity and inhibited cell growth. This study is the first to demonstrate that strict modulation of DKK1 expression in different cell types partially maintains cell survival via tight regulation of the ROS-producing ROMO1 and radiation resistance.

Disturbance of DKK1 level is partly involved in survival of lung cancer cells via regulation of ROMO1 and γ-radiation sensitivity

International Nuclear Information System (INIS)

Kim, In Gyu; Kim, Seo Yoen; Kim, Hyun A; Kim, Jeong Yul; Lee, Jae Ha; Choi, Soo Im; Han, Jeong Ran; Kim, Kug Chan; Cho, Eun Wie

2014-01-01

Highlights: •DKK1 was expressed differently among non-small-cell lung cancer cell lines. •DKK1 negatively regulated ROMO1 gene expression. •Disturbance of DKK1 level induced the imbalance of cellular ROS. •DKK1/ROMO1-induced ROS imbalance is involved in cell survival in NSCLC. -- Abstract: Dickkopf1 (DKK1), a secreted protein involved in embryonic development, is a potent inhibitor of the Wnt signaling pathway and has been postulated to be a tumor suppressor or tumor promoter depending on the tumor type. In this study, we showed that DKK1 was expressed differently among non-small-cell lung cancer cell lines. The DKK1 expression level was much higher in A549 cells than in H460 cells. We revealed that blockage of DKK1 expression by silencing RNA in A549 cells caused up-regulation of intracellular reactive oxygen species (ROS) modulator (ROMO1) protein, followed by partial cell death, cell growth inhibition, and loss of epithelial–mesenchymal transition property caused by ROS, and it also increased γ-radiation sensitivity. DKK1 overexpression in H460 significantly inhibited cell survival with the decrease of ROMO1 level, which induced the decrease of cellular ROS. Thereafter, exogenous N-acetylcysteine, an antioxidant, or hydrogen peroxide, a pro-oxidant, partially rescued cells from death and growth inhibition. In each cell line, both overexpression and blockage of DKK1 not only elevated p-RB activation, which led to cell growth arrest, but also inactivated AKT/NF-kB, which increased radiation sensitivity and inhibited cell growth. This study is the first to demonstrate that strict modulation of DKK1 expression in different cell types partially maintains cell survival via tight regulation of the ROS-producing ROMO1 and radiation resistance

Genetic modification of embryonic stem cells with VEGF enhances cell survival and improves cardiac function.

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Xie, Xiaoyan; Cao, Feng; Sheikh, Ahmad Y; Li, Zongjin; Connolly, Andrew J; Pei, Xuetao; Li, Ren-Ke; Robbins, Robert C; Wu, Joseph C

2007-01-01

Cardiac stem cell therapy remains hampered by acute donor cell death posttransplantation and the lack of reliable methods for tracking cell survival in vivo. We hypothesize that cells transfected with inducible vascular endothelial growth factor 165 (VEGF(165)) can improve their survival as monitored by novel molecular imaging techniques. Mouse embryonic stem (ES) cells were transfected with an inducible, bidirectional tetracycline (Bi-Tet) promoter driving VEGF(165) and renilla luciferase (Rluc). Addition of doxycycline induced Bi-Tet expression of VEGF(165) and Rluc significantly compared to baseline (p<0.05). Expression of VEGF(165) enhanced ES cell proliferation and inhibited apoptosis as determined by Annexin-V staining. For noninvasive imaging, ES cells were transduced with a double fusion (DF) reporter gene consisting of firefly luciferase and enhanced green fluorescence protein (Fluc-eGFP). There was a robust correlation between cell number and Fluc activity (R(2)=0.99). Analysis by immunostaining, histology, and RT-PCR confirmed that expression of Bi-Tet and DF systems did not affect ES cell self-renewal or pluripotency. ES cells were differentiated into beating embryoid bodies expressing cardiac markers such as troponin, Nkx2.5, and beta-MHC. Afterward, 5 x 10(5) cells obtained from these beating embryoid bodies or saline were injected into the myocardium of SV129 mice (n=36) following ligation of the left anterior descending (LAD) artery. Bioluminescence imaging (BLI) and echocardiography showed that VEGF(165) induction led to significant improvements in both transplanted cell survival and cardiac function (p<0.05). This is the first study to demonstrate imaging of embryonic stem cell-mediated gene therapy targeting cardiovascular disease. With further validation, this platform may have broad applications for current basic research and further clinical studies.

Unus pro omnibus, omnes pro uno: A novel, evidence-based, unifying theory for the pathogenesis of endometriosis.

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Laganà, Antonio Simone; Vitale, Salvatore Giovanni; Salmeri, Francesca Maria; Triolo, Onofrio; Ban Frangež, Helena; Vrtačnik-Bokal, Eda; Stojanovska, Lily; Apostolopoulos, Vasso; Granese, Roberta; Sofo, Vincenza

2017-06-01

The theory of retrograde menstruation as aetiopathogenesis of endometriosis formulated by John Sampson in 1927 shows clear shortcomings: this does not explain why retrograde menstruation is a physiological process that affects 90% of women, while endometriosis occurs in only 10% of cases; it also does not explain the endometriotic foci distant from the pelvis, nor explains the cases of endometriosis in male patients. The immunological alterations of the peritoneal fluid explains the effects of disease, such as the inhibition of the physiological processes of cytolysis, but does not explain the cause. There is evidence to support the hypothesis that ectopic müllerian remnants of the endometrium, endocervix and endosalpinx are items from the genital ridge leaked during organogenesis. It is known that tissues derived from coelomatic epithelial and mesenchymal cells have the potential to metaplastically differentiate into epithelium and stroma. In addition, the phenotype of the ectopic endometrial cells is significantly different from those ectopic. There is scientific evidence that, during organogenesis, the genes of the Homeobox and Wingless family play a fundamental role in the differentiation of the ducts of Muller and development of the anatomical structure of the urogenital tract. We present here a hypothesis that deregulation of genes and the Wnt signaling pathway Wnt/β-catenin leads to aberrations and deregulation within the mesoderm, thus, may cause aberrant placement of stem cells. In addition, immune cells, adhesion molecules, extracellular matrix metalloproteinase and pro-inflammatory cytokines activate/alter peritoneal microenvironment, creating the conditions for differentiation, adhesion, proliferation and survival of ectopic endometrial cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

Circulating CXCR5+CD4+ T cells assist in the survival and growth of primary diffuse large B cell lymphoma cells through interleukin 10 pathway

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Cha, Zhanshan; Qian, Guangfang; Zang, Yan; Gu, Haihui; Huang, Yanyan; Zhu, Lishuang; Li, Jinqi; Liu, Yang; Tu, Xiaohua; Song, Haihan; Qian, Baohua

2017-01-01

Diffuse large B cell lymphoma (DLBCL) is a common and aggressive cancer caused by the malignant transformation of B cells. Although it has been established that the follicular helper T (Tfh) cells play a central role in B cell development, little information is available on their involvement in DLBCL pathogenesis. We studied the role of the peripheral Tfh equivalent, the CXCR5"+ CD4"+ T cells, in DLBCL. Data showed that compared to CXCR5"- CD4"+ T cells, CXCR5"+ CD4"+ T cells were significantly more effective at promoting the proliferation as well as inhibiting the apoptosis of primary autologous DLBCL tumor cells. Surprisingly, we found that at equal cell numbers, CXCR5"+ CD4"+ T cells in DLBCL patients secreted significantly less interleukin (IL)-21 than CXCR5"- CD4"+ T cells, while the level of IL-10 secretion was significant elevated in the CXCR5"+ compartment compared to the CXCR5"- compartment. Neutralization of IL-10 in the primary DLBCL-CXCR5"+ CD4"+ T cell coculture compromised the CXCR5"+ CD4"+ T cell-mediated pro-tumor effects, in a manner that was dependent on the concentration of anti-IL-10 antibodies. The CXCR5"+ compartment also contained significantly lower frequencies of cytotoxic CD4"+ T cells than the CXCR5"- compartment. In conclusion, our investigations discovered a previously unknown pro-tumor role of CXCR5-expressing circulating CD4"+ T cells, which assisted the survival and proliferation of primary DLBCL cells through IL-10. - Highlights: • We studied the role of the peripheral Tfh in DLBCL. • Tfh were effective at promoting the proliferation of primary DLBCL tumor cells. • Tfh were effective at inhibiting the apoptosis of primary DLBCL tumor cells. • IL-10 secretion in Tfh was significant elevated in DLBCL. • Neutralization of IL-10 compromised Tfh-mediated pro-tumor effects.

Combining Gene Signatures Improves Prediction of Breast Cancer Survival

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Zhao, Xi; Naume, Bjørn; Langerød, Anita; Frigessi, Arnoldo; Kristensen, Vessela N.; Børresen-Dale, Anne-Lise; Lingjærde, Ole Christian

2011-01-01

Background Several gene sets for prediction of breast cancer survival have been derived from whole-genome mRNA expression profiles. Here, we develop a statistical framework to explore whether combination of the information from such sets may improve prediction of recurrence and breast cancer specific death in early-stage breast cancers. Microarray data from two clinically similar cohorts of breast cancer patients are used as training (n = 123) and test set (n = 81), respectively. Gene sets from eleven previously published gene signatures are included in the study. Principal Findings To investigate the relationship between breast cancer survival and gene expression on a particular gene set, a Cox proportional hazards model is applied using partial likelihood regression with an L2 penalty to avoid overfitting and using cross-validation to determine the penalty weight. The fitted models are applied to an independent test set to obtain a predicted risk for each individual and each gene set. Hierarchical clustering of the test individuals on the basis of the vector of predicted risks results in two clusters with distinct clinical characteristics in terms of the distribution of molecular subtypes, ER, PR status, TP53 mutation status and histological grade category, and associated with significantly different survival probabilities (recurrence: p = 0.005; breast cancer death: p = 0.014). Finally, principal components analysis of the gene signatures is used to derive combined predictors used to fit a new Cox model. This model classifies test individuals into two risk groups with distinct survival characteristics (recurrence: p = 0.003; breast cancer death: p = 0.001). The latter classifier outperforms all the individual gene signatures, as well as Cox models based on traditional clinical parameters and the Adjuvant! Online for survival prediction. Conclusion Combining the predictive strength of multiple gene signatures improves prediction of breast

Combining gene signatures improves prediction of breast cancer survival.

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Xi Zhao

Full Text Available BACKGROUND: Several gene sets for prediction of breast cancer survival have been derived from whole-genome mRNA expression profiles. Here, we develop a statistical framework to explore whether combination of the information from such sets may improve prediction of recurrence and breast cancer specific death in early-stage breast cancers. Microarray data from two clinically similar cohorts of breast cancer patients are used as training (n = 123 and test set (n = 81, respectively. Gene sets from eleven previously published gene signatures are included in the study. PRINCIPAL FINDINGS: To investigate the relationship between breast cancer survival and gene expression on a particular gene set, a Cox proportional hazards model is applied using partial likelihood regression with an L2 penalty to avoid overfitting and using cross-validation to determine the penalty weight. The fitted models are applied to an independent test set to obtain a predicted risk for each individual and each gene set. Hierarchical clustering of the test individuals on the basis of the vector of predicted risks results in two clusters with distinct clinical characteristics in terms of the distribution of molecular subtypes, ER, PR status, TP53 mutation status and histological grade category, and associated with significantly different survival probabilities (recurrence: p = 0.005; breast cancer death: p = 0.014. Finally, principal components analysis of the gene signatures is used to derive combined predictors used to fit a new Cox model. This model classifies test individuals into two risk groups with distinct survival characteristics (recurrence: p = 0.003; breast cancer death: p = 0.001. The latter classifier outperforms all the individual gene signatures, as well as Cox models based on traditional clinical parameters and the Adjuvant! Online for survival prediction. CONCLUSION: Combining the predictive strength of multiple gene signatures improves

High E6 Gene Expression Predicts for Distant Metastasis and Poor Survival in Patients With HPV-Positive Oropharyngeal Squamous Cell Carcinoma

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Khwaja, Shariq S.; Baker, Callie; Haynes, Wesley; Spencer, Christopher R.; Gay, Hiram; Thorstad, Wade [Department of Radiation Oncology, Washington University School of Medicine, St. Louis, Missouri (United States); Adkins, Douglas R. [Division of Medical Oncology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri (United States); Nussenbaum, Brian [Department of Otolaryngology – Head and Neck Surgery, Washington University School of Medicine, St. Louis, Missouri (United States); Chernock, Rebecca D. [Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri (United States); Lewis, James S. [Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee (United States); Wang, Xiaowei, E-mail: xwang@radonc.wustl.edu [Department of Radiation Oncology, Washington University School of Medicine, St. Louis, Missouri (United States)

2016-07-15

Purpose: Patients with human papillomavirus (HPV)–positive oropharyngeal squamous cell carcinoma (OPSCC) have a favorable prognosis. As a result, de-escalation clinical trials are under way. However, approximately 10% of patients will experience distant recurrence even with standard-of-care treatment. Here, we sought to identify novel biomarkers to better risk-stratify HPV-positive patients with OPSCC. Methods and Materials: Gene expression profiling by RNA sequencing (RNA-seq) and quantitative polymerase chain reaction was performed on HPV-positive OPSCC primary tumor specimens from patients with and without distant metastasis (DM). Results: RNA-seq analysis of 39 HPV-positive OPSCC specimens revealed that patients with DM had 2-fold higher E6 gene expression levels than did patients without DM (P=.029). This observation was confirmed in a validation cohort comprising 93 patients with HPV-positive OPSCC. The mean normalized E6 expression level in the 17 recurring primary specimens was 13 ± 2 compared with 8 ± 1 in the remaining 76 nonrecurring primaries (P=.001). Receiver operating characteristic analysis established an E6 expression level of 7.3 as a cutoff for worse recurrence-free survival (RFS). Patients from this cohort with high E6 gene expression (E6-high) (n=51, 55%) had more cancer-related deaths (23% vs 2%, P<.001) and DM (26% vs 5%, P<.001) than did patients with low E6 gene expression (E6-low) (n=42, 45%). Kaplan-Meier survival analysis revealed that E6-high had worse RFS (95% vs 69%, P=.004) and cancer-specific survival (97% vs 79%, P=.007). E6-high maintained statistical significance in multivariate regression models balancing surgery, chemotherapy, nodal stage, and smoking status. Gene set enrichment analysis demonstrated that tumors with high E6 expression were associated with P53, epidermal growth factor receptor, activating transcription factor-2, and transforming growth factor-β signaling pathways. Conclusion: High E6 gene expression

A preliminary study on action mechanisms of surviving expression in cell apoptosis induced by high-LET radiation

International Nuclear Information System (INIS)

Jin Xiaodong; Li Qiang; Gong Li; Wu Qingfeng; Li Ping; Dai Zhongying; Liu Xinguo; Tao Jiajun

2010-01-01

It has been proven that over-expression of surviving in cancerous cell lines is related to the radioresistance of cells to high-LET radiation in previous work. In this study, action mechanisms of surviving gene in apoptosis induced by high-LET radiation were investigated. We found that inhibiting surviving by siRNA had no notable influence on Bcl-2 and Bax expressions induced by carbon ions. Surviving depressed cell apoptosis through the inhibition of the activities of caspase-3 and -9 possibly in cell apoptosis induced by high-LET radiation. (authors)

Combined Gene Expression and RNAi Screening to Identify Alkylation Damage Survival Pathways from Fly to Human.

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Zanotto-Filho, Alfeu; Dashnamoorthy, Ravi; Loranc, Eva; de Souza, Luis H T; Moreira, José C F; Suresh, Uthra; Chen, Yidong; Bishop, Alexander J R

2016-01-01

Alkylating agents are a key component of cancer chemotherapy. Several cellular mechanisms are known to be important for its survival, particularly DNA repair and xenobiotic detoxification, yet genomic screens indicate that additional cellular components may be involved. Elucidating these components has value in either identifying key processes that can be modulated to improve chemotherapeutic efficacy or may be altered in some cancers to confer chemoresistance. We therefore set out to reevaluate our prior Drosophila RNAi screening data by comparison to gene expression arrays in order to determine if we could identify any novel processes in alkylation damage survival. We noted a consistent conservation of alkylation survival pathways across platforms and species when the analysis was conducted on a pathway/process level rather than at an individual gene level. Better results were obtained when combining gene lists from two datasets (RNAi screen plus microarray) prior to analysis. In addition to previously identified DNA damage responses (p53 signaling and Nucleotide Excision Repair), DNA-mRNA-protein metabolism (transcription/translation) and proteasome machinery, we also noted a highly conserved cross-species requirement for NRF2, glutathione (GSH)-mediated drug detoxification and Endoplasmic Reticulum stress (ER stress)/Unfolded Protein Responses (UPR) in cells exposed to alkylation. The requirement for GSH, NRF2 and UPR in alkylation survival was validated by metabolomics, protein studies and functional cell assays. From this we conclude that RNAi/gene expression fusion is a valid strategy to rapidly identify key processes that may be extendable to other contexts beyond damage survival.

Combined Gene Expression and RNAi Screening to Identify Alkylation Damage Survival Pathways from Fly to Human.

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Alfeu Zanotto-Filho

Full Text Available Alkylating agents are a key component of cancer chemotherapy. Several cellular mechanisms are known to be important for its survival, particularly DNA repair and xenobiotic detoxification, yet genomic screens indicate that additional cellular components may be involved. Elucidating these components has value in either identifying key processes that can be modulated to improve chemotherapeutic efficacy or may be altered in some cancers to confer chemoresistance. We therefore set out to reevaluate our prior Drosophila RNAi screening data by comparison to gene expression arrays in order to determine if we could identify any novel processes in alkylation damage survival. We noted a consistent conservation of alkylation survival pathways across platforms and species when the analysis was conducted on a pathway/process level rather than at an individual gene level. Better results were obtained when combining gene lists from two datasets (RNAi screen plus microarray prior to analysis. In addition to previously identified DNA damage responses (p53 signaling and Nucleotide Excision Repair, DNA-mRNA-protein metabolism (transcription/translation and proteasome machinery, we also noted a highly conserved cross-species requirement for NRF2, glutathione (GSH-mediated drug detoxification and Endoplasmic Reticulum stress (ER stress/Unfolded Protein Responses (UPR in cells exposed to alkylation. The requirement for GSH, NRF2 and UPR in alkylation survival was validated by metabolomics, protein studies and functional cell assays. From this we conclude that RNAi/gene expression fusion is a valid strategy to rapidly identify key processes that may be extendable to other contexts beyond damage survival.

The essential role of the Deinococcus radiodurans ssb gene in cell survival and radiation tolerance.

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J Scott Lockhart

Full Text Available Recent evidence has implicated single-stranded DNA-binding protein (SSB expression level as an important factor in microbial radiation resistance. The genome of the extremely radiation resistant bacterium Deinococcus radiodurans contains genes for two SSB homologs: the homodimeric, canonical Ssb, encoded by the gene ssb, and a novel pentameric protein encoded by the gene ddrB. ddrB is highly induced upon exposure to radiation, and deletions result in decreased radiation-resistance, suggesting an integral role of the protein in the extreme resistance exhibited by this organism. Although expression of ssb is also induced after irradiation, Ssb is thought to be involved primarily in replication. In this study, we demonstrate that Ssb in D. radiodurans is essential for cell survival. The lethality of an ssb deletion cannot be complemented by providing ddrB in trans. In addition, the radiation-sensitive phenotype conferred by a ddrB deletion is not alleviated by providing ssb in trans. By altering expression of the ssb gene, we also show that lower levels of transcription are required for optimal growth than are necessary for high radiation resistance. When expression is reduced to that of E. coli, ionizing radiation resistance is similarly reduced. UV resistance is also decreased under low ssb transcript levels where growth is unimpaired. These results indicate that the expression of ssb is a key component of both normal cellular metabolism as well as pathways responsible for the high radiation tolerance of D. radiodurans.

Generation and CRISPR/Cas9 editing of transformed progenitor B cells as a pseudo-physiological system to study DNA repair gene function in V(D)J recombination.

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Lenden Hasse, Hélène; Lescale, Chloé; Bianchi, Joy J; Yu, Wei; Bedora-Faure, Marie; Deriano, Ludovic

2017-12-01

Antigen receptor gene assembly is accomplished in developing lymphocytes by the V(D)J recombination reaction, which can be separated into two steps: DNA cleavage by the recombination-activating gene (RAG) nuclease and joining of DNA double strand breaks (DSBs) by components of the nonhomologous end joining (NHEJ) pathway. Deficiencies for NHEJ factors can result in immunodeficiency and a propensity to accumulate genomic instability, thus highlighting the importance of identifying all players in this process and deciphering their functions. Bcl2 transgenic v-Abl kinase-transformed pro-B cells provide a pseudo-physiological cellular system to study V(D)J recombination. Treatment of v-Abl/Bcl2 pro-B cells with the Abl kinase inhibitor Imatinib leads to G1 cell cycle arrest, the rapid induction of Rag1/2 gene expression and V(D)J recombination. In this system, the Bcl2 transgene alleviates Imatinib-induced apoptosis enabling the analysis of induced V(D)J recombination. Although powerful, the use of mouse models carrying the Bcl2 transgene for the generation of v-Abl pro-B cell lines is time and money consuming. Here, we describe a method for generating v-Abl/Bcl2 pro-B cell lines from wild type mice and for performing gene knock-out using episomal CRISPR/Cas9 targeting vectors. Using this approach, we generated distinct NHEJ-deficient pro-B cell lines and quantified V(D)J recombination levels in these cells. Furthermore, this methodology can be adapted to generate pro-B cell lines deficient for any gene suspected to play a role in V(D)J recombination, and more generally DSB repair. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Autophagy/Xenophagy as a survival strategy of cancer cells. The role of Cathepsins

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Malorni, W.; Matarrese, P.; Ascione, B.; Ciarlo, L.; Zakeri, Z.

2009-01-01

Macroautophagy, often referred as to autophagy (self-cannibalism), designates the genetically determined process by which portions of the cytoplasm, organelles and long-lived proteins are engulfed in double-membraned vacuoles (autophagosomes) and sent for lysosomal degradation. Basal levels of autophagy contribute to the maintenance of intracellular homoeostasis by ensuring the turnover of supernumerary, aged and/or damaged components. Under conditions of starvation, the autophagic pathway operates to supply cells with metabolic substrates, and thus represents an important pro-survival mechanism. In cultured cells, the withdrawal of growth factors, known to represent an experimental condition triggering autophagy, can also enhance xeno-cannibalism (xenophagy; xeno is from ancient greek=foreign)

Analysis of gene expression in fetal and adult cells infected with rubella virus

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Adamo, Maria Pilar; Zapata, Marta; Frey, Teryl K.

2008-01-01

Congenital infection with rubella virus (RUB) leads to persistent infection and congenital defects and we showed previously that primary human fetal fibroblasts did not undergo apoptosis when infected with RUB, which could promote fetal virus persistence [Adamo, P., Asis, L., Silveyra, P., Cuffini, C., Pedranti, M., Zapata, M., 2004. Rubella virus does not induce apoptosis in primary human embryo fibroblasts cultures: a possible way of viral persistence in congenital infection. Viral Immunol. 17, 87-100]. To extend this observation, gene chip analysis was performed on a line of primary human fetal fibroblasts (10 weeks gestation) and a line of human adult lung fibroblasts (which underwent apoptosis in response to RUB infection) to compare gene expression in infected and uninfected cells. A total of 632 and 516 genes were upregulated or downregulated in the infected fetal and adult cells respectively in comparison to uninfected cells, however only 52 genes were regulated in both cell types. Although the regulated genes were different, across functional gene categories the patterns of gene regulation were similar. In general, regulation of pro- and anti-apoptotic genes following infection appeared to favor apoptosis in the adult cells and lack of apoptosis in the fetal cells, however there was a greater relative expression of anti-apoptotic genes and reduced expression of pro-apoptotic genes in uninfected fetal cells versus uninfected adult cells and thus the lack of apoptosis in fetal cells following RUB infection was also due to the prevailing background of gene expression that is antagonistic to apoptosis. In support of this hypothesis, it was found that of a battery of five chemicals known to induce apoptosis, two induced apoptosis in the adult cells, but not in fetal cells, and two induced apoptosis more rapidly in the adult cells than in fetal cells (the fifth did not induce apoptosis in either). A robust interferon-stimulated gene response was induced

Survival curves for irradiated cells

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Gibson, D.K.

1975-01-01

The subject of the lecture is the probability of survival of biological cells which have been subjected to ionising radiation. The basic mathematical theories of cell survival as a function of radiation dose are developed. A brief comparison with observed survival curves is made. (author)

Cyr61/CCN1 and CTGF/CCN2 mediate the pro-angiogenic activity of VHL mutant renal carcinoma cells

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Chintalapudi, Mastan R.; Markiewicz, Margaret; Kose, Nurgun; Dammai, Vincent; Champion, Kristen J.; Hoda, Rana S.; Trojanowska, Maria; Hsu, Tien

2008-01-01

The von Hippel-Lindau (VHL) protein serves as a negative regulator of hypoxia inducible factor-alpha subunit (HIF-α). Since HIF regulates critical angiogenic factors such as vascular endothelial growth factor (VEGF) and lesions in VHL gene are present in a majority of the highly vascularized renal cell carcinoma (RCC), it is believed that deregulation of the VHL-HIF pathway is crucial for the pro-angiogenic activity of RCC. Although VEGF has been confirmed as a critical angiogenic factor up-regulated in VHL mutant cells, the efficacy of anti-angiogenic therapy specifically targeting VEGF signaling remains modest. In this study we developed a three-dimensional in vitro assay to evaluate the ability of RCC cells to promote cord formation by the primary human dermal microvascular endothelial cells (HDMECs). Compared to VHL wild-type cells, VHL mutant RCC cells demonstrated a significantly increased pro-angiogenic activity, which correlated with increased secretion of Cyr61/CCN1, CTGF/CCN2 and VEGF in conditioned culture medium. Both CCN proteins are required for HDMEC cord formation as shown by RNAi knock-down experiments. Importantly, the pro-angiogenic activities conferred by the CCN proteins and VEGF are additive, suggesting non-overlapping functions. Expression of the CCN proteins is at least partly dependent on the HIF-2α function, the dominant HIF-α isoform expressed in RCC. Finally, immunohistochemical staining of Cyr61/CCN1 and CTGF/CCN2 in renal cell carcinoma tissue samples showed that increased expression of these proteins correlates with loss of VHL protein expression. These findings strengthened the notion that the hypervascularized phenotype of RCC is afforded by multiple pro-angiogenic factors that function in parallel pathways. PMID:18212329

Cell-free culture supernatant of Bifidobacterium breve CNCM I-4035 decreases pro-inflammatory cytokines in human dendritic cells challenged with Salmonella typhi through TLR activation.

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Bermudez-Brito, Miriam; Muñoz-Quezada, Sergio; Gomez-Llorente, Carolina; Matencio, Esther; Bernal, Maria J; Romero, Fernando; Gil, Angel

2013-01-01

Dendritic cells (DCs) constitute the first point of contact between gut commensals and our immune system. Despite growing evidence of the immunomodulatory effects of probiotics, the interactions between the cells of the intestinal immune system and bacteria remain largely unknown. Indeed,, the aim of this work was to determine whether the probiotic Bifidobacterium breve CNCM I-4035 and its cell-free culture supernatant (CFS) have immunomodulatory effects in human intestinal-like dendritic cells (DCs) and how they respond to the pathogenic bacterium Salmonella enterica serovar Typhi, and also to elucidate the molecular mechanisms involved in these interactions. Human DCs were directly challenged with B. breve/CFS, S. typhi or a combination of these stimuli for 4 h. The expression pattern of genes involved in Toll-like receptor (TLR) signaling pathway and cytokine secretion was analyzed. CFS decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged with S. typhi. In contrast, the B. breve CNCM I-4035 probiotic strain was a potent inducer of the pro-inflammatory cytokines and chemokines tested, i.e., TNF-α, IL-8 and RANTES, as well as anti-inflammatory cytokines including IL-10. CFS restored TGF-β levels in the presence of Salmonella. Live B.breve and its supernatant enhanced innate immune responses by the activation of TLR signaling pathway. These treatments upregulated TLR9 gene transcription. In addition, CFS was a more potent inducer of TLR9 expression than the probiotic bacteria in the presence of S. typhi. Expression levels of CASP8 and IRAK4 were also increased by CFS, and both treatments induced TOLLIP gene expression. Our results indicate that the probiotic strain B. breve CNCM I-4035 affects the intestinal immune response, whereas its supernatant exerts anti-inflammatory effects mediated by DCs. This supernatant may protect immune system from highly infectious agents such as Salmonella typhi and can down-regulate pro

Cell-free culture supernatant of Bifidobacterium breve CNCM I-4035 decreases pro-inflammatory cytokines in human dendritic cells challenged with Salmonella typhi through TLR activation.

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Miriam Bermudez-Brito

Full Text Available Dendritic cells (DCs constitute the first point of contact between gut commensals and our immune system. Despite growing evidence of the immunomodulatory effects of probiotics, the interactions between the cells of the intestinal immune system and bacteria remain largely unknown. Indeed,, the aim of this work was to determine whether the probiotic Bifidobacterium breve CNCM I-4035 and its cell-free culture supernatant (CFS have immunomodulatory effects in human intestinal-like dendritic cells (DCs and how they respond to the pathogenic bacterium Salmonella enterica serovar Typhi, and also to elucidate the molecular mechanisms involved in these interactions. Human DCs were directly challenged with B. breve/CFS, S. typhi or a combination of these stimuli for 4 h. The expression pattern of genes involved in Toll-like receptor (TLR signaling pathway and cytokine secretion was analyzed. CFS decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged with S. typhi. In contrast, the B. breve CNCM I-4035 probiotic strain was a potent inducer of the pro-inflammatory cytokines and chemokines tested, i.e., TNF-α, IL-8 and RANTES, as well as anti-inflammatory cytokines including IL-10. CFS restored TGF-β levels in the presence of Salmonella. Live B.breve and its supernatant enhanced innate immune responses by the activation of TLR signaling pathway. These treatments upregulated TLR9 gene transcription. In addition, CFS was a more potent inducer of TLR9 expression than the probiotic bacteria in the presence of S. typhi. Expression levels of CASP8 and IRAK4 were also increased by CFS, and both treatments induced TOLLIP gene expression. Our results indicate that the probiotic strain B. breve CNCM I-4035 affects the intestinal immune response, whereas its supernatant exerts anti-inflammatory effects mediated by DCs. This supernatant may protect immune system from highly infectious agents such as Salmonella typhi and can down

Retinoic acid-loaded polymeric nanoparticles enhance vascular regulation of neural stem cell survival and differentiation after ischaemia

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Ferreira, R.; Fonseca, M. C.; Santos, T.; Sargento-Freitas, J.; Tjeng, R.; Paiva, F.; Castelo-Branco, M.; Ferreira, L. S.; Bernardino, L.

2016-04-01

Stroke is one of the leading causes of death and disability worldwide. However, current therapies only reach a small percentage of patients and may cause serious side effects. We propose the therapeutic use of retinoic acid-loaded nanoparticles (RA-NP) to safely and efficiently repair the ischaemic brain by creating a favourable pro-angiogenic environment that enhances neurogenesis and neuronal restitution. Our data showed that RA-NP enhanced endothelial cell proliferation and tubule network formation and protected against ischaemia-induced death. To evaluate the effect of RA-NP on vascular regulation of neural stem cell (NSC) survival and differentiation, endothelial cell-conditioned media (EC-CM) were collected. EC-CM from healthy RA-NP-treated cells reduced NSC death and promoted proliferation while EC-CM from ischaemic RA-NP-treated cells decreased cell death, increased proliferation and neuronal differentiation. In parallel, human endothelial progenitor cells (hEPC), which are part of the endogenous repair response to vascular injury, were collected from ischaemic stroke patients. hEPC treated with RA-NP had significantly higher proliferation, which further highlights the therapeutic potential of this formulation. To conclude, RA-NP protected endothelial cells from ischaemic death and stimulated the release of pro-survival, proliferation-stimulating factors and differentiation cues for NSC. RA-NP were shown to be up to 83-fold more efficient than free RA and to enhance hEPC proliferation. These data serve as a stepping stone to use RA-NP as vasculotrophic and neurogenic agents for vascular disorders and neurodegenerative diseases with compromised vasculature.

The Impact of Drug Metabolism Gene Polymorphisms on Therapeutic Response and Survival in Diffuse Large B-Cell Lymphoma Patients.

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Pál, Ildikó; Illés, Árpád; Gergely, Lajos; Pál, Tibor; Radnay, Zita; Szekanecz, Zoltán; Zilahi, Erika; Váróczy, László

2018-04-01

Diffuse large B-cell lymphoma (DLBCL) accounts for 30% of all non-Hodgkin lymphomas (NHL) and 80% of agressive lymphomas. Besides the traditional International Prognostic Index (IPI), some other factors may also influence the prognosis of DLBCL patients. To study how the genetic polymorphisms in the metabolic pathway influence the event-free and overall survivals and therapeutic responses in DLBCL. The study was comprised of 51 patients (32 men, 19 women). The average age was 53.1 years. DLBCL was diagnosed between 2011 and 2016 and the average follow-up time was 3.78 years. These patients received 1-8 cycles (an average of 6.2 cycles) of rituximab, cyclophosphamide, doxorubicin, vincristin, prednisolon (R-CHOP) immunochemotherapy. Real-time polymerase chain reaction was used to determine the genetic polymorphisms of CYP2E1, GSTP1, NAT1, and NAT2 genes. Our results showed that the polymorphisms of CYP2E1, GSTP1, and NAT1 genes did not influence the prognosis of DLBCL patients significantly. In terms of the NAT2 gene, GG homozygous patients showed slightly better therapeutic response and survival results compared to those bearing an A allele; however, the differences were not statistically significant. Our results could not confirm that genetic polymorphism in metabolic pathways has any predictive role in DLBCL.

Low-Dose Radiation Induces Genes Promoting Cell Survival

International Nuclear Information System (INIS)

Liu, Shu-Zheng; Chen, Dong; Mu, Ying

1999-01-01

Apoptosis is an important process controlling homeostasis of the body. It is influenced by stimuli constantly arising from the external and internal environment of the organism. It is well known that radiation could induce apoptosis of cells in vitro and in vivo. However, the dose-effect relationship of apoptosis extending to the low-dose range has scarcely been studied. Here, the molecular basis of the phenomenon is explored by examining the changes in expression of some of the proapoptotic and antiapoptotic genes

Accumulation of pro-cancer cytokines in the plasma fraction of stored packed red cells.

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Benson, Douglas D; Beck, Adam W; Burdine, Marie S; Brekken, Rolf; Silliman, Christopher C; Barnett, Carlton C

2012-03-01

Perioperative blood transfusion has been linked to decreased survival in pancreatic cancer; however, the exact causal mechanism has not been elucidated. Allogeneic transfusions are known to expose patients to foreign cells and lipid mediators. We hypothesize that stored packed red cells (pRBCs) contain pro-cancer cytokines that augment tumor progression. We analyzed the plasma fraction of stored pRBCs for pro-cancer cytokines and evaluated the affect of both storage time and leukocyte reduction on these mediators. Chemiarray™ analysis for pro-cancer cytokines was performed on the acellular plasma fraction of stored leukocyte-reduced (LR) and non-leukocyte-reduced (NLR) pRBCs at day 1 (D.1-fresh) and day 42 (D.42-outdate) of storage. Elevated expression of monocyte chemotactic protein-1 (MCP-1), regulated on activation, normal T cell expressed and secreted (RANTES), angiogenin, tumor necrosis factor-alpha (TNF-α), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) was found. Specific enzyme-linked immunosorbent assay was performed for each of these factors in LR and NLR blood at D.1, day 28, and D.42. Data were analyzed by ANOVA. A p value ≤ 0.05 was considered significant; N ≥ 4 per group. Migration assays were performed using inhibitors of EGF (gefitinib) and PDGF (imatinib) on murine pancreatic adenocarcinoma cells (Pan02) exposed to D.1 and D.42 LR and NLR plasma. Proliferation assays were performed on Pan02 cells to test the inhibition of PDGF. MCP-1 levels increased with storage time in LR blood, 86.3 ± 6.3 pg/ml at D.1 vs. 121.2 ± 6.1 pg/ml at D.42 (p = 0.007), and NLR blood, 78.2 ± 7.3 pg/ml at D.1 vs. 647.8 ± 220.7 pg/ml at D.42 (p = 0.02). RANTES levels are lower in LR compared to NLR stored blood, 3.0 ± 1.9 vs. 15.8 ± 0.7 pg/ml at D.42 (p Pro-cancer cytokines that can augment tumor progression were identified in pRBCs. Some of these factors are present in fresh blood. The soluble factors identified herein may represent

Impact of Pancreatic Rat Islet Density on Cell Survival during Hypoxia

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A. Rodriguez-Brotons

2016-01-01

Full Text Available In bioartificial pancreases (BP, the number of islets needed to restore normoglycaemia in the diabetic patient is critical. However, the confinement of a high quantity of islets in a limited space may impact islet survival, particularly in regard to the low oxygen partial pressure (PO2 in such environments. The aim of the present study was to evaluate the impact of islet number in a confined space under hypoxia on cell survival. Rat islets were seeded at three different concentrations (150, 300, and 600 Islet Equivalents (IEQ/cm2 and cultured in normal atmospheric pressure (160 mmHg as well as hypoxic conditions (15 mmHg for 24 hours. Cell viability, function, hypoxia-induced changes in gene expression, and cytokine secretion were then assessed. Notably, hypoxia appeared to induce a decrease in viability and increasing islet density exacerbated the observed increase in cellular apoptosis as well as the loss of function. These changes were also associated with an increase in inflammatory gene transcription. Taken together, these data indicate that when a high number of islets are confined to a small space under hypoxia, cell viability and function are significantly impacted. Thus, in order to improve islet survival in this environment during transplantation, oxygenation is of critical importance.

Ortholog-based screening and identification of genes related to intracellular survival.

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Yang, Xiaowen; Wang, Jiawei; Bing, Guoxia; Bie, Pengfei; De, Yanyan; Lyu, Yanli; Wu, Qingmin

2018-04-20

Bioinformatics and comparative genomics analysis methods were used to predict unknown pathogen genes based on homology with identified or functionally clustered genes. In this study, the genes of common pathogens were analyzed to screen and identify genes associated with intracellular survival through sequence similarity, phylogenetic tree analysis and the λ-Red recombination system test method. The total 38,952 protein-coding genes of common pathogens were divided into 19,775 clusters. As demonstrated through a COG analysis, information storage and processing genes might play an important role intracellular survival. Only 19 clusters were present in facultative intracellular pathogens, and not all were present in extracellular pathogens. Construction of a phylogenetic tree selected 18 of these 19 clusters. Comparisons with the DEG database and previous research revealed that seven other clusters are considered essential gene clusters and that seven other clusters are associated with intracellular survival. Moreover, this study confirmed that clusters screened by orthologs with similar function could be replaced with an approved uvrY gene and its orthologs, and the results revealed that the usg gene is associated with intracellular survival. The study improves the current understanding of intracellular pathogens characteristics and allows further exploration of the intracellular survival-related gene modules in these pathogens. Copyright © 2018. Published by Elsevier B.V.

Heat Stress and Lipopolysaccharide Stimulation of Chicken Macrophage-Like Cell Line Activates Expression of Distinct Sets of Genes.

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Anna Slawinska

Full Text Available Acute heat stress requires immediate adjustment of the stressed individual to sudden changes of ambient temperatures. Chickens are particularly sensitive to heat stress due to development of insufficient physiological mechanisms to mitigate its effects. One of the symptoms of heat stress is endotoxemia that results from release of the lipopolysaccharide (LPS from the guts. Heat-related cytotoxicity is mitigated by the innate immune system, which is comprised mostly of phagocytic cells such as monocytes and macrophages. The objective of this study was to analyze the molecular responses of the chicken macrophage-like HD11 cell line to combined heat stress and lipopolysaccharide treatment in vitro. The cells were heat-stressed and then allowed a temperature-recovery period, during which the gene expression was investigated. LPS was added to the cells to mimic the heat-stress-related endotoxemia. Semi high-throughput gene expression analysis was used to study a gene panel comprised of heat shock proteins, stress-related genes, signaling molecules and immune response genes. HD11 cell line responded to heat stress with increased mRNA abundance of the HSP25, HSPA2 and HSPH1 chaperones as well as DNAJA4 and DNAJB6 co-chaperones. The anti-apoptotic gene BAG3 was also highly up-regulated, providing evidence that the cells expressed pro-survival processes. The immune response of the HD11 cell line to LPS in the heat stress environment (up-regulation of CCL4, CCL5, IL1B, IL8 and iNOS was higher than in thermoneutral conditions. However, the peak in the transcriptional regulation of the immune genes was after two hours of temperature-recovery. Therefore, we propose the potential influence of the extracellular heat shock proteins not only in mitigating effects of abiotic stress but also in triggering the higher level of the immune responses. Finally, use of correlation networks for the data analysis aided in discovering subtle differences in the gene

BAP1 has a survival role in cutaneous melanoma.

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Kumar, Raj; Taylor, Michael; Miao, Benchun; Ji, Zhenyu; Njauw, Jenny C-N; Jönsson, Göran; Frederick, Dennie T; Tsao, Hensin

2015-04-01

Although the pattern of BAP1 inactivation in ocular melanoma specimens and in the BAP1 cutaneous melanoma (CM)/ocular melanoma predisposition syndrome suggests a tumor suppressor function, the specific role of this gene in the pathogenesis of CM is not fully understood. We thus set out to characterize BAP1 in CM and discovered an unexpected pro-survival effect of this protein. Tissue and cell lines analysis showed that BAP1 expression was maintained, rather than lost, in primary melanomas compared with nevi and normal skin. Genetic depletion of BAP1 in melanoma cells reduced proliferation and colony-forming capability, induced apoptosis, and inhibited melanoma tumor growth in vivo. On the molecular level, suppression of BAP1 led to a concomitant drop in the protein levels of survivin, a member of anti-apoptotic proteins and a known mediator of melanoma survival. Restoration of survivin in melanoma cells partially rescued the growth-retarding effects of BAP1 loss. In contrast to melanoma cells, stable overexpression of BAP1 into immortalized but non-transformed melanocytes did suppress proliferation and reduce survivin. Taken together, these studies demonstrate that BAP1 may have a growth-sustaining role in melanoma cells, but that its impact on ubiquitination underpins a complex physiology, which is context and cell dependent.

Tenocytes, pro-inflammatory cytokines and leukocytes: a relationship?

OpenAIRE

Al-Sadi, Onays; Schulze-Tanzil, Gundula; Kohl, Benjamin; Lohan, Anke; Lemke, Marion; Ertel, Wolfgang; John, Thilo

2012-01-01

Leukocyte derived pro-inflammatory mediators could be involved in tendon healing and scar formation. Hence, the effect of autologous leukocytes (PBMCs, peripheral blood mononuclear cells and neutrophils) on primary rabbit Achilles tenocytes gene expression was tested in insert assisted co-cultures.

Activation tagging of the two closely linked genes LEP and VAS independently affects vascular cell number

DEFF Research Database (Denmark)

van der Graaff, Eric; Hooykaas, Paul J J; Keller, Beat

2002-01-01

report that in addition to this leafy petiole phenotype, the size of the vascular bundles is increased in all aerial organs in let as a result of an increase in the number of xylem, phloem (pro)cambial and pericycle cells. This vascular phenotype is caused by activation tagging of the two genes VASCULAR......-promoting factor. The activation tagging of VAS only resulted in a specific increase in phloem (pro)cambial and pericycle cells. We conclude that activation tagging of LEP and VAS results in additive phenotypes. Insertional mutants for LEP and VAS display wild-type vascular development, indicating the relevance...... of activation tagging for functional analysis of novel genes involved in plant development....

Inoculum pretreatment affects bacterial survival, activity and catabolic gene expression during phytoremediation of diesel contaminated soil.

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Khan, Sumia; Afzal, Muhammad; Iqbal, Samina; Mirza, Muhammad Sajjad; Khan, Qaiser M

2013-04-01

Plant-bacteria partnership is a promising approach for remediating soil contaminated with organic pollutants. The colonization and metabolic activity of an inoculated microorganism depend not only on environmental conditions but also on the physiological condition of the applied microorganisms. This study assessed the influence of different inoculum pretreatments on survival, gene abundance and catabolic gene expression of an applied strain (Pantoea sp. strain BTRH79) in the rhizosphere of ryegrass vegetated in diesel contaminated soil. Maximum bacterium survival, gene abundance and expression were observed in the soil inoculated with bacterial cells that had been pregrown on complex medium, and hydrocarbon degradation and genotoxicity reduction were also high in this soil. These findings propose that use of complex media for growing plant inocula may enhance bacterial survival and colonization and subsequently the efficiency of pollutant degradation. Copyright © 2013 Elsevier Ltd. All rights reserved.

Gene Expression Profiling of Human Vaginal Cells In Vitro Discriminates Compounds with Pro-Inflammatory and Mucosa-Altering Properties: Novel Biomarkers for Preclinical Testing of HIV Microbicide Candidates.

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Irina A Zalenskaya

Full Text Available Inflammation and immune activation of the cervicovaginal mucosa are considered factors that increase susceptibility to HIV infection. Therefore, it is essential to screen candidate anti-HIV microbicides for potential mucosal immunomodulatory/inflammatory effects prior to further clinical development. The goal of this study was to develop an in vitro method for preclinical evaluation of the inflammatory potential of new candidate microbicides using a microarray gene expression profiling strategy.To this end, we compared transcriptomes of human vaginal cells (Vk2/E6E7 treated with well-characterized pro-inflammatory (PIC and non-inflammatory (NIC compounds. PICs included compounds with different mechanisms of action. Gene expression was analyzed using Affymetrix U133 Plus 2 arrays. Data processing was performed using GeneSpring 11.5 (Agilent Technologies, Santa Clara, CA.Microarraray comparative analysis allowed us to generate a panel of 20 genes that were consistently deregulated by PICs compared to NICs, thus distinguishing between these two groups. Functional analysis mapped 14 of these genes to immune and inflammatory responses. This was confirmed by the fact that PICs induced NFkB pathway activation in Vk2 cells. By testing microbicide candidates previously characterized in clinical trials we demonstrated that the selected PIC-associated genes properly identified compounds with mucosa-altering effects. The discriminatory power of these genes was further demonstrated after culturing vaginal cells with vaginal bacteria. Prevotella bivia, prevalent bacteria in the disturbed microbiota of bacterial vaginosis, induced strong upregulation of seven selected PIC-associated genes, while a commensal Lactobacillus gasseri associated to vaginal health did not cause any changes.In vitro evaluation of the immunoinflammatory potential of microbicides using the PIC-associated genes defined in this study could help in the initial screening of candidates prior

Whole blood gene expression in adolescent chronic fatigue syndrome: an exploratory cross-sectional study suggesting altered B cell differentiation and survival.

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Nguyen, Chinh Bkrong; Alsøe, Lene; Lindvall, Jessica M; Sulheim, Dag; Fagermoen, Even; Winger, Anette; Kaarbø, Mari; Nilsen, Hilde; Wyller, Vegard Bruun

2017-05-11

Chronic fatigue syndrome (CFS) is a prevalent and disabling condition affecting adolescents. The pathophysiology is poorly understood, but immune alterations might be an important component. This study compared whole blood gene expression in adolescent CFS patients and healthy controls, and explored associations between gene expression and neuroendocrine markers, immune markers and clinical markers within the CFS group. CFS patients (12-18 years old) were recruited nation-wide to a single referral center as part of the NorCAPITAL project. A broad case definition of CFS was applied, requiring 3 months of unexplained, disabling chronic/relapsing fatigue of new onset, whereas no accompanying symptoms were necessary. Healthy controls having comparable distribution of gender and age were recruited from local schools. Whole blood samples were subjected to RNA sequencing. Immune markers were blood leukocyte counts, plasma cytokines, serum C-reactive protein and immunoglobulins. Neuroendocrine markers encompassed plasma and urine levels of catecholamines and cortisol, as well as heart rate variability indices. Clinical markers consisted of questionnaire scores for symptoms of post-exertional malaise, inflammation, fatigue, depression and trait anxiety, as well as activity recordings. A total of 29 CFS patients and 18 healthy controls were included. We identified 176 genes as differentially expressed in patients compared to controls, adjusting for age and gender factors. Gene set enrichment analyses suggested impairment of B cell differentiation and survival, as well as enhancement of innate antiviral responses and inflammation in the CFS group. A pattern of co-expression could be identified, and this pattern, as well as single gene transcripts, was significantly associated with indices of autonomic nervous activity, plasma cortisol, and blood monocyte and eosinophil counts. Also, an association with symptoms of post-exertional malaise was demonstrated. Adolescent CFS is

Stimulated human fibroblast cell survival

International Nuclear Information System (INIS)

Smith, B.P.; Gale, K.L.; Einspenner, M.; Greenstock, C.L.; Gentner, N.E.

1992-01-01

Techniques for cloning cultured mammalian cells have supported the most universally-accepted method for measuring the induction of lethality by geno-toxicants such as ionizing radiation: the 'survival of colony-forming ability (CFA)' assay. Since most cultured human cell lines exhibit plating efficiency (i.e. the percentage of cells that are capable of reproductively surviving and dividing to form visible colonies) well below 100%, such assays are in essence 'survival of plating efficiency' assays, since they are referred to the plating (or cloning) efficiency of control (i.e. unirradiated) cells. (author). 8 refs., 2 figs

BAG3 protects bovine papillomavirus type 1-transformed equine fibroblasts against pro-death signals.

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Cotugno, Roberta; Gallotta, Dario; d'Avenia, Morena; Corteggio, Annunziata; Altamura, Gennaro; Roperto, Franco; Belisario, Maria Antonietta; Borzacchiello, Giuseppe

2013-07-22

In human cancer cells, BAG3 protein is known to sustain cell survival. Here, for the first time, we demonstrate the expression of BAG3 protein both in equine sarcoids in vivo and in EqS04b cells, a sarcoid-derived fully transformed cell line harbouring bovine papilloma virus (BPV)-1 genome. Evidence of a possible involvement of BAG3 in equine sarcoid carcinogenesis was obtained by immunohistochemistry analysis of tumour samples. We found that most tumour samples stained positive for BAG3, even though to a different grade, while normal dermal fibroblasts from healthy horses displayed very weak staining pattern for BAG3 expression. By siRNA technology, we demonstrate in EqS04b the role of BAG3 in counteracting basal as well as chemical-triggered pro-death signals. BAG3 down-modulation was indeed shown to promote cell death and cell cycle arrest in G0/G1. In addition, we found that BAG3 silencing sensitized EqS04b cells to phenethylisothiocyanate (PEITC), a promising cancer chemopreventive/chemotherapeutic agent present in edible cruciferous vegetables. Notably, such a pro-survival role of BAG3 was less marked in E. Derm cells, an equine BPV-negative fibroblast cell line taken as a normal counterpart. Altogether our findings might suggest a mutual cooperation between BAG3 and viral oncoproteins to sustain cell survival.

Expression of Genes Involved in Cellular Adhesion and Extracellular Matrix Remodeling Correlates with Poor Survival of Patients with Renal Cancer.

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Boguslawska, Joanna; Kedzierska, Hanna; Poplawski, Piotr; Rybicka, Beata; Tanski, Zbigniew; Piekielko-Witkowska, Agnieszka

2016-06-01

Renal cell carcinoma is the most common highly metastatic kidney malignancy. Adhesion has a crucial role in the metastatic process. TGF (transforming growth factor)-β1 is a pleiotropic cytokine that influences cancerous transformation. We hypothesized that 1) changes in the expression of adhesion related genes may influence survival rate of patients with renal cell carcinoma and 2) TGF-β1 may contribute to changed expression of adhesion related genes. Two-step quantitative real-time polymerase chain reaction arrays were used to analyze the expression of adhesion related genes in 77 tumors and matched pair controls. The prognostic significance of genes was evaluated in TCGA (The Cancer Genome Atlas) data on 468 patients with renal cell carcinoma. Quantitative real-time polymerase chain reaction and Western blot were applied for TGF-β1 analysis. TGF-β1 mediated regulation of gene expression was analyzed by TGF-β1 supplementation of Caki-2 cells and quantitative real-time polymerase chain reaction. The expression of 19 genes related to adhesion and extracellular matrix remodeling was statistically significantly disturbed in renal cell carcinoma compared with controls. The 10-gene expression signature (COL1A1, COL5A1, COL11A1, FN1, ICAM1, ITGAL, ITGAM, ITGB2, THBS2 and TIMP1) correlated with poor survival (HR 2.85, p = 5.7e-10). TGF-β1 expression was 22 times higher in renal cell carcinoma than in controls (p adhesion and extracellular matrix remodeling develops early during renal cell carcinoma carcinogenesis and correlates with poor survival. TGF-β1 contributes to changed expression of extracellular matrix and adhesion related genes. Bioinformatic analysis performed on a broad panel of cancers of nonkidney origin suggests that disturbed expression of genes related to extracellular matrix and adhesion may be a universal feature of cancerous progression. Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All

Network analysis of genes involved in the enhancement of hyperthermia sensitivity by the knockdown of BAG3 in human oral squamous cell carcinoma cells.

Science.gov (United States)

Yunoki, Tatsuya; Tabuchi, Yoshiaki; Hayashi, Atsushi; Kondo, Takashi

2016-07-01

BCL2-associated athanogene 3 (BAG3), a co-chaperone of the heat shock 70 kDa protein (HSPA) family of proteins, is a cytoprotective protein that acts against various stresses, including heat stress. The aim of the present study was to identify gene networks involved in the enhancement of hyperthermia (HT) sensitivity by the knockdown (KD) of BAG3 in human oral squamous cell carcinoma (OSCC) cells. Although a marked elevation in the protein expression of BAG3 was detected in human the OSCC HSC-3 cells exposed to HT at 44˚C for 90 min, its expression was almost completely suppressed in the cells transfected with small interfering RNA against BAG3 (siBAG) under normal and HT conditions. The silencing of BAG3 also enhanced the cell death that was increased in the HSC-3 cells by exposure to HT. Global gene expression analysis revealed many genes that were differentially expressed by >2-fold in the cells exposed to HT and transfected with siBAG. Moreover, Ingenuity® pathways analysis demonstrated two unique gene networks, designated as Pro-cell death and Anti-cell death, which were obtained from upregulated genes and were mainly associated with the biological functions of induction and the prevention of cell death, respectively. Of note, the expression levels of genes in the Pro-cell death and Anti-cell death gene networks were significantly elevated and reduced in the HT + BAG3-KD group compared to those in the HT control group, respectively. These results provide further insight into the molecular mechanisms involved in the enhancement of HT sensitivity by the silencing of BAG3 in human OSCC cells.

Chitosan drives anti-inflammatory macrophage polarisation and pro-inflammatory dendritic cell stimulation

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MI Oliveira

2012-07-01

Full Text Available Macrophages and dendritic cells (DC share the same precursor and play key roles in immunity. Modulation of their behaviour to achieve an optimal host response towards an implanted device is still a challenge. Here we compare the differentiation process and polarisation of these related cell populations and show that they exhibit different responses to chitosan (Ch, with human monocyte-derived macrophages polarising towards an anti-inflammatory phenotype while their DC counterparts display pro-inflammatory features. Macrophages and DC, whose interactions with biomaterials are frequently analysed using fully differentiated cells, were cultured directly on Ch films, rather than exposed to the polymer after complete differentiation. Ch was the sole stimulating factor and activated both macrophages and DC, without leading to significant T cell proliferation. After 10 d on Ch, macrophages significantly down-regulated expression of pro-inflammatory markers, CD86 and MHCII. Production of pro-inflammatory cytokines, particularly TNF-α, decreased with time for cells cultured on Ch, while anti-inflammatory IL-10 and TGF-β1, significantly increased. Altogether, these results suggest an M2c polarisation. Also, macrophage matrix metalloproteinase activity was augmented and cell motility was stimulated by Ch. Conversely, DC significantly enhanced CD86 expression, reduced IL-10 secretion and increased TNF-α and IL-1β levels. Our findings indicate that cells with a common precursor may display different responses, when challenged by the same biomaterial. Moreover, they help to further comprehend macrophage/DC interactions with Ch and the balance between pro- and anti-inflammatory signals associated with implant biomaterials. We propose that an overall pro-inflammatory reaction may hide the expression of anti-inflammatory cytokines, likely relevant for tissue repair/regeneration.

Role of X-ray-inducible genes and proteins in adaptive survival responses

International Nuclear Information System (INIS)

Meyers, M.; Schea, R.A.; Petrowski, A.E.; Seabury, H.; McLaughlin, P.W.; Lee, I.; Lee, S.W.; Boothman, D.A.

1992-01-01

Certain X-ray-inducible genes and their corresponding protein products, appearing following low priming doses of ionizing radiation may subsequently give rise to an adaptive survival response, ultimately leading to increased radioresistance. Further, this adaptive radioresistance may be due to increased DNA repair (or misrepair) processes. Ultimately, the function of low-dose-induced cDNA clones within the cell is hoped to elucidate to follow the effects of specific gene turn-off on adaptive responses. Future research must determine the various functions of adaptive response gene products so that the beneficial or deleterious consequences of adaptive responses, which increases resistance to ionizing radiation, can be determined. (author). 19 refs., 1 fig

Suppression of pro-inflammatory T-cell responses by human mesothelial cells.

Science.gov (United States)

Lin, Chan-Yu; Kift-Morgan, Ann; Moser, Bernhard; Topley, Nicholas; Eberl, Matthias

2013-07-01

Human γδ T cells reactive to the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) contribute to acute inflammatory responses. We have previously shown that peritoneal dialysis (PD)-associated infections with HMB-PP producing bacteria are characterized by locally elevated γδ T-cell frequencies and poorer clinical outcome compared with HMB-PP negative infections, implying that γδ T cells may be of diagnostic, prognostic and therapeutic value in acute disease. The regulation by local tissue cells of these potentially detrimental γδ T-cell responses remains to be investigated. Freshly isolated γδ or αβ T cells were cultured with primary mesothelial cells derived from omental tissue, or with mesothelial cell-conditioned medium. Stimulation of cytokine production and proliferation by peripheral T cells in response to HMB-PP or CD3/CD28 beads was assessed by flow cytometry. Resting mesothelial cells were potent suppressors of pro-inflammatory γδ T cells as well as CD4+ and CD8+ αβ T cells. The suppression of γδ T-cell responses was mediated through soluble factors released by primary mesothelial cells and could be counteracted by SB-431542, a selective inhibitor of TGF-β and activin signalling. Recombinant TGF-β1 but not activin-A mimicked the mesothelial cell-mediated suppression of γδ T-cell responses to HMB-PP. The present findings indicate an important regulatory function of mesothelial cells in the peritoneal cavity by dampening pro-inflammatory T-cell responses, which may help preserve the tissue integrity of the peritoneal membrane in the steady state and possibly during the resolution of acute inflammation.

The effect of S1P receptor signaling pathway on the survival and drug resistance in multiple myeloma cells.

Science.gov (United States)

Fu, Di; Li, Yingchun; Li, Jia; Shi, Xiaoyan; Yang, Ronghui; Zhong, Yuan; Wang, Huihan; Liao, Aijun

2017-01-01

Multiple myeloma (MM) remains incurable by conventional chemotherapy. Sphingosine-1-phosphate (S1P) receptor-mediated signaling has been recently demonstrated to have critical roles in cell survival and drug resistance in a number of hematological malignancies. To dissect the roles of S1P receptor pathway in MM, we systematically examined cell viability and protein expression associated with cell survival and drug resistance in MM cell lines upon treatment with either pathway activator (S1P) or inhibitor (FTY720). Our results reveal that FTY720 inhibits cell proliferation by downregulating expression of target genes, while S1P has an opposite effect. Knocking down of S1P receptor S1P5R results in a reduction of cell survival-related gene expression; however, it does not have impacts on expression of drug resistance genes. These results suggest that S1P signaling plays a role in cell proliferation and drug resistance in MM, and targeting this pathway will provide a new therapeutic direction for MM management.

Explorative data analysis of MCL reveals gene expression networks implicated in survival and prognosis supported by explorative CGH analysis

International Nuclear Information System (INIS)

Blenk, Steffen; Engelmann, Julia C; Pinkert, Stefan; Weniger, Markus; Schultz, Jörg; Rosenwald, Andreas; Müller-Hermelink, Hans K; Müller, Tobias; Dandekar, Thomas

2008-01-01

Mantle cell lymphoma (MCL) is an incurable B cell lymphoma and accounts for 6% of all non-Hodgkin's lymphomas. On the genetic level, MCL is characterized by the hallmark translocation t(11;14) that is present in most cases with few exceptions. Both gene expression and comparative genomic hybridization (CGH) data vary considerably between patients with implications for their prognosis. We compare patients over and below the median of survival. Exploratory principal component analysis of gene expression data showed that the second principal component correlates well with patient survival. Explorative analysis of CGH data shows the same correlation. On chromosome 7 and 9 specific genes and bands are delineated which improve prognosis prediction independent of the previously described proliferation signature. We identify a compact survival predictor of seven genes for MCL patients. After extensive re-annotation using GEPAT, we established protein networks correlating with prognosis. Well known genes (CDC2, CCND1) and further proliferation markers (WEE1, CDC25, aurora kinases, BUB1, PCNA, E2F1) form a tight interaction network, but also non-proliferative genes (SOCS1, TUBA1B CEBPB) are shown to be associated with prognosis. Furthermore we show that aggressive MCL implicates a gene network shift to higher expressed genes in late cell cycle states and refine the set of non-proliferative genes implicated with bad prognosis in MCL. The results from explorative data analysis of gene expression and CGH data are complementary to each other. Including further tests such as Wilcoxon rank test we point both to proliferative and non-proliferative gene networks implicated in inferior prognosis of MCL and identify suitable markers both in gene expression and CGH data

Circulating CXCR5+CD4+ T cells assist in the survival and growth of primary diffuse large B cell lymphoma cells through interleukin 10 pathway

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Cha, Zhanshan [Department of Transfusion, Changhai Hospital, Second Military Medical University, Shanghai 200433 (China); Qian, Guangfang [Department of Endocrinology, Zhangqiu Municipal Hospital of Traditional Chinese Medicine, Zhangqiu, Shandong 250200 (China); Zang, Yan; Gu, Haihui; Huang, Yanyan; Zhu, Lishuang; Li, Jinqi; Liu, Yang; Tu, Xiaohua [Department of Transfusion, Changhai Hospital, Second Military Medical University, Shanghai 200433 (China); Song, Haihan [Emergency Center, East Hospital, Shanghai 200120 (China); Qian, Baohua, E-mail: qianbhl963@163.com [Department of Transfusion, Changhai Hospital, Second Military Medical University, Shanghai 200433 (China)

2017-01-01

Diffuse large B cell lymphoma (DLBCL) is a common and aggressive cancer caused by the malignant transformation of B cells. Although it has been established that the follicular helper T (Tfh) cells play a central role in B cell development, little information is available on their involvement in DLBCL pathogenesis. We studied the role of the peripheral Tfh equivalent, the CXCR5{sup +} CD4{sup +} T cells, in DLBCL. Data showed that compared to CXCR5{sup -} CD4{sup +} T cells, CXCR5{sup +} CD4{sup +} T cells were significantly more effective at promoting the proliferation as well as inhibiting the apoptosis of primary autologous DLBCL tumor cells. Surprisingly, we found that at equal cell numbers, CXCR5{sup +} CD4{sup +} T cells in DLBCL patients secreted significantly less interleukin (IL)-21 than CXCR5{sup -} CD4{sup +} T cells, while the level of IL-10 secretion was significant elevated in the CXCR5{sup +} compartment compared to the CXCR5{sup -} compartment. Neutralization of IL-10 in the primary DLBCL-CXCR5{sup +} CD4{sup +} T cell coculture compromised the CXCR5{sup +} CD4{sup +} T cell-mediated pro-tumor effects, in a manner that was dependent on the concentration of anti-IL-10 antibodies. The CXCR5{sup +} compartment also contained significantly lower frequencies of cytotoxic CD4{sup +} T cells than the CXCR5{sup -} compartment. In conclusion, our investigations discovered a previously unknown pro-tumor role of CXCR5-expressing circulating CD4{sup +} T cells, which assisted the survival and proliferation of primary DLBCL cells through IL-10. - Highlights: • We studied the role of the peripheral Tfh in DLBCL. • Tfh were effective at promoting the proliferation of primary DLBCL tumor cells. • Tfh were effective at inhibiting the apoptosis of primary DLBCL tumor cells. • IL-10 secretion in Tfh was significant elevated in DLBCL. • Neutralization of IL-10 compromised Tfh-mediated pro-tumor effects.

Hypertension-Related Gene Polymorphisms of G-Protein-Coupled Receptor Kinase 4 Are Associated with NT-proBNP Concentration in Normotensive Healthy Adults

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Junichi Yatabe

2012-01-01

Full Text Available G protein-coupled receptor kinase 4 (GRK4 with activating polymorphisms desensitize the natriuric renal tubular D1 dopamine receptor, and these GRK4 polymorphisms are strongly associated with salt sensitivity and hypertension. Meanwhile, N-terminal pro-B-type natriuretic peptide (NT-proBNP may be useful in detecting slight volume expansion. However, relations between hypertension-related gene polymorphisms including GRK4 and cardiovascular indices such as NT-proBNP are not clear, especially in healthy subjects. Therefore, various hypertension-related polymorphisms and cardiovascular indices were analyzed in 97 normotensive, healthy Japanese adults. NT-proBNP levels were significantly higher in subjects with two or more GRK4 polymorphic alleles. Other hypertension-related gene polymorphisms, such as those of renin-angiotensin-aldosterone system genes, did not correlate with NT-proBNP. There was no significant association between any of the hypertension-related gene polymorphisms and central systolic blood pressure, cardioankle vascular index, augmentation index, plasma aldosterone concentration, or an oxidative stress marker, urinary 8-OHdG. Normotensive individuals with GRK4 polymorphisms show increased serum NT-proBNP concentration and may be at a greater risk of developing hypertension and cardiovascular disease.

Intrinsic, pro-apoptotic effects of IGFBP-3 on breast cancer cells are reversible: Involvement of PKA, Rho and ceramide.

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Claire M Perks

2011-05-01

Full Text Available We established previously that IGFBP-3 could exert positive or negative effects on cell function depending upon the extracellular matrix composition and by interacting with integrin signalling. To elicit its pro-apoptotic effects IGFBP-3 bound to caveolin-1 and the beta 1 integrin receptor and increased their association culminating in MAPK activation. Disruption of these complexes or blocking the beta 1 integrin receptor reversed these intrinsic actions of IGFBP-3. In this study we have examined the signalling pathway between integrin receptor binding and MAPK activation that mediates the intrinsic, pro-apoptotic actions of IGFBP-3. We found on inhibiting protein kinase A(PKA, Rho associated kinase (ROCK and ceramide, the accentuating effects of IGFBP-3 on apoptotic triggers were reversed, such that IGFBP-3 then conferred cell survival. We established that IGFBP-3 activated Rho, the upstream regulator of ROCK and that beta1 integrin and PKA were upstream of Rho activation, whereas the involvement of ceramide was downstream. The beta 1 integrin, PKA, Rho and ceramide were all upstream of MAPK activation. These data highlight key components involved in the pro-apoptotic effects of IGFBP-3 and that inhibiting them leads to a reversal in the action of IGFBP-3.

Microsporidia infection impacts the host cell's cycle and reduces host cell apoptosis

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Higes, Mariano; Sagastume, Soledad; Juarranz, Ángeles; Dias-Almeida, Joyce; Budge, Giles E.; Meana, Aránzazu; Boonham, Neil

2017-01-01

Intracellular parasites can alter the cellular machinery of host cells to create a safe haven for their survival. In this regard, microsporidia are obligate intracellular fungal parasites with extremely reduced genomes and hence, they are strongly dependent on their host for energy and resources. To date, there are few studies into host cell manipulation by microsporidia, most of which have focused on morphological aspects. The microsporidia Nosema apis and Nosema ceranae are worldwide parasites of honey bees, infecting their ventricular epithelial cells. In this work, quantitative gene expression and histology were studied to investigate how these two parasites manipulate their host’s cells at the molecular level. Both these microsporidia provoke infection-induced regulation of genes involved in apoptosis and the cell cycle. The up-regulation of buffy (which encodes a pro-survival protein) and BIRC5 (belonging to the Inhibitor Apoptosis protein family) was observed after infection, shedding light on the pathways that these pathogens use to inhibit host cell apoptosis. Curiously, different routes related to cell cycle were modified after infection by each microsporidia. In the case of N. apis, cyclin B1, dacapo and E2F2 were up-regulated, whereas only cyclin E was up-regulated by N. ceranae, in both cases promoting the G1/S phase transition. This is the first report describing molecular pathways related to parasite-host interactions that are probably intended to ensure the parasite’s survival within the cell. PMID:28152065

An Integrative Analysis to Identify Driver Genes in Esophageal Squamous Cell Carcinoma.

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Genta Sawada

Full Text Available Few driver genes have been well established in esophageal squamous cell carcinoma (ESCC. Identification of the genomic aberrations that contribute to changes in gene expression profiles can be used to predict driver genes.We searched for driver genes in ESCC by integrative analysis of gene expression microarray profiles and copy number data. To narrow down candidate genes, we performed survival analysis on expression data and tested the genetic vulnerability of each genes using public RNAi screening data. We confirmed the results by performing RNAi experiments and evaluating the clinical relevance of candidate genes in an independent ESCC cohort.We found 10 significantly recurrent copy number alterations accompanying gene expression changes, including loci 11q13.2, 7p11.2, 3q26.33, and 17q12, which harbored CCND1, EGFR, SOX2, and ERBB2, respectively. Analysis of survival data and RNAi screening data suggested that GRB7, located on 17q12, was a driver gene in ESCC. In ESCC cell lines harboring 17q12 amplification, knockdown of GRB7 reduced the proliferation, migration, and invasion capacities of cells. Moreover, siRNA targeting GRB7 had a synergistic inhibitory effect when combined with trastuzumab, an anti-ERBB2 antibody. Survival analysis of the independent cohort also showed that high GRB7 expression was associated with poor prognosis in ESCC.Our integrative analysis provided important insights into ESCC pathogenesis. We identified GRB7 as a novel ESCC driver gene and potential new therapeutic target.

Hepatitis Bx Antigen Stimulates Expression of a Novel Cellular Gene, URG4, that Promotes Hepatocellular Growth and Survival

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N. Lale Satiroglu Tufan

2002-01-01

Full Text Available Hepatitis B virus encoded X antigen (HBxAg may contribute to the development of hepatocellular carcinoma (HCC by up-or downregulating the expression of cellular genes that promote cell growth and survival. To test this hypothesis, HBxAg-positive and-negative HepG2 cells were constructed, and the patterns of cellular gene expression compared by polymerase chain reaction select cDNA subtraction. The full-length clone of one of these upregulated genes (URG, URG4, encoded a protein of about 104 kDa. URG4 was strongly expressed in hepatitis 13-infected liver and in HCC cells, where it costained with HBxAg, and was weakly expressed in uninfected liver, suggesting URG4 was an effector of HBxAg in vivo. Overexpression of URG4 in HepG2 cells promoted hepatocellular growth and survival in tissue culture and in soft agar, and accelerated tumor development in nude mice. Hence, URG4 may be a natural effector of HBxAg that contributes importantly to multistep hepatocarcinogenesis.

Ser80Ile mutation and a concurrent Pro25Leu variant of the VHL gene in an extended Hungarian von Hippel-Lindau family

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Fazakas Ferenc

2008-04-01

Full Text Available Abstract Von Hippel-Lindau disease (VHL is a rare autosomal dominant disease characterized by development of cystic and tumorous lesions at multiple sites, including the brain, spinal cord, kidneys, adrenals, pancreas, epididymis and eyes. The clinical phenotype results from molecular abnormalities of the VHL tumor suppressor gene, mapped to human chromosome 3p25-26. The VHL gene encodes two functionally active VHL proteins due to the presence of two translational initiation sites separated by 53 codons. The majority of disease-causing mutations have been detected downstream of the second translational initiation site, but there are conflicting data as to whether few mutations located in the first 53 codons, such as the Pro25Leu could have a pathogenic role. In this paper we report a large Hungarian VHL type 2 family consisting of 32 members in whom a disease-causing AGT80AAT (Ser80Ile c.239G>A, p.Ser80Ile mutation, but not the concurrent CCT25CTT (Pro25Leu c.74C>T, p.Pro25Leu variant co-segregated with the disease. To our knowledge, the Ser80Ile mutation has not been previously described in VHL type 2 patients with high risk of pheochromocytoma and renal cell cancer. Therefore, this finding represents a novel genotype-phenotype association and VHL kindreds with Ser80Ile mutation will require careful surveillance for pheochromocytoma. We concluded that the Pro25Leu variant is a rare, neutral variant, but the presence such a rare gene variant may make genetic counseling difficult.

Encapsulation of rat bone marrow stromal cells using a poly-ion complex gel of chitosan and succinylated poly(Pro-Hyp-Gly).

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Kusumastuti, Yuni; Shibasaki, Yoshiaki; Hirohara, Shiho; Kobayashi, Mime; Terada, Kayo; Ando, Tsuyoshi; Tanihara, Masao

2017-03-01

Encapsulation of stem cells into a three-dimensional (3D) scaffold is necessary to achieve tissue regeneration. Prefabricated 3D scaffolds, such as fibres or porous sponges, have limitations regarding homogeneous cell distribution. Hydrogels that can encapsulate cells such as animal-derived collagen gels need adjustment of the pH and/or temperature upon cell mixing. In this report, we fabricated a poly-ion complex (PIC) hydrogel of chitosan and succinylated poly(Pro-Hyp-Gly) and assessed its effect on cell viability after encapsulation of rat bone marrow stromal cells. PIC hydrogels were obtained successfully with a concentration of each precursor as low as 3.0-3.8 mg/ml. The maximum gelation and swelling ratios were achieved with an equal molar ratio (1:1) of anionic and cationic groups. Using chitosan acetate as a cationic precursor produced a PIC hydrogel with both a significantly greater gelation ratio and a better swelling ratio than chitosan chloride. Ammonium succinylated poly(Pro-Hyp-Gly) as an anionic precursor gave similar gelation and swelling ratios to those of sodium succinylated poly(Pro-Hyp-Gly). Cell encapsulation was also achieved successfully by mixing rat bone marrow stromal cells with the PIC hydrogel simultaneously during its formation. The PIC hydrogel was maintained in the culture medium for 7 days at 37°C and the encapsulated cells survived and proliferated in it. Although it is necessary to improve its functionality, this PIC hydrogel has the potential to act as a 3D scaffold for cell encapsulation and tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Global gene expression response to telomerase in bovine adrenocortical cells

International Nuclear Information System (INIS)

Perrault, Steven D.; Hornsby, Peter J.; Betts, Dean H.

2005-01-01

The infinite proliferative capability of most immortalized cells is dependent upon the presence of the enzyme telomerase and its ability to maintain telomere length and structure. However, telomerase may be involved in a greater system than telomere length regulation, as recent evidence has shown it capable of increasing wound healing in vivo, and improving cellular proliferation rate and survival from apoptosis in vitro. Here, we describe the global gene expression response to ectopic telomerase expression in an in vitro bovine adrenocortical cell model. Telomerase-immortalized cells showed an increased ability for proliferation and survival in minimal essential medium above cells transgenic for GFP. cDNA microarray analyses revealed an altered cell state indicative of increased adrenocortical cell proliferation regulated by the IGF2 pathway and alterations in members of the TGF-B family. As well, we identified alterations in genes associated with development and wound healing that support a model that high telomerase expression induces a highly adaptable, progenitor-like state

Dimethyl sulfoxide-caused changes in pro- and anti-angiogenic factor levels could contribute to an anti-angiogenic response in HeLa cells.

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Şimşek, Ece; Aydemir, Esra Arslan; İmir, Nilüfer; Koçak, Orhan; Kuruoğlu, Aykut; Fışkın, Kayahan

2015-10-01

Dimethyl sulfoxide (DMSO) is widely used in biological research as a general solvent. While it has been previously demonstrated that DMSO possesses a wide range of pharmacological effects, there is no published work regarding the effects of DMSO on pro-angiogenic factor levels. This study was designed to investigate the possible effects of DMSO on the levels of three pro-angiogenic factors released from HeLa cells in vitro. Cells were treated with two different and previously determined concentrations of DMSO. The cytotoxic effects of DMSO concentrations on HeLa cells were determined via MTT. Survival rates of DMSO-treated cells were determined by Invitrogen live/dead viability/cytotoxicity kit and trypan blue exclusion assay. Changes in the pro-angiogenic levels in media were evaluated by Cayman's Substance P Enzyme Immunoassay ELISA kit. Vascular endothelial growth factor ELISA kit and interferon gamma ELISA kit for substance P, VEGF and IFNγ respectively. Changes in substance P levels were corrected by standard western blotting. Changes in VEGF and IFNγ levels were corrected both by western blot and real time PCR. Treatment with 1.4 μM DMSO caused a time-dependent inhibition of cell proliferation at 24, 48 and 72 h. 1.4 μM DMSO caused a significant reduction in VEGF levels at 72 h of incubation and sharp increases in IFNγ levels at both 48 and 72 h of incubation. According to real time PCR analyses, DMSO (1.4 μM) exhibited an inhibitory effect on VEGF but acted as an augmenter of IFNγ release on HeLa cells in vitro. This is the first report showing that the general solvent DMSO suppressed HeLa cell proliferation, decreased the levels of two pro-angiogenic factors (substance P and VEGF) and increased the release of an anti-angiogenic factor IFNγ in vitro. Copyright © 2015 Elsevier Ltd. All rights reserved.

The Immunomodulatory Small Molecule Imiquimod Induces Apoptosis in Devil Facial Tumour Cell Lines.

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Amanda L Patchett

Full Text Available The survival of the Tasmanian devil (Sarcophilus harrisii is threatened by devil facial tumour disease (DFTD. This transmissible cancer is usually fatal, and no successful treatments have been developed. In human studies, the small immunomodulatory molecule imiquimod is a successful immunotherapy, activating anti-tumour immunity via stimulation of toll-like receptor-7 (TLR7 signaling pathways. In addition, imiquimod is a potent inducer of apoptosis in human tumour cell lines via TLR7 independent mechanisms. Here we investigate the potential of imiquimod as a DFTD therapy through analysis of treated DFTD cell lines and Tasmanian devil fibroblasts. WST-8 proliferation assays and annexin V apoptosis assays were performed to monitor apoptosis, and changes to the expression of pro- and anti-apoptotic genes were analysed using qRT-PCR. Our results show that DFTD cell lines, but not Tasmanian devil fibroblasts, are sensitive to imiquimod-induced apoptosis in a time and concentration dependent manner. Induction of apoptosis was accompanied by down-regulation of the anti-apoptotic BCL2 and BCLXL genes, and up-regulation of the pro-apoptotic BIM gene. Continuous imiquimod treatment was required for these effects to occur. These results demonstrate that imiquimod can deregulate DFTD cell growth and survival in direct and targeted manner. In vivo, this may increase DFTD vulnerability to imiquimod-induced TLR7-mediated immune responses. Our findings have improved the current knowledge of imiquimod action in tumour cells for application to both DFTD and human cancer therapy.

A hybrid approach of gene sets and single genes for the prediction of survival risks with gene expression data.

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Seok, Junhee; Davis, Ronald W; Xiao, Wenzhong

2015-01-01

Accumulated biological knowledge is often encoded as gene sets, collections of genes associated with similar biological functions or pathways. The use of gene sets in the analyses of high-throughput gene expression data has been intensively studied and applied in clinical research. However, the main interest remains in finding modules of biological knowledge, or corresponding gene sets, significantly associated with disease conditions. Risk prediction from censored survival times using gene sets hasn't been well studied. In this work, we propose a hybrid method that uses both single gene and gene set information together to predict patient survival risks from gene expression profiles. In the proposed method, gene sets provide context-level information that is poorly reflected by single genes. Complementarily, single genes help to supplement incomplete information of gene sets due to our imperfect biomedical knowledge. Through the tests over multiple data sets of cancer and trauma injury, the proposed method showed robust and improved performance compared with the conventional approaches with only single genes or gene sets solely. Additionally, we examined the prediction result in the trauma injury data, and showed that the modules of biological knowledge used in the prediction by the proposed method were highly interpretable in biology. A wide range of survival prediction problems in clinical genomics is expected to benefit from the use of biological knowledge.

Enhanced B-Raf-mediated NRF2 gene transcription and HATs-mediated NRF2 protein acetylation contributes to ABCC1-mediated chemoresistance and glutathione-mediated survival in acquired topoisomerase II poison-resistant cancer cells.

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Chen, Huang-Hui; Chang, Hsin-Huei; Chang, Jang-Yang; Tang, Ya-Chu; Cheng, Yung-Chi; Lin, Li-Mei; Cheng, Shu-Ying; Huang, Chih-Hsiang; Sun, Man-Wu; Chen, Chiung-Tong; Kuo, Ching-Chuan

2017-12-01

Nuclear factor erythroid-2-related factor 2 (NRF2) mainly regulates transcriptional activation through antioxidant-responsive elements (AREs) present in the promoters of NRF2 target genes. Recently, we found that NRF2 was overexpressed in a KB-derived drug-resistant cancer cell panel. In this panel, KB-7D cells, which show acquired resistance to topoisomerase II (Top II) poisons, exhibited the highest NRF2 activation. To investigate whether NRF2 directly contributed to acquired resistance against Top II poisons, we manipulated NRF2 by genetic and pharmacological approaches. The result demonstrated that silencing of NRF2 by RNA interference increased the sensitivity and treatment with NRF2 activator decreased the sensitivity of KB and KB-7D cells toward Top II poisons. Further, increased B-Raf-mediated NRF2 gene transcription and HATs-mediated NRF2 protein acetylation activated NRF2 signaling in KB-7D cells. Moreover, increased binding of NRF2 to an ARE in the promoter of ATP-binding cassette subfamily C member 1 (ABCC1) directly contributed to Top II poison resistance. In addition, activation of NRF2 increased glutathione level and antioxidant capacity in KB-7D cells compared with that in KB cells; moreover, high glutathione level provided survival advantage to KB-7D cells. Our study is the first to show that aberrant NRF2 activation is via increased B-Raf-mediated NRF2 gene transcription and HATs-mediated NRF2 protein acetylation, which increases the acquired resistance and promote the survival of Top II poison-resistant cancer cells. Importantly, NRF2 downstream effectors ABCC1 and glutathione directly contribute to acquired resistance and survival, respectively. These results suggest that blockade of NRF2 signaling may enhance therapeutic efficacy and reduce the survival of Top II poison-refractory tumors in clinical. Copyright © 2017 Elsevier Inc. All rights reserved.

AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells

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Lorenzato, Annalisa; Biolatti, Marta [Department of Oncology, University of Torino School of Medicine, Torino (Italy); Institute for Cancer Research at Candiolo, Candiolo, Torino (Italy); Delogu, Giuseppe [Department of Biomedical Sciences-Histology, University of Sassari, Sassari (Italy); Capobianco, Giampiero [Department of Surgical, Microsurgical and Medical Sciences, University of Sassari, Sassari (Italy); Farace, Cristiano [Department of Biomedical Sciences-Histology, University of Sassari, Sassari (Italy); Dessole, Salvatore; Cossu, Antonio; Tanda, Francesco [Department of Surgical, Microsurgical and Medical Sciences, University of Sassari, Sassari (Italy); Madeddu, Roberto [Department of Biomedical Sciences-Histology, University of Sassari, Sassari (Italy); National Institute of Biostructures and Biosystems, Rome (Italy); Olivero, Martina [Department of Oncology, University of Torino School of Medicine, Torino (Italy); Institute for Cancer Research at Candiolo, Candiolo, Torino (Italy); Di Renzo, Maria Flavia, E-mail: mariaflavia.direnzo@unito.it [Department of Oncology, University of Torino School of Medicine, Torino (Italy); Institute for Cancer Research at Candiolo, Candiolo, Torino (Italy)

2013-10-15

The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals. - highlights: • CSE1L is a key player in nucleocytoplasmic traffic by forming complex with Ran. • AKT phosphorylates RanBP3 that regulates the nucleocytoplasmic gradient of Ran. • The activated oncogenic AKT drives the nuclear accumulation of CSE1L. • CSE1L in the nucleus up-regulates genes conveying pro-oncogenic signals. • CSE1L might contribute to tumor progression driven by the activated oncogenic AKT.

AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells

International Nuclear Information System (INIS)

Lorenzato, Annalisa; Biolatti, Marta; Delogu, Giuseppe; Capobianco, Giampiero; Farace, Cristiano; Dessole, Salvatore; Cossu, Antonio; Tanda, Francesco; Madeddu, Roberto; Olivero, Martina; Di Renzo, Maria Flavia

2013-01-01

The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals. - highlights: • CSE1L is a key player in nucleocytoplasmic traffic by forming complex with Ran. • AKT phosphorylates RanBP3 that regulates the nucleocytoplasmic gradient of Ran. • The activated oncogenic AKT drives the nuclear accumulation of CSE1L. • CSE1L in the nucleus up-regulates genes conveying pro-oncogenic signals. • CSE1L might contribute to tumor progression driven by the activated oncogenic AKT

Radionuclide blood cell survival studies

International Nuclear Information System (INIS)

Bentley, S.A.; Miller, D.T.

1986-01-01

Platelet and red cell survival studies are reviewed. The use of 51 Cr and di-isopropylfluoridate labelled with tritium or 32 P is discussed for red cell survival study and 51 Cr and 111 In-oxine are considered as platelet labels. (UK)

Screen for genes involved in radiation survival of Escherichia coli and construction of a reference database

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Sargentini, Neil J., E-mail: nsargentini@atsu.edu; Gularte, Nicholas P.; Hudman, Deborah A.

2016-11-15

-3 for 39 common mutants (P = 0.010). Comparing gene functions using MultiFun terms, uncommon genes tended to show less involvement in DNA repair-relevant categories (information transfer and cell processes), but greater involvement in seven other categories. Our analysis of 455 genes suggests cell survival and DNA repair processes are more complex than previously understood, and may be compromised by deficiencies in other processes.

Telomerase gene therapy rescues telomere length, bone marrow aplasia, and survival in mice with aplastic anemia.

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Bär, Christian; Povedano, Juan Manuel; Serrano, Rosa; Benitez-Buelga, Carlos; Popkes, Miriam; Formentini, Ivan; Bobadilla, Maria; Bosch, Fatima; Blasco, Maria A

2016-04-07

Aplastic anemia is a fatal bone marrow disorder characterized by peripheral pancytopenia and marrow hypoplasia. The disease can be hereditary or acquired and develops at any stage of life. A subgroup of the inherited form is caused by replicative impairment of hematopoietic stem and progenitor cells due to very short telomeres as a result of mutations in telomerase and other telomere components. Abnormal telomere shortening is also described in cases of acquired aplastic anemia, most likely secondary to increased turnover of bone marrow stem and progenitor cells. Here, we test the therapeutic efficacy of telomerase activation by using adeno-associated virus (AAV)9 gene therapy vectors carrying the telomerase Tert gene in 2 independent mouse models of aplastic anemia due to short telomeres (Trf1- and Tert-deficient mice). We find that a high dose of AAV9-Tert targets the bone marrow compartment, including hematopoietic stem cells. AAV9-Tert treatment after telomere attrition in bone marrow cells rescues aplastic anemia and mouse survival compared with mice treated with the empty vector. Improved survival is associated with a significant increase in telomere length in peripheral blood and bone marrow cells, as well as improved blood counts. These findings indicate that telomerase gene therapy represents a novel therapeutic strategy to treat aplastic anemia provoked or associated with short telomeres. © 2016 by The American Society of Hematology.

Gene Expression during Survival of Escherichia coli O157:H7 in Soil and Water

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Ashley D. Duffitt

2011-01-01

Full Text Available The in vitro survival of Escherichia coli O157:H7 at 15∘C under two experimental conditions (sterile soil and sterile natural water was examined. DNA microarrays of the entire set of E. coli O157:H7 genes were used to measure the genomic expression patterns after 14 days. Although the populations declined, some E. coli O157:H7 cells survived in sterile stream water up to 234 days and in sterile soil for up to 179 days. Cells incubated in soil microcosms for 14 days expressed genes for antibiotic resistance, biosynthesis, DNA replication and modification, metabolism, phages, transposons, plasmids, pathogenesis and virulence, antibiotic resistance, ribosomal proteins, the stress response, transcription, translation, and transport and binding proteins at significantly higher levels than cells grown in Luria broth. These results suggest that E. coli O157:H7 may develop a different phenotype during transport through the environment. Furthermore, this pathogen may become more resistant to antibiotics making subsequent infections more difficult to treat.

The scaffold protein MEK Partner 1 is required for the survival of estrogen receptor positive breast cancer cells

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Marina Mihaela

2012-07-01

Full Text Available Abstract MEK Partner 1 (MP1 or MAPKSP1 is a scaffold protein that has been reported to function in multiple signaling pathways, including the ERK, PAK and mTORC pathways. Several of these pathways influence the biology of breast cancer, but MP1’s functional significance in breast cancer cells has not been investigated. In this report, we demonstrate a requirement for MP1 expression in estrogen receptor (ER positive breast cancer cells. MP1 is widely expressed in both ER-positive and negative breast cancer cell lines, and in non-tumorigenic mammary epithelial cell lines. However, inhibition of its expression using siRNA duplexes resulted in detachment and apoptosis of several ER-positive breast cancer cell lines, but not ER-negative breast cancer cells or non-tumorigenic mammary epithelial cells. Inhibition of MP1 expression in ER-positive MCF-7 cells did not affect ERK activity, but resulted in reduced Akt1 activity and reduced ER expression and activity. Inhibition of ER expression did not result in cell death, suggesting that decreased ER expression is not the cause of cell death. In contrast, pharmacological inhibition of PI3K signaling did induce cell death in MCF-7 cells, and expression of a constitutively active form of Akt1 partially rescued the cell death observed when the MP1 gene was silenced in these cells. Together, these results suggest that MP1 is required for pro-survival signaling from the PI3K/Akt pathway in ER-positive breast cancer cells.

Neonatal maternal separation up-regulates protein signalling for cell survival in rat hypothalamus.

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Irles, Claudine; Nava-Kopp, Alicia T; Morán, Julio; Zhang, Limei

2014-05-01

We have previously reported that in response to early life stress, such as maternal hyperthyroidism and maternal separation (MS), the rat hypothalamic vasopressinergic system becomes up-regulated, showing enlarged nuclear volume and cell number, with stress hyperresponsivity and high anxiety during adulthood. The detailed signaling pathways involving cell death/survival, modified by adverse experiences in this developmental window remains unknown. Here, we report the effects of MS on cellular density and time-dependent fluctuations of the expression of pro- and anti-apoptotic factors during the development of the hypothalamus. Neonatal male rats were exposed to 3 h-daily MS from postnatal days 2 to 15 (PND 2-15). Cellular density was assessed in the hypothalamus at PND 21 using methylene blue staining, and neuronal nuclear specific protein and glial fibrillary acidic protein immunostaining at PND 36. Expression of factors related to apoptosis and cell survival in the hypothalamus was examined at PND 1, 3, 6, 9, 12, 15, 20 and 43 by Western blot. Rats subjected to MS exhibited greater cell-density and increased neuronal density in all hypothalamic regions assessed. The time course of protein expression in the postnatal brain showed: (1) decreased expression of active caspase 3; (2) increased Bcl-2/Bax ratio; (3) increased activation of ERK1/2, Akt and inactivation of Bad; PND 15 and PND 20 were the most prominent time-points. These data indicate that MS can induce hypothalamic structural reorganization by promoting survival, suppressing cell death pathways, increasing cellular density which may alter the contribution of these modified regions to homeostasis.

Mindfulness-Based Stress Reduction training reduces loneliness and pro-inflammatory gene expression in older adults: a small randomized controlled trial.

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Creswell, J David; Irwin, Michael R; Burklund, Lisa J; Lieberman, Matthew D; Arevalo, Jesusa M G; Ma, Jeffrey; Breen, Elizabeth Crabb; Cole, Steven W

2012-10-01

Lonely older adults have increased expression of pro-inflammatory genes as well as increased risk for morbidity and mortality. Previous behavioral treatments have attempted to reduce loneliness and its concomitant health risks, but have had limited success. The present study tested whether the 8-week Mindfulness-Based Stress Reduction (MBSR) program (compared to a Wait-List control group) reduces loneliness and downregulates loneliness-related pro-inflammatory gene expression in older adults (N = 40). Consistent with study predictions, mixed effect linear models indicated that the MBSR program reduced loneliness, compared to small increases in loneliness in the control group (treatment condition × time interaction: F(1,35) = 7.86, p = .008). Moreover, at baseline, there was an association between reported loneliness and upregulated pro-inflammatory NF-κB-related gene expression in circulating leukocytes, and MBSR downregulated this NF-κB-associated gene expression profile at post-treatment. Finally, there was a trend for MBSR to reduce C Reactive Protein (treatment condition × time interaction: (F(1,33) = 3.39, p = .075). This work provides an initial indication that MBSR may be a novel treatment approach for reducing loneliness and related pro-inflammatory gene expression in older adults. Copyright © 2012 Elsevier Inc. All rights reserved.

BAP1 PLAYS A SURVIVAL ROLE IN CUTANEOUS MELANOMA

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Kumar, Raj; Taylor, Michael; Miao, Benchun; Ji, Zhenyu; Njauw, Jenny Ching-Ni; Jönsson, Göran; Frederick, Dennie Tompers; Tsao, Hensin

2014-01-01

Although the pattern of BAP1 inactivation in ocular melanoma specimens and in the BAP1 cutaneous/ocular melanoma (CM/OM) predisposition syndrome suggests a tumor suppressor function, the specific role of this gene in the pathogenesis of cutaneous melanoma is not fully understood. We thus set out to characterize BAP1 in cutaneous melanoma and discovered an unexpected pro-survival effect of this protein. Tissue and cell lines analysis showed that BAP1 expression was maintained, rather than lost, in primary melanomas compared to nevi and normal skin. Genetic depletion of BAP1 in melanoma cells reduced proliferation and colony forming capability, induced apoptosis and inhibited melanoma tumor growth in vivo. On the molecular level, suppression of BAP1 led to a concomitant drop in the protein levels of survivin a member of anti-apoptotic proteins and a known mediator of melanoma survival. Restoration of survivin in melanoma cells partially rescued the growth-retarding effects of BAP1 loss. In contrast to melanoma cells, stable overexpression of BAP1 into immortalized but non-transformed melanocytes did suppress proliferation and reduce survivin. Taken together, these studies demonstrate that BAP1 may play a growth-sustaining role in melanoma cells, but that its impact on ubiquitination underpins a complex physiology which is context and cell dependent. PMID:25521456

Histone deacetylase 2 is decreased in peripheral blood pro-inflammatory CD8+ T and NKT-like lymphocytes following lung transplant.

Science.gov (United States)

Hodge, Greg; Hodge, Sandra; Holmes-Liew, Chien-Li; Reynolds, Paul N; Holmes, Mark

2017-02-01

Immunosuppression therapy following lung transplantation fails to prevent chronic rejection in many patients, which is associated with lack of suppression of cytotoxic mediators and pro-inflammatory cytokines in peripheral blood T and natural killer T (NKT)-like cells. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) upregulate/downregulate pro-inflammatory gene expression, respectively; however, differences in the activity of these enzymes following lung transplant are unknown. We hypothesized decreased HDAC2 expression and increased HAT expression in pro-inflammatory lymphocytes following lung transplant. Blood was collected from 18 stable lung transplant patients and 10 healthy age-matched controls. Intracellular pro-inflammatory cytokines and HAT/HDAC2 expression were determined in lymphocyte subsets following culture using flow cytometry. A loss of HDAC2 in cluster of differentiation (CD) 8+ T and NKT-like cells in transplant patients compared with controls was noted (CD8+ T: 28 ± 10 (45 ± 10), CD8+NKT-like: 30 ± 13 (54 ± 16) (mean ± SD transplant) (control)). Loss of HDAC2 was associated with an increased percentage of CD8+ T and NKT-like cells expressing perforin, granzyme b, interferon gamma (IFN-γ) and TNF-α (no change in HAT expression in any lymphocyte subset). There was a negative correlation between loss of HDAC2 expression by CD8+ T cells with cumulative dose of prednisolone and time post-transplant. Treatment with 10 mg/L theophylline + 1 µmol/L prednisolone or 2.5 ng/mL cyclosporine A synergistically upregulated HDAC2 and inhibited IFN-γ and TNF-α production by CD8+ T and NKT-like lymphocytes. HDAC2 is decreased in CD8+ T and NKT-like pro-inflammatory lymphocytes following lung transplant. Treatment options that increase HDAC2 may improve graft survival. © 2016 Asian Pacific Society of Respirology.

Selective survival of peripheral blood lymphocytes in children with HIV-1 following delivery of an anti-HIV gene to bone marrow CD34(+) cells.

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Podsakoff, Greg M; Engel, Barbara C; Carbonaro, Denise A; Choi, Chris; Smogorzewska, Elzbieta M; Bauer, Gerhard; Selander, David; Csik, Susan; Wilson, Kathy; Betts, Michael R; Koup, Richard A; Nabel, Gary J; Bishop, Keith; King, Steven; Schmidt, Manfred; von Kalle, Christof; Church, Joseph A; Kohn, Donald B

2005-07-01

Two HIV-1-infected children on antiretroviral therapy were enrolled into a clinical study of retroviral-mediated transfer of a gene that inhibits replication of HIV-1, targeting bone marrow CD34+ hematopoietic stem/progenitor cells. Two retroviral vectors were used, one encoding a "humanized" dominant-negative REV protein (huM10) that is a potent inhibitor of HIV-1 replication and one encoding a nontranslated marker gene (FX) to serve as an internal control for the level of gene marking. Peripheral blood mononuclear cells (PBMC) containing the huM10 gene or FX gene were detected by quantitative PCR at frequencies of approximately 1/10,000 in both subjects for the first 1-3 months following re-infusion of the gene-transduced bone marrow, but then were at or below the limits of detection (<1/1,000,000) at most times over 2 years. In one patient, a reappearance of PBMC containing the huM10 gene, but not the FX gene, occurred concomitant with a rise in the HIV-1 viral load during a period of nonadherence to the antiretroviral regimen. Unique clones of gene-marked PBMC were detected by LAM-PCR during the time of elevated HIV-1 levels. These findings indicate that there was a selective survival advantage for PBMC containing the huM10 gene during the time of increased HIV-1 load.

Born to be Alive: A Role for the BCL-2 Family in Melanoma Tumor Cell Survival, Apoptosis, and Treatment

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Anvekar, Rina A.; Asciolla, James J.; Missert, Derek J.; Chipuk, Jerry E., E-mail: jerry.chipuk@mssm.edu [Department of Oncological Sciences, Mount Sinai School of Medicine, New York, NY (United States); Department of Dermatology, Mount Sinai School of Medicine, New York, NY (United States); The Tisch Cancer Institute, Mount Sinai Medical Center, New York, NY (United States)

2011-10-13

The global incidence of melanoma has dramatically increased during the recent decades, yet the advancement of primary and adjuvant therapies has not kept a similar pace. The development of melanoma is often centered on cellular signaling that hyper-activates survival pathways, while inducing a concomitant blockade to cell death. Aberrations in cell death signaling not only promote tumor survival and enhanced metastatic potential, but also create resistance to anti-tumor strategies. Chemotherapeutic agents target melanoma tumor cells by inducing a form of cell death called apoptosis, which is governed by the BCL-2 family of proteins. The BCL-2 family is comprised of anti-apoptotic proteins (e.g., BCL-2, BCL-xL, and MCL-1) and pro-apoptotic proteins (e.g., BAK, BAX, and BIM), and their coordinated regulation and function are essential for optimal responses to chemotherapeutics. Here we will discuss what is currently known about the mechanisms of BCL-2 family function with a focus on the signaling pathways that maintain melanoma tumor cell survival. Importantly, we will critically evaluate the literature regarding how chemotherapeutic strategies directly impact on BCL-2 family function and offer several suggestions for future regimens to target melanoma and enhance patient survival.

[Influence of tissue-specific superoxide dismutase genes expression in brain cells on Drosophila melanogaster sensitivity to oxidative stress and viability].

Science.gov (United States)

Vitushynska, M V; Matiytsiv, N P; Chernyk, Y

2015-01-01

The study has shown that both functional gene knockout Sodl and Sod2 and their overexpression in neurons and glial tissue increase the sensitivity of Drosophila melanogaster to oxidative stress (OS) conditions. The lowest survival rate was only 20.5% in insects with Sod2 knockout in neurons. Comparative analysis of the survival curves showed that adults with altered tissue-specific expression of the studied genes had reduced average and maximum life span. Under OS conditions induced by 5% hydrogen peroxide the life spans of wild type Oregon R and transgenic insects were significantly reduced. Altered Sod gene expression in glial tissue leads to degenerative changes in Drosophila brain at the young age. During the aging of insects and the action of pro-oxidants increasing of neurodegenerative phenotype is observed.

Changes in proHB-EGF expression after functional activation of the immune system cells

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T. O. Chudina

2017-12-01

Full Text Available The level of proHB-EGF expression on J774, Raji, KG-1 cells derived from different types of human and mouse immune system cells under the standard in vitro culture conditions and during functional activation of these cells was investigated. Changes in the proHB-EGF expression on the cell surface were found to depend on the density of cell population, the content of fetal bovine serum in the culture medium, the effect of mitogenic factors – bacterial lipopolysaccharide, an inactive full-size form of diphtheria toxin (CRM197 and recombinant soluble HB-EGF – rsHB-EGF. The results obtained are important for the understanding of the functional role of proHB-EGF receptor on the surface of macrophage-like cells and B lymphocytes and indicate the involvement of this receptor in immune response regulation in an organism.

EMX2 gene expression predicts liver metastasis and survival in colorectal cancer.

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Aykut, Berk; Ochs, Markus; Radhakrishnan, Praveen; Brill, Adrian; Höcker, Hermine; Schwarz, Sandra; Weissinger, Daniel; Kehm, Roland; Kulu, Yakup; Ulrich, Alexis; Schneider, Martin

2017-08-22

The Empty Spiracles Homeobox (EMX-) 2 gene has been associated with regulation of growth and differentiation in neuronal development. While recent studies provide evidence that EMX2 regulates tumorigenesis of various solid tumors, its role in colorectal cancer remains unknown. We aimed to assess the prognostic significance of EMX2 expression in stage III colorectal adenocarcinoma. Expression levels of EMX2 in human colorectal cancer and adjacent mucosa were assessed by qRT-PCR technology, and results were correlated with clinical and survival data. siRNA-mediated knockdown and adenoviral delivery-mediated overexpression of EMX2 were performed in order to investigate its effects on the migration of colorectal cancer cells in vitro. Compared to corresponding healthy mucosa, colorectal tumor samples had decreased EMX2 expression levels. Furthermore, EMX2 down-regulation in colorectal cancer tissue was associated with distant metastasis (M1) and impaired overall patient survival. In vitro knockdown of EMX2 resulted in increased tumor cell migration. Conversely, overexpression of EMX2 led to an inhibition of tumor cell migration. EMX2 is frequently down-regulated in human colorectal cancer, and down-regulation of EMX2 is a prognostic marker for disease-free and overall survival. EMX2 might thus represent a promising therapeutic target in colorectal cancer.

Sustainability of keratinocyte gene transfer and cell survival in vivo.

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Choate, K A; Khavari, P A

1997-05-20

The epidermis is an attractive site for therapeutic gene delivery because it is accessible and capable of delivering polypeptides to the systemic circulation. A number of difficulties, however, have emerged in attempts at cutaneous gene delivery, and central among these is an inability to sustain therapeutic gene production. We have examined two major potential contributing factors, viral vector stamina and involvement of long-lived epidermal progenitor cells. Human keratinocytes were either untreated or transduced with a retroviral vector for beta-galactosidase (beta-Gal) at > 99% efficiency and then grafted onto immunodeficient mice to regenerate human epidermis. Human epidermis was monitored in vivo after grafting for clinical and histologic appearance as well as for gene expression. Although integrated vector sequences persisted unchanged in engineered epidermis at 10 weeks post-grafting, retroviral long terminal repeat (LTR)-driven beta-Gal expression ceased in vivo after approximately 4 weeks. Endogenous cellular promoters, however, maintained consistently normal gene expression levels without evidence of time-dependent decline, as determined by immunostaining with species-specific antibodies for human involucrin, filaggrin, keratinocyte transglutaminase, keratin 10, type VII collagen, and Laminin 5 proteins out to week 14 post-grafting. Transduced human keratinocytes generated multilayer epidermis sustained through multiple epidermal turnover cycles; this epidermis demonstrated retention of a spatially appropriate pattern of basal and suprabasal epidermal marker gene expression. These results confirm previous findings suggesting that viral promoter-driven gene expression is not durable and demonstrate that keratinocytes passaged in vitro can regenerate and sustain normal epidermis for prolonged periods.

Regulatory dendritic cell infusion prolongs kidney allograft survival in nonhuman primates.

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Ezzelarab, M B; Zahorchak, A F; Lu, L; Morelli, A E; Chalasani, G; Demetris, A J; Lakkis, F G; Wijkstrom, M; Murase, N; Humar, A; Shapiro, R; Cooper, D K C; Thomson, A W

2013-08-01

We examined the influence of regulatory dendritic cells (DCreg), generated from cytokine-mobilized donor blood monocytes in vitamin D3 and IL-10, on renal allograft survival in a clinically relevant rhesus macaque model. DCreg expressed low MHC class II and costimulatory molecules, but comparatively high levels of programmed death ligand-1 (B7-H1), and were resistant to pro-inflammatory cytokine-induced maturation. They were infused intravenously (3.5-10 × 10(6) /kg), together with the B7-CD28 costimulation blocking agent CTLA4Ig, 7 days before renal transplantation. CTLA4Ig was given for up to 8 weeks and rapamycin, started on Day -2, was maintained with tapering of blood levels until full withdrawal at 6 months. Median graft survival time was 39.5 days in control monkeys (no DC infusion; n = 6) and 113.5 days (p DCreg-treated animals (n = 6). No adverse events were associated with DCreg infusion, and there was no evidence of induction of host sensitization based on circulating donor-specific alloantibody levels. Immunologic monitoring also revealed regulation of donor-reactive memory CD95(+) T cells and reduced memory/regulatory T cell ratios in DCreg-treated monkeys compared with controls. Termination allograft histology showed moderate combined T cell- and Ab-mediated rejection in both groups. These findings justify further preclinical evaluation of DCreg therapy and their therapeutic potential in organ transplantation. © Copyright 2013 The American Society of Transplantation and the American Society of Transplant Surgeons.

Promotion of Survival and Engraftment of Transplanted Adipose Tissue-Derived Stromal and Vascular Cells by Overexpression of Manganese Superoxide Dismutase

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Silvia Baldari

2016-07-01

Full Text Available Short-term persistence of transplanted cells during early post-implant period limits clinical efficacy of cell therapy. Poor cell survival is mainly due to the harsh hypoxic microenvironment transplanted cells face at the site of implantation and to anoikis, driven by cell adhesion loss. We evaluated the hypothesis that viral-mediated expression of a gene conferring hypoxia resistance to cells before transplant could enhance survival of grafted cells in early stages after implant. We used adipose tissue as cell source because it consistently provides high yields of adipose-tissue-derived stromal and vascular cells (ASCs, suitable for regenerative purposes. Luciferase positive cells were transduced with lentiviral vectors expressing either green fluorescent protein as control or human manganese superoxide dismutase (SOD2. Cells were then exposed in vitro to hypoxic conditions, mimicking cell transplantation into an ischemic site. Cells overexpressing SOD2 displayed survival rates significantly greater compared to mock transduced cells. Similar results were also obtained in vivo after implantation into syngeneic mice and assessment of cell engraftment by in vivo bioluminescent imaging. Taken together, these findings suggest that ex vivo gene transfer of SOD2 into ASCs before implantation confers a cytoprotective effect leading to improved survival and engraftment rates, therefore enhancing cell therapy regenerative potential.

Basic fibroblast growth factor is pro-adipogenic in rat skeletal muscle progenitor clone, 2G11 cells.

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Nakano, Shin-ichi; Nakamura, Katsuyuki; Teramoto, Naomi; Yamanouchi, Keitaro; Nishihara, Masugi

2016-01-01

Intramuscular adipose tissue (IMAT) formation is a hallmark of marbling in cattle. IMAT is considered to originate from skeletal muscle progenitor cells with adipogenic potential. However, the mechanism involved in IMAT formation from these progenitor cells in vivo remains unclear. In the present study, among the growth factors tested, which were known to be expressed in skeletal muscle, we found only basic fibroblast growth factor (bFGF) has a pro-adipogenic effect on skeletal muscle derived adipogenic progenitor clone, 2G11 cells. Pre-exposure of 2G11 cells to bFGF did not affect initial gene expressions of CCAAT/enhancer-binding protein (C/EBP)β and C/EBPδ, while resulting in an enhancement of subsequent expressions of C/EBPα and proliferator-activated receptor gamma (PPARγ) during adipogenesis, indicating that bFGF is acting on the transcriptional regulation of C/EBPα and PPARγ. In addition, the effect of bFGF is mediated via two types of FGF receptor (FGFR) isoforms: FGFR1 and FGFR2 IIIc, and both receptors are prerequisite for bFGF to express its pro-adipogenic effect. These results suggest that bFGF plays an important role as a key trigger of IMAT formation in vivo. © 2015 Japanese Society of Animal Science.

Activation of CHK1 in Supporting Cells Indirectly Promotes Hair Cell Survival

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Azadeh Jadali

2017-05-01

Full Text Available The sensory hair cells of the inner ear are exquisitely sensitive to ototoxic insults. Loss of hair cells after exposure to ototoxic agents causes hearing loss. Chemotherapeutic agents such as cisplatin causes hair cell loss. Cisplatin forms DNA mono-adducts as well as intra- and inter-strand DNA crosslinks. DNA cisplatin adducts are repaired through the DNA damage response. The decision between cell survival and cell death following DNA damage rests on factors that are involved in determining damage tolerance, cell survival and apoptosis. Cisplatin damage on hair cells has been the main focus of many ototoxic studies, yet the effect of cisplatin on supporting cells has been largely ignored. In this study, the effects of DNA damage response in cochlear supporting cells were interrogated. Supporting cells play a major role in the development, maintenance and oto-protection of hair cells. Loss of supporting cells may indirectly affect hair cell survival or maintenance. Activation of the Phosphoinositide 3-Kinase (PI3K signaling was previously shown to promote hair cell survival. To test whether activating PI3K signaling promotes supporting cell survival after cisplatin damage, cochlear explants from the neural subset (NS Cre Pten conditional knockout mice were employed. Deletion of Phosphatase and Tensin Homolog (PTEN activates PI3K signaling in multiple cell types within the cochlea. Supporting cells lacking PTEN showed increased cell survival after cisplatin damage. Supporting cells lacking PTEN also showed increased phosphorylation of Checkpoint Kinase 1 (CHK1 levels after cisplatin damage. Nearest neighbor analysis showed increased numbers of supporting cells with activated PI3K signaling in close proximity to surviving hair cells in cisplatin damaged cochleae. We propose that increased PI3K signaling promotes supporting cell survival through phosphorylation of CHK1 and increased survival of supporting cells indirectly increases hair cell

A track-event theory of cell survival

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Besserer, Juergen; Schneider, Uwe [Zuerich Univ. (Switzerland). Inst. of Physics; Radiotherapy Hirslanden, Zuerich (Switzerland)

2015-09-01

When fractionation schemes for hypofractionation and stereotactic body radiotherapy are considered, a reliable cell survival model at high dose is needed for calculating doses of similar biological effectiveness. In this work a simple model for cell survival which is valid also at high dose is developed from Poisson statistics. An event is defined by two double strand breaks (DSB) on the same or different chromosomes. An event is always lethal due to direct lethal damage or lethal binary misrepair by the formation of chromosome aberrations. Two different mechanisms can produce events: one-track events (OTE) or two-track-events (TTE). The target for an OTE is always a lethal event, the target for an TTE is one DSB. At least two TTEs on the same or different chromosomes are necessary to produce an event. Both, the OTE and the TTE are statistically independent. From the stochastic nature of cell kill which is described by the Poisson distribution the cell survival probability was derived. It was shown that a solution based on Poisson statistics exists for cell survival. It exhibits exponential cell survival at high dose and a finite gradient of cell survival at vanishing dose, which is in agreement with experimental cell studies. The model fits the experimental data nearly as well as the three-parameter formula of Hug-Kellerer and is only based on two free parameters. It is shown that the LQ formalism is an approximation of the model derived in this work. It could be also shown that the derived model predicts a fractionated cell survival experiment better than the LQ-model. It was shown that cell survival can be described with a simple analytical formula on the basis of Poisson statistics. This solution represents in the limit of large dose the typical exponential behavior and predicts cell survival after fractionated dose application better than the LQ-model.

A track-event theory of cell survival

International Nuclear Information System (INIS)

Besserer, Juergen; Schneider, Uwe

2015-01-01

When fractionation schemes for hypofractionation and stereotactic body radiotherapy are considered, a reliable cell survival model at high dose is needed for calculating doses of similar biological effectiveness. In this work a simple model for cell survival which is valid also at high dose is developed from Poisson statistics. An event is defined by two double strand breaks (DSB) on the same or different chromosomes. An event is always lethal due to direct lethal damage or lethal binary misrepair by the formation of chromosome aberrations. Two different mechanisms can produce events: one-track events (OTE) or two-track-events (TTE). The target for an OTE is always a lethal event, the target for an TTE is one DSB. At least two TTEs on the same or different chromosomes are necessary to produce an event. Both, the OTE and the TTE are statistically independent. From the stochastic nature of cell kill which is described by the Poisson distribution the cell survival probability was derived. It was shown that a solution based on Poisson statistics exists for cell survival. It exhibits exponential cell survival at high dose and a finite gradient of cell survival at vanishing dose, which is in agreement with experimental cell studies. The model fits the experimental data nearly as well as the three-parameter formula of Hug-Kellerer and is only based on two free parameters. It is shown that the LQ formalism is an approximation of the model derived in this work. It could be also shown that the derived model predicts a fractionated cell survival experiment better than the LQ-model. It was shown that cell survival can be described with a simple analytical formula on the basis of Poisson statistics. This solution represents in the limit of large dose the typical exponential behavior and predicts cell survival after fractionated dose application better than the LQ-model.

A pro-inflammatory role for Th22 cells in Helicobacter pylori-associated gastritis.

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Zhuang, Yuan; Cheng, Ping; Liu, Xiao-fei; Peng, Liu-sheng; Li, Bo-sheng; Wang, Ting-ting; Chen, Na; Li, Wen-hua; Shi, Yun; Chen, Weisan; Pang, Ken C; Zeng, Ming; Mao, Xu-hu; Yang, Shi-ming; Guo, Hong; Guo, Gang; Liu, Tao; Zuo, Qian-fei; Yang, Hui-jie; Yang, Liu-yang; Mao, Fang-yuan; Lv, Yi-pin; Zou, Quan-ming

2015-09-01

Helper T (Th) cell responses are critical for the pathogenesis of Helicobacter pylori-induced gastritis. Th22 cells represent a newly discovered Th cell subset, but their relevance to H. pylori-induced gastritis is unknown. Flow cytometry, real-time PCR and ELISA analyses were performed to examine cell, protein and transcript levels in gastric samples from patients and mice infected with H. pylori. Gastric tissues from interleukin (IL)-22-deficient and wild-type (control) mice were also examined. Tissue inflammation was determined for pro-inflammatory cell infiltration and pro-inflammatory protein production. Gastric epithelial cells and myeloid-derived suppressor cells (MDSC) were isolated, stimulated and/or cultured for Th22 cell function assays. Th22 cells accumulated in gastric mucosa of both patients and mice infected with H. pylori. Th22 cell polarisation was promoted via the production of IL-23 by dendritic cells (DC) during H. pylori infection, and resulted in increased inflammation within the gastric mucosa. This inflammation was characterised by the CXCR2-dependent influx of MDSCs, whose migration was induced via the IL-22-dependent production of CXCL2 by gastric epithelial cells. Under the influence of IL-22, MDSCs, in turn, produced pro-inflammatory proteins, such as S100A8 and S100A9, and suppressed Th1 cell responses, thereby contributing to the development of H. pylori-associated gastritis. This study, therefore, identifies a novel regulatory network involving H. pylori, DCs, Th22 cells, gastric epithelial cells and MDSCs, which collectively exert a pro-inflammatory effect within the gastric microenvironment. Efforts to inhibit this Th22-dependent pathway may therefore prove a valuable strategy in the therapy of H. pylori-associated gastritis. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

The action of hyperthermia on gene expression in Friend erythroleukemia cells by dimethyl sulfoxide or X-rays

International Nuclear Information System (INIS)

Raaphorst, G.P.; Azzam, E.I.; Einspenner, M.; Ewing, D.; Borsa, J.

1982-02-01

The effect of heat on gene control and on cell killing by X-rays or dimethyl sulfoxide (DMSO) was studied in cultured Friend erythroleukemia cells (FELC). FELC are very sensitive to heat and X-rays in terms of survival, as measured by the colony-forming assay. Heat inactivation kinetics are similar for FELC and Chinese hamster cells. Thermal enhancement of cell inactivation by irradiation was observed at 42.0 and 45.0deg C, and increased as a function of heating time. The simultaneous application of heat and X-rays had a greater effect in terms of cell inactivation. Dimethyl sulfoxide could induce FELC to synthesize hemoglobin, and hyperthermia could inhibit this response. Likewise, hyperthermia could affect induction of heme synthesis by irradiation. Heating before irradiation enhanced production of heme synthesis, whereas heating after irradiation inhibited induction of heme synthesis. The effects of hyperthermia on the survival and gene induction endpoints were compared. Thus, heat can affect both cell survival and gene induction by irradiation or DMSO. The two endpoints of gene induction and survival (proliferative capacity) responded differently, both quantitatively and qualitatively, to heat and X-rays, implying that different cellular targets are affected for each of these endpoints

Human immune cells' behavior and survival under bioenergetically restricted conditions in an in vitro fracture hematoma model

Science.gov (United States)

Hoff, Paula; Maschmeyer, Patrick; Gaber, Timo; Schütze, Tabea; Raue, Tobias; Schmidt-Bleek, Katharina; Dziurla, René; Schellmann, Saskia; Lohanatha, Ferenz Leonard; Röhner, Eric; Ode, Andrea; Burmester, Gerd-Rüdiger; Duda, Georg N; Perka, Carsten; Buttgereit, Frank

2013-01-01

The initial inflammatory phase of bone fracture healing represents a critical step for the outcome of the healing process. However, both the mechanisms initiating this inflammatory phase and the function of immune cells present at the fracture site are poorly understood. In order to study the early events within a fracture hematoma, we established an in vitro fracture hematoma model: we cultured hematomas forming during an osteotomy (artificial bone fracture) of the femur during total hip arthroplasty (THA) in vitro under bioenergetically controlled conditions. This model allowed us to monitor immune cell populations, cell survival and cytokine expression during the early phase following a fracture. Moreover, this model enabled us to change the bioenergetical conditions in order to mimic the in vivo situation, which is assumed to be characterized by hypoxia and restricted amounts of nutrients. Using this model, we found that immune cells adapt to hypoxia via the expression of angiogenic factors, chemoattractants and pro-inflammatory molecules. In addition, combined restriction of oxygen and nutrient supply enhanced the selective survival of lymphocytes in comparison with that of myeloid derived cells (i.e., neutrophils). Of note, non-restricted bioenergetical conditions did not show any similar effects regarding cytokine expression and/or different survival rates of immune cell subsets. In conclusion, we found that the bioenergetical conditions are among the crucial factors inducing the initial inflammatory phase of fracture healing and are thus a critical step for influencing survival and function of immune cells in the early fracture hematoma. PMID:23396474

System-level analysis of genes and functions affecting survival during nutrient starvation in Saccharomyces cerevisiae.

Science.gov (United States)

Gresham, David; Boer, Viktor M; Caudy, Amy; Ziv, Naomi; Brandt, Nathan J; Storey, John D; Botstein, David

2011-01-01

An essential property of all cells is the ability to exit from active cell division and persist in a quiescent state. For single-celled microbes this primarily occurs in response to nutrient deprivation. We studied the genetic requirements for survival of Saccharomyces cerevisiae when starved for either of two nutrients: phosphate or leucine. We measured the survival of nearly all nonessential haploid null yeast mutants in mixed populations using a quantitative sequencing method that estimates the abundance of each mutant on the basis of frequency of unique molecular barcodes. Starvation for phosphate results in a population half-life of 337 hr whereas starvation for leucine results in a half-life of 27.7 hr. To measure survival of individual mutants in each population we developed a statistical framework that accounts for the multiple sources of experimental variation. From the identities of the genes in which mutations strongly affect survival, we identify genetic evidence for several cellular processes affecting survival during nutrient starvation, including autophagy, chromatin remodeling, mRNA processing, and cytoskeleton function. In addition, we found evidence that mitochondrial and peroxisome function is required for survival. Our experimental and analytical methods represent an efficient and quantitative approach to characterizing genetic functions and networks with unprecedented resolution and identified genotype-by-environment interactions that have important implications for interpretation of studies of aging and quiescence in yeast.

The Pro-inflammatory Effects of Glucocorticoids in the Brain

Science.gov (United States)

Duque, Erica de Almeida; Munhoz, Carolina Demarchi

2016-01-01

Glucocorticoids are a class of steroid hormones derived from cholesterol. Their actions are mediated by the glucocorticoid and mineralocorticoid receptors, members of the superfamily of nuclear receptors, which, once bound to their ligands, act as transcription factors that can directly modulate gene expression. Through protein–protein interactions with other transcription factors, they can also regulate the activity of many genes in a composite or tethering way. Rapid non-genomic signaling was also demonstrated since glucocorticoids can act through membrane receptors and activate signal transduction pathways, such as protein kinases cascades, to modulate other transcriptions factors and activate or repress various target genes. By all these different mechanisms, glucocorticoids regulate numerous important functions in a large variety of cells, not only in the peripheral organs but also in the central nervous system during development and adulthood. In general, glucocorticoids are considered anti-inflammatory and protective agents due to their ability to inhibit gene expression of pro-inflammatory mediators and other possible damaging molecules. Nonetheless, recent studies have uncovered situations in which these hormones can act as pro-inflammatory agents depending on the dose, chronicity of exposure, and the structure/organ analyzed. In this review, we will provide an overview of the conditions under which these phenomena occur, a discussion that will serve as a basis for exploring the mechanistic foundation of glucocorticoids pro-inflammatory gene regulation in the brain. PMID:27445981

SAG/ROC-SCFβ-TrCP E3 Ubiquitin Ligase Promotes Pro-Caspase-3 Degradation as a Mechanism of Apoptosis Protection

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Mingjia Tan

2006-12-01

Full Text Available Skp1-cullin-F-box protein (SCF is a multicomponent E3 ubiquitin (Ub ligase that ubiquitinates a number of important biologic molecules such as p27, β-catenin, and lκB for proteasomal degradation, thus regulating cell proliferation and survival. One SCF component, SAG/ROC2/Rbx2/Hrt2, a RING finger protein, was first identified as a redox-inducible protein, which, when overexpressed, inhibited apoptosis both in vitro and in vivo. We report here that sensitive to apoptosis gene (SAG, as well as its family member ROC1/Rbxi, bound to the proinactive form of caspase-3 (pro-caspase-3. Binding was likely mediated through F-box protein, β-transducin repeat-containing protein (β-TrCP, which binds to the first 38 amino acids of pro-caspase-3. Importantly, β-TrCP1 expression significantly shortened the protein half-life of pro-caspase-3, whereas expression of a dominant-negative β-TrCP1 mutant with the F-box domain deleted extended it. An in vitro ubiquitination assay showed that SAG/ROC-SCF -Trcp promoted ubiquitination of pro-caspase-3. Furthermore, endogenous levels of pro-caspase-3 were decreased by overexpression of SAG/ROC-SCFβ-TrCP E3 Ub ligases, but increased on siRNA silencing of SAG, regulator of cullin-1 (ROC1, or β-TrCPs, leading to increased apoptosis by etoposide and TNF-related apoptosis-inducing ligand through increased activation of caspase-3. Thus, pro-caspase-3 appears to be a substrate of SAG/ROC-SCFβ-TrCP E3 Ub ligase, which protects cells from apoptosis through increased apoptosis threshold by reducing the basal level of pro-caspase-3.

A multi-stage process including transient polyploidization and EMT precedes the emergence of chemoresistent ovarian carcinoma cells with a dedifferentiated and pro-inflammatory secretory phenotype.

Science.gov (United States)

Rohnalter, Verena; Roth, Katrin; Finkernagel, Florian; Adhikary, Till; Obert, Julia; Dorzweiler, Kristina; Bensberg, Maike; Müller-Brüsselbach, Sabine; Müller, Rolf

2015-11-24

DNA-damaging drugs induce a plethora of molecular and cellular alterations in tumor cells, but their interrelationship is largely obscure. Here, we show that carboplatin treatment of human ovarian carcinoma SKOV3 cells triggers an ordered sequence of events, which precedes the emergence of mitotic chemoresistant cells. The initial phase of cell death after initiation of carboplatin treatment is followed around day 14 by the emergence of a mixed cell population consisting of cycling, cell cycle-arrested and senescent cells. At this stage, giant cells make up >80% of the cell population, p21 (CDKN1A) in strongly induced, and cell numbers remain nearly static. Subsequently, cell death decreases, p21 expression drops to a low level and cell divisions increase, including regular mitoses of giant cells and depolyploidization by multi-daughter divisions. These events are accompanied by the upregulation of stemness markers and a pro-inflammatory secretory phenotype, peaking after approximately 14 days of treatment. At the same time the cells initiate epithelial to mesenchymal transition, which over the subsequent weeks continuously increases, concomitantly with the emergence of highly proliferative, migratory, dedifferentiated, pro-inflammatory and chemoresistant cells (SKOV3-R). These cells are anchorage-independent and grow in a 3D collagen matrix, while cells on day 14 do not survive under these conditions, indicating that SKOV3-R cells were generated thereafter by the multi-stage process described above. This process was essentially recapitulated with the ovarian carcinoma cell line IGROV-1. Our observations suggest that transitory cells characterized by polyploidy, features of stemness and a pro-inflammatory secretory phenotype contribute to the acquisition of chemoresistance.

Glucocorticoid-induced reversal of interleukin-1β-stimulated inflammatory gene expression in human oviductal cells.

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Stéphanie Backman

Full Text Available Studies indicate that high-grade serous ovarian carcinoma (HGSOC, the most common epithelial ovarian carcinoma histotype, originates from the fallopian tube epithelium (FTE. Risk factors for this cancer include reproductive parameters associated with lifetime ovulatory events. Ovulation is an acute inflammatory process during which the FTE is exposed to follicular fluid containing both pro- and anti-inflammatory molecules, such as interleukin-1 (IL1, tumor necrosis factor (TNF, and cortisol. Repeated exposure to inflammatory cytokines may contribute to transforming events in the FTE, with glucocorticoids exerting a protective effect. The global response of FTE cells to inflammatory cytokines or glucocorticoids has not been investigated. To examine the response of FTE cells and the ability of glucocorticoids to oppose this response, an immortalized human FTE cell line, OE-E6/E7, was treated with IL1β, dexamethasone (DEX, IL1β and DEX, or vehicle and genome-wide gene expression profiling was performed. IL1β altered the expression of 47 genes of which 17 were reversed by DEX. DEX treatment alone altered the expression of 590 genes, whereas combined DEX and IL1β treatment altered the expression of 784 genes. Network and pathway enrichment analysis indicated that many genes altered by DEX are involved in cytokine, chemokine, and cell cycle signaling, including NFκΒ target genes and interacting proteins. Quantitative real time RT-PCR studies validated the gene array data for IL8, IL23A, PI3 and TACC2 in OE-E6/E7 cells. Consistent with the array data, Western blot analysis showed increased levels of PTGS2 protein induced by IL1β that was blocked by DEX. A parallel experiment using primary cultured human FTE cells indicated similar effects on PTGS2, IL8, IL23A, PI3 and TACC2 transcripts. These findings support the hypothesis that pro-inflammatory signaling is induced in FTE cells by inflammatory mediators and raises the possibility that

Gene expression profiles in BCL11B-siRNA treated malignant T cells

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Grabarczyk Piotr

2011-05-01

Full Text Available Abstract Background Downregulation of the B-cell chronic lymphocytic leukemia (CLL/lymphoma11B (BCL11B gene by small interfering RNA (siRNA leads to growth inhibition and apoptosis of the human T-cell acute lymphoblastic leukemia (T-ALL cell line Molt-4. To further characterize the molecular mechanism, a global gene expression profile of BCL11B-siRNA -treated Molt-4 cells was established. The expression profiles of several genes were further validated in the BCL11B-siRNA -treated Molt-4 cells and primary T-ALL cells. Results 142 genes were found to be upregulated and 109 genes downregulated in the BCL11B-siRNA -treated Molt-4 cells by microarray analysis. Among apoptosis-related genes, three pro-apoptotic genes, TNFSF10, BIK, BNIP3, were upregulated and one anti-apoptotic gene, BCL2L1 was downregulated. Moreover, the expression of SPP1 and CREBBP genes involved in the transforming growth factor (TGF-β pathway was down 16-fold. Expression levels of TNFSF10, BCL2L1, SPP1, and CREBBP were also examined by real-time PCR. A similar expression pattern of TNFSF10, BCL2L1, and SPP1 was identified. However, CREBBP was not downregulated in the BLC11B-siRNA -treated Molt-4 cells. Conclusion BCL11B-siRNA treatment altered expression profiles of TNFSF10, BCL2L1, and SPP1 in both Molt-4 T cell line and primary T-ALL cells.

Tracking plasma cell differentiation and survival.

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Roth, Katrin; Oehme, Laura; Zehentmeier, Sandra; Zhang, Yang; Niesner, Raluca; Hauser, Anja E

2014-01-01

Plasma cells play a crucial role for the humoral immune response as they represent the body's factories for antibody production. The differentiation from a B cell into a plasma cell is controlled by a complex transcriptional network and happens within secondary lymphoid organs. Based on their lifetime, two types of antibody secreting cells can be distinguished: Short-lived plasma cells are located in extrafollicular sites of secondary lymphoid organs such as lymph node medullary cords and the splenic red pulp. A fraction of plasmablasts migrate from secondary lymphoid organs to the bone marrow where they can become long-lived plasma cells. Bone marrow plasma cells reside in special microanatomical environments termed survival niches, which provide factors promoting their longevity. Reticular stromal cells producing the chemokine CXCL12, which is known to attract plasmablasts to the bone marrow but also to promote plasma cell survival, play a crucial role in the maintenance of these niches. In addition, hematopoietic cells are contributing to the niches by providing other soluble survival factors. Here, we review the current knowledge on the factors involved in plasma cell differentiation, their localization and migration. We also give an overview on what is known regarding the maintenance of long lived plasma cells in survival niches of the bone marrow. © 2013 International Society for Advancement of Cytometry.

Ras and Rheb Signaling in Survival and Cell Death

International Nuclear Information System (INIS)

Ehrkamp, Anja; Herrmann, Christian; Stoll, Raphael; Heumann, Rolf

2013-01-01

One of the most obvious hallmarks of cancer is uncontrolled proliferation of cells partly due to independence of growth factor supply. A major component of mitogenic signaling is Ras, a small GTPase. It was the first identified human protooncogene and is known since more than three decades to promote cellular proliferation and growth. Ras was shown to support growth factor-independent survival during development and to protect from chemical or mechanical lesion-induced neuronal degeneration in postmitotic neurons. In contrast, for specific patho-physiological cases and cellular systems it has been shown that Ras may also promote cell death. Proteins from the Ras association family (Rassf, especially Rassf1 and Rassf5) are tumor suppressors that are activated by Ras-GTP, triggering apoptosis via e.g., activation of mammalian sterile 20-like (MST1) kinase. In contrast to Ras, their expression is suppressed in many types of tumours, which makes Rassf proteins an exciting model for understanding the divergent effects of Ras activity. It seems likely that the outcome of Ras signaling depends on the balance between the activation of its various downstream effectors, thus determining cellular fate towards either proliferation or apoptosis. Ras homologue enriched in brain (Rheb) is a protein from the Ras superfamily that is also known to promote proliferation, growth, and regeneration through the mammalian target of rapamycin (mTor) pathway. However, recent evidences indicate that the Rheb-mTor pathway may switch its function from a pro-growth into a cell death pathway, depending on the cellular situation. In contrast to Ras signaling, for Rheb, the cellular context is likely to modulate the whole Rheb-mTor pathway towards cellular death or survival, respectively

Ras and Rheb Signaling in Survival and Cell Death

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Ehrkamp, Anja [Molecular Neurobiochemistry, Ruhr University of Bochum, 44780 Bochum (Germany); Herrmann, Christian [Department of Physical Chemistry1, Protein Interaction, Ruhr University of Bochum, 44780 Bochum (Germany); Stoll, Raphael [Biomolecular NMR, Ruhr University of Bochum, 44780 Bochum (Germany); Heumann, Rolf, E-mail: rolf.heumann@rub.de [Molecular Neurobiochemistry, Ruhr University of Bochum, 44780 Bochum (Germany)

2013-05-28

One of the most obvious hallmarks of cancer is uncontrolled proliferation of cells partly due to independence of growth factor supply. A major component of mitogenic signaling is Ras, a small GTPase. It was the first identified human protooncogene and is known since more than three decades to promote cellular proliferation and growth. Ras was shown to support growth factor-independent survival during development and to protect from chemical or mechanical lesion-induced neuronal degeneration in postmitotic neurons. In contrast, for specific patho-physiological cases and cellular systems it has been shown that Ras may also promote cell death. Proteins from the Ras association family (Rassf, especially Rassf1 and Rassf5) are tumor suppressors that are activated by Ras-GTP, triggering apoptosis via e.g., activation of mammalian sterile 20-like (MST1) kinase. In contrast to Ras, their expression is suppressed in many types of tumours, which makes Rassf proteins an exciting model for understanding the divergent effects of Ras activity. It seems likely that the outcome of Ras signaling depends on the balance between the activation of its various downstream effectors, thus determining cellular fate towards either proliferation or apoptosis. Ras homologue enriched in brain (Rheb) is a protein from the Ras superfamily that is also known to promote proliferation, growth, and regeneration through the mammalian target of rapamycin (mTor) pathway. However, recent evidences indicate that the Rheb-mTor pathway may switch its function from a pro-growth into a cell death pathway, depending on the cellular situation. In contrast to Ras signaling, for Rheb, the cellular context is likely to modulate the whole Rheb-mTor pathway towards cellular death or survival, respectively.

Androgen-Sensitized Apoptosis of HPr-1AR Human Prostate Epithelial Cells.

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Congcong Chen

Full Text Available Androgen receptor (AR signaling is crucial to the development and homeostasis of the prostate gland, and its dysregulation mediates common prostate pathologies. The mechanisms whereby AR regulates growth suppression and differentiation of luminal epithelial cells in the prostate gland and proliferation of malignant versions of these cells have been investigated in human and rodent adult prostate. However, the cellular stress response of human prostate epithelial cells is not well understood, though it is central to prostate health and pathology. Here, we report that androgen sensitizes HPr-1AR and RWPE-AR human prostate epithelial cells to cell stress agents and apoptotic cell death. Although 5α-dihydrotestosterone (DHT treatment alone did not induce cell death, co-treatment of HPr-1AR cells with DHT and an apoptosis inducer, such as staurosporine (STS, TNFt, or hydrogen peroxide, synergistically increased cell death in comparison to treatment with each apoptosis inducer by itself. We found that the synergy between DHT and apoptosis inducer led to activation of the intrinsic/mitochondrial apoptotic pathway, which is supported by robust cleavage activation of caspase-9 and caspase-3. Further, the dramatic depolarization of the mitochondrial membrane potential that we observed upon co-treatment with DHT and STS is consistent with increased mitochondrial outer membrane permeabilization (MOMP in the pro-apoptotic mechanism. Interestingly, the synergy between DHT and apoptosis inducer was abolished by AR antagonists and inhibitors of transcription and protein synthesis, suggesting that AR mediates pro-apoptotic synergy through transcriptional regulation of MOMP genes. Expression analysis revealed that pro-apoptotic genes (BCL2L11/BIM and AIFM2 were DHT-induced, whereas pro-survival genes (BCL2L1/BCL-XL and MCL1 were DHT-repressed. Hence, we propose that the net effect of these AR-mediated expression changes shifts the balance of BCL2-family proteins

Association of MTHFR gene polymorphisms with breast cancer survival

International Nuclear Information System (INIS)

Martin, Damali N; Boersma, Brenda J; Howe, Tiffany M; Goodman, Julie E; Mechanic, Leah E; Chanock, Stephen J; Ambs, Stefan

2006-01-01

Two functional single nucleotide polymorphisms (SNPs) in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, C677T and A1298C, lead to decreased enzyme activity and affect chemosensitivity of tumor cells. We investigated whether these MTHFR SNPs were associated with breast cancer survival in African-American and Caucasian women. African-American (n = 143) and Caucasian (n = 105) women, who had incident breast cancer with surgery, were recruited between 1993 and 2003 from the greater Baltimore area, Maryland, USA. Kaplan-Meier survival and multivariate Cox proportional hazards regression analyses were used to examine the relationship between MTHFR SNPs and disease-specific survival. We observed opposite effects of the MTHFR polymorphisms A1298C and C677T on breast cancer survival. Carriers of the variant allele at codon 1298 (A/C or C/C) had reduced survival when compared to homozygous carriers of the common A allele [Hazard ratio (HR) = 2.05; 95% confidence interval (CI), 1.05–4.00]. In contrast, breast cancer patients with the variant allele at codon 677 (C/T or T/T) had improved survival, albeit not statistically significant, when compared to individuals with the common C/C genotype (HR = 0.65; 95% CI, 0.31–1.35). The effects were stronger in patients with estrogen receptor-negative tumors (HR = 2.70; 95% CI, 1.17–6.23 for A/C or C/C versus A/A at codon 1298; HR = 0.36; 95% CI, 0.12–1.04 for C/T or T/T versus C/C at codon 677). Interactions between the two MTHFR genotypes and race/ethnicity on breast cancer survival were also observed (A1298C, p interaction = 0.088; C677T, p interaction = 0.026). We found that the MTHFR SNPs, C677T and A1298C, were associated with breast cancer survival. The variant alleles had opposite effects on disease outcome in the study population. Race/ethnicity modified the association between the two SNPs and breast cancer survival

Radiobilogical cell survival models

International Nuclear Information System (INIS)

Zackrisson, B.

1992-01-01

A central issue in clinical radiobiological research is the prediction of responses to different radiation qualities. The choice of cell survival and dose-response model greatly influences the results. In this context the relationship between theory and model is emphasized. Generally, the interpretations of experimental data depend on the model. Cell survival models are systematized with respect to their relations to radiobiological theories of cell kill. The growing knowlegde of biological, physical, and chemical mechanisms is reflected in the formulation of new models. The present overview shows that recent modelling has been more oriented towards the stochastic fluctuations connected to radiation energy deposition. This implies that the traditional cell surivival models ought to be complemented by models of stochastic energy deposition processes and repair processes at the intracellular level. (orig.)

Induction of Protective Genes Leads to Islet Survival and Function

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Hongjun Wang

2011-01-01

Full Text Available Islet transplantation is the most valid approach to the treatment of type 1 diabetes. However, the function of transplanted islets is often compromised since a large number of β cells undergo apoptosis induced by stress and the immune rejection response elicited by the recipient after transplantation. Conventional treatment for islet transplantation is to administer immunosuppressive drugs to the recipient to suppress the immune rejection response mounted against transplanted islets. Induction of protective genes in the recipient (e.g., heme oxygenase-1 (HO-1, A20/tumor necrosis factor alpha inducible protein3 (tnfaip3, biliverdin reductase (BVR, Bcl2, and others or administration of one or more of the products of HO-1 to the donor, the islets themselves, and/or the recipient offers an alternative or synergistic approach to improve islet graft survival and function. In this perspective, we summarize studies describing the protective effects of these genes on islet survival and function in rodent allogeneic and xenogeneic transplantation models and the prevention of onset of diabetes, with emphasis on HO-1, A20, and BVR. Such approaches are also appealing to islet autotransplantation in patients with chronic pancreatitis after total pancreatectomy, a procedure that currently only leads to 1/3 of transplanted patients being diabetes-free.

Bimodal CD40/Fas-Dependent Crosstalk between iNKT Cells and Tumor-Associated Macrophages Impairs Prostate Cancer Progression.

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Cortesi, Filippo; Delfanti, Gloria; Grilli, Andrea; Calcinotto, Arianna; Gorini, Francesca; Pucci, Ferdinando; Lucianò, Roberta; Grioni, Matteo; Recchia, Alessandra; Benigni, Fabio; Briganti, Alberto; Salonia, Andrea; De Palma, Michele; Bicciato, Silvio; Doglioni, Claudio; Bellone, Matteo; Casorati, Giulia; Dellabona, Paolo

2018-03-13

Heterotypic cellular and molecular interactions in the tumor microenvironment (TME) control cancer progression. Here, we show that CD1d-restricted invariant natural killer (iNKT) cells control prostate cancer (PCa) progression by sculpting the TME. In a mouse PCa model, iNKT cells restrained the pro-angiogenic and immunosuppressive capabilities of tumor-infiltrating immune cells by reducing pro-angiogenic TIE2 + , M2-like macrophages (TEMs), and sustaining pro-inflammatory M1-like macrophages. iNKT cells directly contacted macrophages in the PCa stroma, and iNKT cell transfer into tumor-bearing mice abated TEMs, delaying tumor progression. iNKT cells modulated macrophages through the cooperative engagement of CD1d, Fas, and CD40, which promoted selective killing of M2-like and survival of M1-like macrophages. Human PCa aggressiveness associate with reduced intra-tumoral iNKT cells, increased TEMs, and expression of pro-angiogenic genes, underscoring the clinical significance of this crosstalk. Therefore, iNKT cells may control PCa through mechanisms involving differential macrophage modulation, which may be harnessed for therapeutically reprogramming the TME. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Immunohistochemical detection of cdc2 is useful in predicting survival in patients with mantle cell lymphoma.

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Hui, David; Reiman, Tony; Hanson, John; Linford, Rick; Wong, Winson; Belch, Andrew; Lai, Raymond

2005-09-01

Recent cDNA microarray studies have reported the prognostic value of several genes in mantle cell lymphoma patients. We aimed to validate the prognostic significance of three of these genes: alpha-tubulin, cdc2, and CENP-F. The protein expression of alpha-tubulin, cdc2, and CENP-F was assessed using immunohistochemistry. Their immunoreactivity in 48 formalin-fixed/paraffin-embedded mantle cell lymphoma tumors was determined by estimating the percentage of positive cells. These results were correlated with the expression of proliferation marker Ki67 and survival. Of these 48 mantle cell lymphoma patients, 41 were men and seven were women. The median age at time of diagnosis was 64.5 years, and the overall median survival was 40 months. In benign lymph nodes, the expression of cdc2 and alpha-tubulin was restricted to the germinal centers; mantle zones were negative. Expression of CENP-F was more uniformly distributed. In mantle cell lymphoma, Ki67 significantly correlated with all three markers (P50%) and cdc2 (>25%) significantly correlated with shorter survival (Por=2 correlated with worse clinical outcome, and high clinical stage (ie 4 vs survival. The prognostic significance of cdc2 and Ki67 was independent of international prognostic index and clinical stage. We have validated the prognostic value of cdc2, and confirmed that of Ki67, in a cohort of mantle cell lymphoma patients. Immunohistochemical detection of cdc2 and Ki67 may be a useful and simple method in evaluating the prognosis of mantle cell lymphoma patients.

Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells.

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Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. Copyright © 2015 Elsevier Inc. All rights reserved.

Phosphatidylinositol 3-kinase is essential for kit ligand-mediated survival, whereas interleukin-3 and flt3 ligand induce expression of antiapoptotic Bcl-2 family genes

DEFF Research Database (Denmark)

Karlsson, Richard; Engström, Maria; Jönsson, Maria

2003-01-01

Cytokines such as interleukin 3 (IL-3), kit ligand (KL), and flt3 ligand (FL) promote survival of hematopoietic stem cells and myeloid progenitor cells. In many cell types, members of the Bcl-2 gene family are major regulators of survival, but the mediating mechanisms are not fully understood....... Using two myeloid progenitor cell lines, FDCP-mix and FDC-P1, as well as primary mouse bone marrow progenitors, we demonstrate that KL-mediated survival is dependent on the activation of phosphatidylinositol-3 (PI-3) kinase. The inhibitor LY294002 was able to completely abolish survival mediated by KL...

Integration of gene dosage and gene expression in non-small cell lung cancer, identification of HSP90 as potential target.

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Mariëlle I Gallegos Ruiz

Full Text Available BACKGROUND: Lung cancer causes approximately 1.2 million deaths per year worldwide, and non-small cell lung cancer (NSCLC represents 85% of all lung cancers. Understanding the molecular events in non-small cell lung cancer (NSCLC is essential to improve early diagnosis and treatment for this disease. METHODOLOGY AND PRINCIPAL FINDINGS: In an attempt to identify novel NSCLC related genes, we performed a genome-wide screening of chromosomal copy number changes affecting gene expression using microarray based comparative genomic hybridization and gene expression arrays on 32 radically resected tumor samples from stage I and II NSCLC patients. An integrative analysis tool was applied to determine whether chromosomal copy number affects gene expression. We identified a deletion on 14q32.2-33 as a common alteration in NSCLC (44%, which significantly influenced gene expression for HSP90, residing on 14q32. This deletion was correlated with better overall survival (P = 0.008, survival was also longer in patients whose tumors had low expression levels of HSP90. We extended the analysis to three independent validation sets of NSCLC patients, and confirmed low HSP90 expression to be related with longer overall survival (P = 0.003, P = 0.07 and P = 0.04. Furthermore, in vitro treatment with an HSP90 inhibitor had potent antiproliferative activity in NSCLC cell lines. CONCLUSIONS: We suggest that targeting HSP90 will have clinical impact for NSCLC patients.

Exosomes confer pro-survival signals to alter the phenotype of prostate cells in their surrounding environment

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Hosseini-Beheshti, Elham; Choi, Wendy; Weiswald, Louis-Bastien; Kharmate, Geetanjali; Ghaffari, Mazyar; Roshan-Moniri, Mani; Hassona, Mohamed D.; Chan, Leslie; Chin, Mei Yieng; Tai, Isabella T.; Rennie, Paul S.; Fazli, Ladan; Guns, Emma S. Tomlinson

2016-01-01

Prostate cancer (PCa) is the most frequently diagnosed cancer in men. Current research on tumour-related extracellular vesicles (EVs) suggests that exosomes play a significant role in paracrine signaling pathways, thus potentially influencing cancer progression via multiple mechanisms. In fact, during the last decade numerous studies have revealed the role of EVs in the progression of various pathological conditions including cancer. Moreover, differences in the proteomic, lipidomic, and cholesterol content of exosomes derived from PCa cell lines versus benign prostate cell lines confirm that exosomes could be excellent biomarker candidates. As such, as part of an extensive proteomic analysis using LCMS we previously described a potential role of exosomes as biomarkers for PCa. Current evidence suggests that uptake of EV's into the local tumour microenvironment encouraging us to further examine the role of these vesicles in distinct mechanisms involved in the progression of PCa and castration resistant PCa. For the purpose of this study, we hypothesized that exosomes play a pivotal role in cell-cell communication in the local tumour microenvironment, conferring activation of numerous survival mechanisms during PCa progression and development of therapeutic resistance. Our in vitro results demonstrate that PCa derived exosomes significantly reduce apoptosis, increase cancer cell proliferation and induce cell migration in LNCaP and RWPE-1 cells. In conjunction with our in vitro findings, we have also demonstrated that exosomes increased tumor volume and serum PSA levels in vivo when xenograft bearing mice were administered DU145 cell derived exosomes intravenously. This research suggests that, regardless of androgen receptor phenotype, exosomes derived from PCa cells significantly enhance multiple mechanisms that contribute to PCa progression. PMID:26840259

Regulatory dendritic cell infusion prolongs kidney allograft survival in non-human primates

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Ezzelarab, M.; Zahorchak, A.F.; Lu, L.; Morelli, A.E.; Chalasani, G.; Demetris, A.J.; Lakkis, F.G.; Wijkstrom, M.; Murase, N.; Humar, A.; Shapiro, R.; Cooper, D.K.C.; Thomson, A.W.

2014-01-01

We examined the influence of regulatory dendritic cells (DCreg), generated from cytokine-mobilized donor blood monocytes in vitamin D3 and IL-10, on renal allograft survival in a clinically-relevant rhesus macaque model. DCreg expressed low MHC class II and costimulatory molecules, but comparatively high levels of programmed death ligand-1 (B7-H1), and were resistant to pro-inflammatory cytokine-induced maturation. They were infused intravenously (3.5–10×106/kg), together with the B7-CD28 costimulation blocking agent CTLA4Ig, 7 days before renal transplantation. CTLA4Ig was given for up to 8 weeks and rapamycin, started on day −2, was maintained with tapering of blood levels until full withdrawal at 6 months. Median graft survival time was 39.5 days in control monkeys (no DC infusion; n=6) and 113.5 days (pDCreg-treated animals (n=6). No adverse events were associated with DCreg infusion, and there was no evidence of induction of host sensitization based on circulating donor-specific alloantibody levels. Immunologic monitoring also revealed regulation of donor-reactive memory CD95+ T cells and reduced memory/regulatory T cell ratios in DCreg-treated monkeys compared with controls. Termination allograft histology showed moderate combined T cell- and Ab-mediated rejection in both groups. These findings justify further pre-clinical evaluation of DCreg therapy and their therapeutic potential in organ transplantation. PMID:23758811

Transitional-2 B cells acquire regulatory function during tolerance induction and contribute to allograft survival.

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Moreau, Aurélie; Blair, Paul A; Chai, Jian-Guo; Ratnasothy, Kulachelvy; Stolarczyk, Emilie; Alhabbab, Rowa; Rackham, Chloe L; Jones, Peter M; Smyth, Lesley; Elgueta, Raul; Howard, Jane K; Lechler, Robert I; Lombardi, Giovanna

2015-03-01

In humans, tolerance to renal transplants has been associated with alterations in B-cell gene transcription and maintenance of the numbers of circulating transitional B cells. Here, we use a mouse model of transplantation tolerance to investigate the contribution of B cells to allograft survival. We demonstrate that transfer of B cells from mice rendered tolerant to MHC class I mismatched skin grafts can prolong graft survival in a dose-dependent and antigen-specific manner to a degree similar to that afforded by graft-specific regulatory T (Treg) cells. Tolerance in this model was associated with an increase in transitional-2 (T2) B cells. Only T2 B cells from tolerized mice, not naïve T2 nor alloantigen experienced T2, were capable of prolonging skin allograft survival, and suppressing T-cell activation. Tolerized T2 B cells expressed lower levels of CD86, increased TIM-1, and demonstrated a preferential survival in vivo. Furthermore, we demonstrate a synergistic effect between tolerized B cells and graft-specific Treg cells. IL-10 production by T2 B cells did not contribute to tolerance, as shown by transfer of B cells from IL-10(-/-) mice. These results suggest that T2 B cells in tolerant patients may include a population of regulatory B cells that directly inhibit graft rejection. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Survival and SOS response induction in ultraviolet B irradiated Escherichia coli cells with defective repair mechanisms.

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Prada Medina, Cesar Augusto; Aristizabal Tessmer, Elke Tatjana; Quintero Ruiz, Nathalia; Serment-Guerrero, Jorge; Fuentes, Jorge Luis

2016-06-01

Purpose In this paper, the contribution of different genes involved in DNA repair for both survival and SOS induction in Escherichia coli mutants exposed to ultraviolet B radiation (UVB, [wavelength range 280-315 nm]) was evaluated. Materials and methods E. coli strains defective in uvrA, oxyR, recO, recN, recJ, exoX, recB, recD or xonA genes were used to determine cell survival. All strains also had the genetic sulA::lacZ fusion, which allowed for the quantification of SOS induction through the SOS Chromotest. Results Five gene products were particularly important for survival, as follows: UvrA > RecB > RecO > RecJ > XonA. Strains defective in uvrA and recJ genes showed elevated SOS induction compared with the wild type, which remained stable for up to 240 min after UVB-irradiation. In addition, E. coli strains carrying the recO or recN mutation showed no SOS induction. Conclusions The nucleotide excision and DNA recombination pathways were equally used to repair UVB-induced DNA damage in E. coli cells. The sulA gene was not turned off in strains defective in UvrA and RecJ. RecO protein was essential for processing DNA damage prior to SOS induction. In this study, the roles of DNA repair proteins and their contributions to the mechanisms that induce SOS genes in E. coli are proposed.

Over-expression of Eph and ephrin genes in advanced ovarian cancer: ephrin gene expression correlates with shortened survival

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Lincoln Douglas

2006-06-01

Full Text Available Abstract Background Increased expression of Eph receptor tyrosine kinases and their ephrin ligands has been implicated in tumor progression in a number of malignancies. This report describes aberrant expression of these genes in ovarian cancer, the commonest cause of death amongst gynaecological malignancies. Methods Eph and ephrin expression was determined using quantitative real time RT-PCR. Correlation of gene expression was measured using Spearman's rho statistic. Survival was analysed using log-rank analysis and (was visualised by Kaplan-Meier survival curves. Results Greater than 10 fold over-expression of EphA1 and a more modest over-expression of EphA2 were observed in partially overlapping subsets of tumors. Over-expression of EphA1 strongly correlated (r = 0.801; p Conclusion These data imply that increased levels of ephrins A1 and A5 in the presence of high expression of Ephs A1 and A2 lead to a more aggressive tumor phenotype. The known functions of Eph/ephrin signalling in cell de-adhesion and movement may explain the observed correlation of ephrin expression with poor prognosis.

Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells

International Nuclear Information System (INIS)

Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

2015-01-01

Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. - Highlights: • TLX overexpression in MIN6 cell causes significant expression changes of 225 genes. • TLX overexpression promotes MIN6 cell proliferation and decreases cell apoptosis. • TLX overexpression does not cause impairment of insulin secretion.

Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells

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Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng, E-mail: oxyccc@163.com

2015-12-04

Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. - Highlights: • TLX overexpression in MIN6 cell causes significant expression changes of 225 genes. • TLX overexpression promotes MIN6 cell proliferation and decreases cell apoptosis. • TLX overexpression does not cause impairment of insulin secretion.

EVALUATION OF CYTOKINE GENE POLYMORPHISM IN B CELL LYMPHOID MALIGNANCIES

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E. L. Nazarova

2014-01-01

Full Text Available Previous studies with some solid tumors has shown that polymorphisms of certain cytokine genes may be used as predictors of clinical outcome in the patients. It seemed important to evaluate potential correlations between production of certain pro- and anti-inflammatory cytokines and co-receptor molecules, and promoter polymorphism of the cytokine genes involved into regulation of cell proliferation, differentiation, apoptosis, lipid metabolism and blood clotting in the patients with hematological malignancies. The article contains our results concerning associations between of IL-1β, -2, -4, -10, -17, TNFα, and allelic polymorphisms of their genes in 62 patients with B cell lymphoid malignancies in an ethnically homogenous group (self-identified as Russians. We have shown that the GА and AA genotypes of the G-308A polymorphism in TNFα gene are significantly associated with increased production of this cytokine, being more common in aggressive non-Hodgkin lymphomas, more rare in multiple myeloma and in indolent non-Hodgkin lymphomas.

Genomic instability induced by 137Cs γ-ray irradiation in CHL surviving cells

International Nuclear Information System (INIS)

Yue Jingyin; Liu Bingchen; Wu Hongying; Zhou Jiwen; Mu Chuanjie

1999-01-01

Objective: To study in parallel several possible manifestations of instability of surviving CHL cells after irradiation, namely the frequencies of mutation at locus, micronuclei and apoptosis. Methods: The frequencies of mutation at HGPRT locus, micronuclei and apoptosis were assayed at various times in surviving cells irradiated with γ-rays. Results: The surviving cells showed a persistently increased frequency of mutation at the HGPRT locus after irradiation until 53 days. Mutant fraction as high as 10 -4 was scored, tens of times higher than those assayed in control cells studied in parallel. The frequency of bi nucleated cells with micronuclei determined within 24 hours after irradiation increased with dose and reached a peak value of (26.58 +- 2.48)% at 3 Gy, decreasing at higher doses to a plateau around 20%. The micronucleus frequency decreased steeply to about (14.47 +- 2.39)% within the first 3 days post-irradiation, and fluctuated at around 10% up to 56 days post-irradiation. The delayed efficiency of irradiated cells was significantly decreased. The frequency of apoptosis peaked about (24.90 +- 4.72)% at 10 Gy 48 h post-irradiation (γ-ray dose between 3-10 Gy) and then decreased to about 12% within 3 days. It was significantly higher than in control cells until 14 days. Conclusions: It shows that genomic instability induced by radiation can be transmitted to the progeny of surviving cells and may take many forms of expression such as lethal mutation, chromosome aberrations, gene mutation, etc

Anti-apoptotic effect of hyperglycemia can allow survival of potentially autoreactive T cells.

Science.gov (United States)

Ramakrishnan, P; Kahn, D A; Baltimore, D

2011-04-01

Thymocyte development is a tightly controlled multi-step process involving selective elimination of self-reactive and non-functional T cells by apoptosis. This developmental process depends on signaling by Notch, IL-7 and active glucose metabolism. In this study, we explored the requirement of glucose for thymocyte survival and found that in addition to metabolic regulation, glucose leads to the expression of anti-apoptotic genes. Under hyperglycemic conditions, both mouse and human thymocytes demonstrate enhanced survival. We show that glucose-induced anti-apoptotic genes are dependent on NF-κB p65 because high glucose is unable to attenuate normal ongoing apoptosis of thymocytes isolated from p65 knockout mice. Furthermore, we demonstrate that in vivo hyperglycemia decreases apoptosis of thymocytes allowing for survival of potentially self-reactive thymocytes. These results imply that hyperglycemic conditions could contribute to the development of autoimmunity through dysregulated thymic selection. © 2011 Macmillan Publishers Limited

Evaluation of Gene Therapy as an Intervention Strategy to Treat Brain Injury from Stroke

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Amanda J Craig

2016-05-01

Full Text Available Stroke is a leading cause of death and disability, with a lack of treatments available to prevent cell death, regenerate damaged cells and pathways, or promote neurogenesis. The extended period of hours to weeks over which tissue damage continues to occur makes this disorder a candidate for gene therapy. This review highlights the development of gene therapy in the area of stroke, with the evolution of viral administration, in experimental stroke models, from pre-injury to clinically relevant timeframes of hours to days post-stroke. The putative therapeutic proteins being examined include anti-apoptotic, pro-survival, anti-inflammatory, and guidance proteins, targeting multiple pathways within the complex pathology, with promising results. The balance of findings from animal models suggests that gene therapy provides a viable translational platform for treatment of ischaemic brain injury arising from stroke.

Oxidative Stress Promotes Peroxiredoxin Hyperoxidation and Attenuates Pro-survival Signaling in Aging Chondrocytes*

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Collins, John A.; Wood, Scott T.; Nelson, Kimberly J.; Rowe, Meredith A.; Carlson, Cathy S.; Chubinskaya, Susan; Poole, Leslie B.; Furdui, Cristina M.; Loeser, Richard F.

2016-01-01

Oxidative stress-mediated post-translational modifications of redox-sensitive proteins are postulated as a key mechanism underlying age-related cellular dysfunction and disease progression. Peroxiredoxins (PRX) are critical intracellular antioxidants that also regulate redox signaling events. Age-related osteoarthritis is a common form of arthritis that has been associated with mitochondrial dysfunction and oxidative stress. The objective of this study was to determine the effect of aging and oxidative stress on chondrocyte intracellular signaling, with a specific focus on oxidation of cytosolic PRX2 and mitochondrial PRX3. Menadione was used as a model to induce cellular oxidative stress. Compared with chondrocytes isolated from young adult humans, chondrocytes from older adults exhibited higher levels of PRX1–3 hyperoxidation basally and under conditions of oxidative stress. Peroxiredoxin hyperoxidation was associated with inhibition of pro-survival Akt signaling and stimulation of pro-death p38 signaling. These changes were prevented in cultured human chondrocytes by adenoviral expression of catalase targeted to the mitochondria (MCAT) and in cartilage explants from MCAT transgenic mice. Peroxiredoxin hyperoxidation was observed in situ in human cartilage sections from older adults and in osteoarthritic cartilage. MCAT transgenic mice exhibited less age-related osteoarthritis. These findings demonstrate that age-related oxidative stress can disrupt normal physiological signaling and contribute to osteoarthritis and suggest peroxiredoxin hyperoxidation as a potential mechanism. PMID:26797130

Promoter hypermethylation of the RECK gene is associated with its low expression and poor survival of esophageal squamous cell carcinoma

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Zhu, Jing; Ling, Yang; Xu, Yun; Lu, Mingzhu; Liu, Yongping; Zhang, Changsong

2017-01-01

The present study aimed to investigate the association between the methylation status of the reversion-inducing cysteine-rich protein with kazal motifs (RECK) gene and its mRNA expression levels in patients with esophageal squamous cell carcinoma (ESCC). The methylation status of RECK was analyzed by methylation-specific polymerase chain reaction (PCR), and RECK mRNA expression levels were analyzed by quantitative PCR, in 310 paired ESCC tissues. The mean RECK methylation index (MI) was 0.65 in ESCCs and 0.49 in non-tumor samples. There was a significant association between RECK methylation and the American Joint Committee on Cancer stage and lymph node metastasis in ESCC (P0.16; mean-∆∆Cq=−2.85) compared with those with hypomethylation of the RECK gene (∆MI ≤0.16; mean-∆∆Ct=−0.83), and there was a significant difference in the mRNA expression levels of RECK between those with N0–1 and N2–3 lymph node metastasis (P<0.0001). A significant correlation was observed between RECK mRNA expression levels, the MI of RECK and poor postoperative survival (P=0.0003; P<0.0001). The results of the present study suggested that promoter hypermethylation may be an important factor for loss of RECK mRNA expression and may be an indicator of poor survival in ESCC. PMID:28454343

Pathway-focused PCR array profiling of enriched populations of laser capture microdissected hippocampal cells after traumatic brain injury.

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Deborah R Boone

Full Text Available Cognitive deficits in survivors of traumatic brain injury (TBI are associated with irreversible neurodegeneration in brain regions such as the hippocampus. Comparative gene expression analysis of dying and surviving neurons could provide insight into potential therapeutic targets. We used two pathway-specific PCR arrays (RT2 Profiler Apoptosis and Neurotrophins & Receptors PCR arrays to identify and validate TBI-induced gene expression in dying (Fluoro-Jade-positive or surviving (Fluoro-Jade-negative pyramidal neurons obtained by laser capture microdissection (LCM. In the Apoptosis PCR array, dying neurons showed significant increases in expression of genes associated with cell death, inflammation, and endoplasmic reticulum (ER stress compared with adjacent, surviving neurons. Pro-survival genes with pleiotropic functions were also significantly increased in dying neurons compared to surviving neurons, suggesting that even irreversibly injured neurons are able to mount a protective response. In the Neurotrophins & Receptors PCR array, which consists of genes that are normally expected to be expressed in both groups of hippocampal neurons, only a few genes were expressed at significantly different levels between dying and surviving neurons. Immunohistochemical analysis of selected, differentially expressed proteins supported the gene expression data. This is the first demonstration of pathway-focused PCR array profiling of identified populations of dying and surviving neurons in the brain after TBI. Combining precise laser microdissection of identifiable cells with pathway-focused PCR array analysis is a practical, low-cost alternative to microarrays that provided insight into neuroprotective signals that could be therapeutically targeted to ameliorate TBI-induced neurodegeneration.

Anti-apoptotic A1 is not essential for lymphoma development in Eµ-Myc mice but helps sustain transplanted Eµ-Myc tumour cells.

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Mensink, Mark; Anstee, Natasha S; Robati, Mikara; Schenk, Robyn L; Herold, Marco J; Cory, Suzanne; Vandenberg, Cassandra J

2018-03-01

The transcription factor c-MYC regulates a multiplicity of genes involved in cellular growth, proliferation, metabolism and DNA damage response and its overexpression is a hallmark of many tumours. Since MYC promotes apoptosis under conditions of stress, such as limited availability of nutrients or cytokines, MYC-driven cells are very much dependent on signals that inhibit cell death. Stress signals trigger apoptosis via the pathway regulated by opposing fractions of the BCL-2 protein family and previous genetic studies have shown that the development of B lymphoid tumours in Eµ-Myc mice is critically dependent on expression of pro-survival BCL-2 relatives MCL-1, BCL-W and, to a lesser extent, BCL-X L , but not BCL-2 itself, and that sustained growth of these lymphomas is dependent on MCL-1. Using recently developed mice that lack expression of all three functional pro-survival A1 genes, we show here that the kinetics of lymphoma development in Eµ-Myc mice and the competitive repopulation capacity of Eµ-Myc haemopoietic stem and progenitor cells is unaffected by the absence of A1. However, conditional loss of a single remaining functional A1 gene from transplanted A1-a -/- A1-b fl/fl A1-c -/- Eµ-Myc lymphomas slowed their expansion, significantly extending the life of the transplant recipients. Thus, A1 contributes to the survival of malignant Eµ-Myc-driven B lymphoid cells. These results strengthen the case for BFL-1, the human homologue of A1, being a valid target for drug development for MYC-driven tumours.

Genes Associated With Prognosis After Surgery For Malignant Pleural Mesothelioma Promote Tumor Cell Survival In Vitro

International Nuclear Information System (INIS)

Gordon, Gavin J; Bueno, Raphael; Sugarbaker, David J

2011-01-01

Mesothelioma is an aggressive neoplasm with few effective treatments, one being cytoreductive surgery. We previously described a test, based on differential expression levels of four genes, to predict clinical outcome in prospectively consented mesothelioma patients after surgery. In this study, we determined whether any of these four genes could be linked to a cancer relevant phenotype. We conducted a high-throughput RNA inhibition screen to knockdown gene expression levels of the four genes comprising the test (ARHGDIA, COBLL1, PKM2, TM4SF1) in both a human lung-derived normal and a tumor cell line using three different small inhibitory RNA molecules per gene. Successful knockdown was confirmed using quantitative RT-PCR. Detection of statistically significant changes in apoptosis and mitosis was performed using immunological assays and quantified using video-assisted microscopy at a single time-point. Changes in nuclear shape, size, and numbers were used to provide additional support of initial findings. Each experiment was conducted in triplicate. Specificity was assured by requiring that at least 2 different siRNAs produced the observed change in each cell line/time-point/gene/assay combination. Knockdown of ARHGDIA, COBLL1, and TM4SF1 resulted in 2- to 4-fold increased levels of apoptosis in normal cells (ARHGDIA only) and tumor cells (all three genes). No statistically significant changes were observed in apoptosis after knockdown of PKM2 or for mitosis after knockdown of any gene. We provide evidence that ARHGDIA, COBLL1, and TM4SF1 are negative regulators of apoptosis in cultured tumor cells. These genes, and their related intracellular signaling pathways, may represent potential therapeutic targets in mesothelioma

Association of pro-ghrelin and GHS-R1A gene polymorphisms and haplotypes with heavy alcohol use and body mass.

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Landgren, Sara; Jerlhag, Elisabet; Zetterberg, Henrik; Gonzalez-Quintela, Arturo; Campos, Joaquin; Olofsson, Ulrica; Nilsson, Staffan; Blennow, Kaj; Engel, Jörgen A

2008-12-01

Ghrelin, an orexigenic peptide, acts on growth hormone secretagogue receptors (GHS-R1A), expressed in the hypothalamus as well as in important reward nodes such as the ventral tegmental area. Interestingly, ghrelin has been found to activate an important part of the reward systems, i.e., the cholinergic-dopaminergic reward link. Additionally, the rewarding and neurochemical properties of alcohol are, at least in part, mediated via this reward link. There is comorbidity between alcohol dependence and eating disorders. Thus, plasma levels of ghrelin are altered in patients with addictive behaviors such as alcohol and nicotine dependence and in binge eating disorder. This overlap prompted as to investigate the pro-ghrelin and GHS-R1A genes in a haplotype analysis of heavy alcohol-using individuals. A total of 417 Spanish individuals (abstainers, moderate, and heavy alcohol drinkers) were investigated in a haplotype analysis of the pro-ghrelin and GHS-R1A genes. Tag SNPs were chosen using HapMap data and the Tagger and Haploview softwares. These SNPs were then genotyped using TaqMan Allelic Discrimination. SNP rs2232165 of the GHS-R1A gene was associated with heavy alcohol consumption and SNP rs2948694 of the same gene as well as haplotypes of both the pro-ghrelin and the GHS-R1A genes were associated with body mass in heavy alcohol consuming individuals. The present findings are the first to disclose an association between the pro-ghrelin and GHS-R1A genes and heavy alcohol use, further strengthening the role of the ghrelin system in addictive behaviors and brain reward.

Cytokine responses in primary chicken embryo intestinal cells infected with Campylobacter jejuni strains of human and chicken origin and the expression of bacterial virulence-associated genes

DEFF Research Database (Denmark)

Li, Yiping; Ingmer, Hanne; Madsen, Mogens

2008-01-01

of the bacterial genes. We have investigated the invasiveness of primary chicken embryo intestinal cells (CEICs) by C. jejuni strains of human and chicken origins and the production of pro-inflammatory cytokines as well as the expression of the bacterial virulence-associated genes during co-cultivation. Results C......-free media from another co-cultivation experiment also increased the expression of the virulence-associated genes in the C. jejuni chicken isolate, indicating that the expression of bacterial genes is regulated by component(s) secreted upon co-cultivation of bacteria and CEICs. Conclusion We show that under...... in vitro culture condition C. jejuni strains of both human and chicken origins can invade avian host cells with a pro-inflammatory response and that the virulence-associated genes of C. jejuni may play a role in this process....

Latexin Inactivation Enhances Survival and Long-Term Engraftment of Hematopoietic Stem Cells and Expands the Entire Hematopoietic System in Mice

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Yi Liu

2017-04-01

Full Text Available Summary: Natural genetic diversity offers an important yet largely untapped resource to decipher the molecular mechanisms regulating hematopoietic stem cell (HSC function. Latexin (Lxn is a negative stem cell regulatory gene identified on the basis of genetic diversity. By using an Lxn knockout mouse model, we found that Lxn inactivation in vivo led to the physiological expansion of the entire hematopoietic hierarchy. Loss of Lxn enhanced the competitive repopulation capacity and survival of HSCs in a cell-intrinsic manner. Gene profiling of Lxn-null HSCs showed altered expression of genes enriched in cell-matrix and cell-cell interactions. Thrombospondin 1 (Thbs1 was a potential downstream target with a dramatic downregulation in Lxn-null HSCs. Enforced expression of Thbs1 restored the Lxn inactivation-mediated HSC phenotypes. This study reveals that Lxn plays an important role in the maintenance of homeostatic hematopoiesis, and it may lead to development of safe and effective approaches to manipulate HSCs for clinical benefit. : In this article, Liang and colleagues show that loss of latexin in vivo expands the HSC population and increases their survival and engraftment. Latexin regulates HSC function and hematopoiesis via the Thbs1 signaling pathway. Keywords: latexin, hematopoietic stem cell, repopulating advantage, expansion, survival, thrombospondin 1

Effect of Amaranthus on Advanced Glycation End-Products Induced Cytotoxicity and Proinflammatory Cytokine Gene Expression in SH-SY5Y Cells

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Warisa Amornrit

2015-09-01

Full Text Available Amaranthus plants, or spinach, are used extensively as a vegetable and are known to possess medicinal properties. Neuroinflammation and oxidative stress play a major role in the pathogenesis of many neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease. Advanced glycation end-products (AGEs cause cell toxicity in the human neuronal cell line, SH-SY5Y, through an increase in oxidative stress, as shown by reducing cell viability and increasing cell toxicity in a dose-dependent manner. We found that preincubation of SH-SY5Y cells with either petroleum ether, dichloromethane or methanol extracts of A. lividus and A. tricolor dose-dependently attenuated the neuron toxicity caused by AGEs treatment. Moreover, the results showed that A. lividus and A. tricolor extracts significantly downregulated the gene expression of the pro-inflammatory cytokines, TNF-α, IL-1 and IL-6 genes in AGEs-induced cells. We concluded that A. lividus and A. tricolor extracts not only have a neuroprotective effect against AGEs toxicity, but also have anti-inflammatory activity by reducing pro-inflammatory cytokine gene expression. This suggests that Amaranthus may be useful for treating chronic inflammation associated with neurodegenerative disorders.

Genes Associated With Prognosis After Surgery For Malignant Pleural Mesothelioma Promote Tumor Cell Survival In Vitro

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Sugarbaker David J

2011-05-01

Full Text Available Abstract Background Mesothelioma is an aggressive neoplasm with few effective treatments, one being cytoreductive surgery. We previously described a test, based on differential expression levels of four genes, to predict clinical outcome in prospectively consented mesothelioma patients after surgery. In this study, we determined whether any of these four genes could be linked to a cancer relevant phenotype. Methods We conducted a high-throughput RNA inhibition screen to knockdown gene expression levels of the four genes comprising the test (ARHGDIA, COBLL1, PKM2, TM4SF1 in both a human lung-derived normal and a tumor cell line using three different small inhibitory RNA molecules per gene. Successful knockdown was confirmed using quantitative RT-PCR. Detection of statistically significant changes in apoptosis and mitosis was performed using immunological assays and quantified using video-assisted microscopy at a single time-point. Changes in nuclear shape, size, and numbers were used to provide additional support of initial findings. Each experiment was conducted in triplicate. Specificity was assured by requiring that at least 2 different siRNAs produced the observed change in each cell line/time-point/gene/assay combination. Results Knockdown of ARHGDIA, COBLL1, and TM4SF1 resulted in 2- to 4-fold increased levels of apoptosis in normal cells (ARHGDIA only and tumor cells (all three genes. No statistically significant changes were observed in apoptosis after knockdown of PKM2 or for mitosis after knockdown of any gene. Conclusions We provide evidence that ARHGDIA, COBLL1, and TM4SF1 are negative regulators of apoptosis in cultured tumor cells. These genes, and their related intracellular signaling pathways, may represent potential therapeutic targets in mesothelioma.

PRO40 is a scaffold protein of the cell wall integrity pathway, linking the MAP kinase module to the upstream activator protein kinase C.

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Ines Teichert

2014-09-01

Full Text Available Mitogen-activated protein kinase (MAPK pathways are crucial signaling instruments in eukaryotes. Most ascomycetes possess three MAPK modules that are involved in key developmental processes like sexual propagation or pathogenesis. However, the regulation of these modules by adapters or scaffolds is largely unknown. Here, we studied the function of the cell wall integrity (CWI MAPK module in the model fungus Sordaria macrospora. Using a forward genetic approach, we found that sterile mutant pro30 has a mutated mik1 gene that encodes the MAPK kinase kinase (MAPKKK of the proposed CWI pathway. We generated single deletion mutants lacking MAPKKK MIK1, MAPK kinase (MAPKK MEK1, or MAPK MAK1 and found them all to be sterile, cell fusion-deficient and highly impaired in vegetative growth and cell wall stress response. By searching for MEK1 interaction partners via tandem affinity purification and mass spectrometry, we identified previously characterized developmental protein PRO40 as a MEK1 interaction partner. Although fungal PRO40 homologs have been implicated in diverse developmental processes, their molecular function is currently unknown. Extensive affinity purification, mass spectrometry, and yeast two-hybrid experiments showed that PRO40 is able to bind MIK1, MEK1, and the upstream activator protein kinase C (PKC1. We further found that the PRO40 N-terminal disordered region and the central region encompassing a WW interaction domain are sufficient to govern interaction with MEK1. Most importantly, time- and stress-dependent phosphorylation studies showed that PRO40 is required for MAK1 activity. The sum of our results implies that PRO40 is a scaffold protein for the CWI pathway, linking the MAPK module to the upstream activator PKC1. Our data provide important insights into the mechanistic role of a protein that has been implicated in sexual and asexual development, cell fusion, symbiosis, and pathogenicity in different fungal systems.

Lung cells support osteosarcoma cell migration and survival.

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Yu, Shibing; Fourman, Mitchell Stephen; Mahjoub, Adel; Mandell, Jonathan Brendan; Crasto, Jared Anthony; Greco, Nicholas Giuseppe; Weiss, Kurt Richard

2017-01-25

Osteosarcoma (OS) is the most common primary bone tumor, with a propensity to metastasize to the lungs. Five-year survival for metastatic OS is below 30%, and has not improved for several decades despite the introduction of multi-agent chemotherapy. Understanding OS cell migration to the lungs requires an evaluation of the lung microenvironment. Here we utilized an in vitro lung cell and OS cell co-culture model to explore the interactions between OS and lung cells, hypothesizing that lung cells would promote OS cell migration and survival. The impact of a novel anti-OS chemotherapy on OS migration and survival in the lung microenvironment was also examined. Three human OS cell lines (SJSA-1, Saos-2, U-2) and two human lung cell lines (HULEC-5a, MRC-5) were cultured according to American Type Culture Collection recommendations. Human lung cell lines were cultured in growth medium for 72 h to create conditioned media. OS proliferation was evaluated in lung co-culture and conditioned media microenvironment, with a murine fibroblast cell line (NIH-3 T3) in fresh growth medium as controls. Migration and invasion were measured using a real-time cell analysis system. Real-time PCR was utilized to probe for Aldehyde Dehydrogenase (ALDH1) expression. Osteosarcoma cells were also transduced with a lentivirus encoding for GFP to permit morphologic analysis with fluorescence microscopy. The anti-OS efficacy of Disulfiram, an ALDH-inhibitor previously shown to inhibit OS cell proliferation and metastasis in vitro, was evaluated in each microenvironment. Lung-cell conditioned medium promoted osteosarcoma cell migration, with a significantly higher attractive effect on all three osteosarcoma cell lines compared to basic growth medium, 10% serum containing medium, and NIH-3 T3 conditioned medium (p cell conditioned medium induced cell morphologic changes, as demonstrated with GFP-labeled cells. OS cells cultured in lung cell conditioned medium had increased alkaline

MSCs ameliorates DPN induced cellular pathology via [Ca2+ ]i homeostasis and scavenging the pro-inflammatory cytokines.

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Chandramoorthy, Harish C; Bin-Jaliah, Ismaeel; Karari, Hussian; Rajagopalan, Prasanna; Ahmed Shariff, Mohammed Eajaz; Al-Hakami, Ahmed; Al-Humayad, Suliman M; Baptain, Fawzi A; Ahmed, Humeda Suekit; Yassin, Hanaa Z; Haidara, Mohamed A

2018-02-01

The MSCs of various origins are known to ameliorate or modulate cell survival strategies. We investigated, whether UCB MSCs could improve the survival of the human neuronal cells and/or fibroblast assaulted with DPN sera. The results showed, the co-culture of UCB MSCs with human neuronal cells and/or fibroblasts could effectively scavenge the pro-inflammatory cytokines TNF-α, IL-1β, IFN-ɤ and IL - 12 and control the pro-apoptotic expression of p53/Bax. Further co-culture of UCB MSCs have shown to induce anti-inflammatory cytokines like IL-4, IL-10 and TGF-β and anti-apoptotic Bclxl/Bcl2 expression in the DPN sera stressed cells. Amelioration of elevated [Ca 2+ ] i and cROS, the portent behind the NFκB/Caspase-3 mediated inflammation in DPN rescued the cells from apoptosis. The results of systemic administration of BM MSCs improved DPN pathology in rat as extrapolated from human cell model. The BM MSCs ameliorated prolonged distal motor latency (control: 0.70 ± 0.06, DPN: 1.29 ± 0.13 m/s DPN + BM MSCs: 0.89 ± 0.02 m/s, p glucose levels. Together, all these results showed that administration of BM or UCB MSCs improved the DPN via ameliorating pro-inflammatory cytokine signaling and [Ca 2+ ] i homeostasis. © 2017 Wiley Periodicals, Inc.

Stem Cell Gene Therapy for Fanconi Anemia: Report from the 1st International Fanconi Anemia Gene Therapy Working Group Meeting

Science.gov (United States)

Tolar, Jakub; Adair, Jennifer E; Antoniou, Michael; Bartholomae, Cynthia C; Becker, Pamela S; Blazar, Bruce R; Bueren, Juan; Carroll, Thomas; Cavazzana-Calvo, Marina; Clapp, D Wade; Dalgleish, Robert; Galy, Anne; Gaspar, H Bobby; Hanenberg, Helmut; Von Kalle, Christof; Kiem, Hans-Peter; Lindeman, Dirk; Naldini, Luigi; Navarro, Susana; Renella, Raffaele; Rio, Paula; Sevilla, Julián; Schmidt, Manfred; Verhoeyen, Els; Wagner, John E; Williams, David A; Thrasher, Adrian J

2011-01-01

Survival rates after allogeneic hematopoietic cell transplantation (HCT) for Fanconi anemia (FA) have increased dramatically since 2000. However, the use of autologous stem cell gene therapy, whereby the patient's own blood stem cells are modified to express the wild-type gene product, could potentially avoid the early and late complications of allogeneic HCT. Over the last decades, gene therapy has experienced a high degree of optimism interrupted by periods of diminished expectation. Optimism stems from recent examples of successful gene correction in several congenital immunodeficiencies, whereas diminished expectations come from the realization that gene therapy will not be free of side effects. The goal of the 1st International Fanconi Anemia Gene Therapy Working Group Meeting was to determine the optimal strategy for moving stem cell gene therapy into clinical trials for individuals with FA. To this end, key investigators examined vector design, transduction method, criteria for large-scale clinical-grade vector manufacture, hematopoietic cell preparation, and eligibility criteria for FA patients most likely to benefit. The report summarizes the roadmap for the development of gene therapy for FA. PMID:21540837

Effect of passage number on cellular response to DNA-damaging agents: Cell survival and gene expression

International Nuclear Information System (INIS)

Chang-Liu, C.M.; Woloschak, G.E.

1997-01-01

The effect of different passage numbers on plating efficiency, doubling time, cell growth, and radiation sensitivity was assessed in Syrian hamster embryo (SHE) cells. Changes in gene expression after UV or γ-ray irradiation at different passage numbers were also examined. The SHE cells were maintained in culture medium for up to 64 passages. Cells were exposed to 60 Co γ rays or 254-nm UV radiation. Differential display of cDNAs and northern blots were used for the study of gene expression. With increasing passage number, SHE cells demonstrated decreased doubling time, increased plating efficiency, and a decreased yield in the number of cells per plate. Between passages 41 and 48 a crisis period was evident during which time cell growth in high serum was no longer optimal, and serum concentrations were reduced to maintain cell growth. Sensitivity to ionizing radiation was no different between early- and intermediate-passage cells. However, after UV exposure at low passages (passage 3), confluent cells were more sensitive to the killing effects of UV than were log-phase cells. At intermediate passages (passages 43, 48), confluent cells were slightly more radioresistant than were log-phase cells. By passage 64, however, both confluent and log-phase cells showed similar patterns of UV sensitivity. Expression of γ-actin, PCNA, and p53 transcripts did not change following UV exposure. p53 mRNA was induced following γ-ray exposure of the intermediate (passage 45) epithelial cells. The observed differences in radiation sensitivity associated with increasing passage number may be influenced by radiation-induced gene expression. The authors are conducted experiments to identify these genes

T cell activation inhibitors reduce CD8+ T cell and pro-inflammatory macrophage accumulation in adipose tissue of obese mice.

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Vince N Montes

Full Text Available Adipose tissue inflammation and specifically, pro-inflammatory macrophages are believed to contribute to insulin resistance (IR in obesity in humans and animal models. Recent studies have invoked T cells in the recruitment of pro-inflammatory macrophages and the development of IR. To test the role of the T cell response in adipose tissue of mice fed an obesogenic diet, we used two agents (CTLA-4 Ig and anti-CD40L antibody that block co-stimulation, which is essential for full T cell activation. C57BL/6 mice were fed an obesogenic diet for 16 weeks, and concomitantly either treated with CTLA-4 Ig, anti-CD40L antibody or an IgG control (300 µg/week. The treatments altered the immune cell composition of adipose tissue in obese mice. Treated mice demonstrated a marked reduction in pro-inflammatory adipose tissue macrophages and activated CD8+ T cells. Mice treated with anti-CD40L exhibited reduced weight gain, which was accompanied by a trend toward improved IR. CTLA-4 Ig treatment, however, was not associated with improved IR. These data suggest that the presence of pro-inflammatory T cells and macrophages can be altered with co-stimulatory inhibitors, but may not be a significant contributor to the whole body IR phenotype.

Amyloid precursor protein regulates migration and metalloproteinase gene expression in prostate cancer cells

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Miyazaki, Toshiaki; Ikeda, Kazuhiro; Horie-Inoue, Kuniko [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama 350-1241 (Japan); Inoue, Satoshi, E-mail: INOUE-GER@h.u-tokyo.ac.jp [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama 350-1241 (Japan); Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655 (Japan); Department of Anti-Aging Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655 (Japan)

2014-09-26

Highlights: • APP knockdown reduced proliferation and migration of prostate cancer cells. • APP knockdown reduced expression of metalloproteinase and EMT-related genes. • APP overexpression promoted LNCaP cell migration. • APP overexpression increased expression of metalloproteinase and EMT-related genes. - Abstract: Amyloid precursor protein (APP) is a type I transmembrane protein, and one of its processed forms, β-amyloid, is considered to play a central role in the development of Alzheimer’s disease. We previously showed that APP is a primary androgen-responsive gene in prostate cancer and that its increased expression is correlated with poor prognosis for patients with prostate cancer. APP has also been implicated in several human malignancies. Nevertheless, the mechanism underlying the pro-proliferative effects of APP on cancers is still not well-understood. In the present study, we explored a pathophysiological role for APP in prostate cancer cells using siRNA targeting APP (siAPP). The proliferation and migration of LNCaP and DU145 prostate cancer cells were significantly suppressed by siAPP. Differentially expressed genes in siAPP-treated cells compared to control siRNA-treated cells were identified by microarray analysis. Notably, several metalloproteinase genes, such as ADAM10 and ADAM17, and epithelial–mesenchymal transition (EMT)-related genes, such as VIM, and SNAI2, were downregulated in siAPP-treated cells as compared to control cells. The expression of these genes was upregulated in LNCaP cells stably expressing APP when compared with control cells. APP-overexpressing LNCaP cells exhibited enhanced migration in comparison to control cells. These results suggest that APP may contribute to the proliferation and migration of prostate cancer cells by modulating the expression of metalloproteinase and EMT-related genes.

MiR-7 triggers cell cycle arrest at the G1/S transition by targeting multiple genes including Skp2 and Psme3.

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Noelia Sanchez

Full Text Available MiR-7 acts as a tumour suppressor in many cancers and abrogates proliferation of CHO cells in culture. In this study we demonstrate that miR-7 targets key regulators of the G1 to S phase transition, including Skp2 and Psme3, to promote increased levels of p27(KIP and temporary growth arrest of CHO cells in the G1 phase. Simultaneously, the down-regulation of DNA repair-specific proteins via miR-7 including Rad54L, and pro-apoptotic regulators such as p53, combined with the up-regulation of anti-apoptotic factors like p-Akt, promoted cell survival while arrested in G1. Thus miR-7 can co-ordinate the levels of multiple genes and proteins to influence G1 to S phase transition and the apoptotic response in order to maintain cellular homeostasis. This work provides further mechanistic insight into the role of miR-7 as a regulator of cell growth in times of cellular stress.

Signal transducer and activator of transcription 3 activation is associated with bladder cancer cell growth and survival

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Hsieh Fu-Chuan

2008-10-01

Full Text Available Abstract Background Constitutive activation of signal transducer and activator of transcription 3 (Stat3 signaling pathway plays an important role in several human cancers. Activation of Stat3 is dependent on the phosphorylation at the tyrosine residue 705 by upstream kinases and subsequent nuclear translocation after dimerization. It remains unclear whether oncogenic Stat3 signaling pathway is involved in the oncogenesis of bladder cancer. Results We found that elevated Stat3 phosphorylation in 19 of 100 (19% bladder cancer tissues as well as bladder cancer cell lines, WH, UMUC-3 and 253J. To explore whether Stat3 activation is associated with cell growth and survival of bladder cancer, we targeted the Stat3 signaling pathway in bladder cancer cells using an adenovirus-mediated dominant-negative Stat3 (Y705F and a small molecule compound, STA-21. Both prohibited cell growth and induction of apoptosis in these bladder cancer cell lines but not in normal bladder smooth muscle cell (BdSMC. The survival inhibition might be mediated through apoptotic caspase 3, 8 and 9 pathways. Moreover, down-regulation of anti-apoptotic genes (Bcl-2, Bcl-xL and survivin and a cell cycle regulating gene (cyclin D1 was associated with the cell growth inhibition and apoptosis. Conclusion These results indicated that activation of Stat3 is crucial for bladder cancer cell growth and survival. Therefore, interference of Stat3 signaling pathway emerges as a potential therapeutic approach for bladder cancer.

The novel ER membrane protein PRO41 is essential for sexual development in the filamentous fungus Sordaria macrospora.

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Nowrousian, Minou; Frank, Sandra; Koers, Sandra; Strauch, Peter; Weitner, Thomas; Ringelberg, Carol; Dunlap, Jay C; Loros, Jennifer J; Kück, Ulrich

2007-05-01

The filamentous fungus Sordaria macrospora develops complex fruiting bodies (perithecia) to propagate its sexual spores. Here, we present an analysis of the sterile mutant pro41 that is unable to produce mature fruiting bodies. The mutant carries a deletion of 4 kb and is complemented by the pro41 open reading frame that is contained within the region deleted in the mutant. In silico analyses predict PRO41 to be an endoplasmic reticulum (ER) membrane protein, and a PRO41-EGFP fusion protein colocalizes with ER-targeted DsRED. Furthermore, Western blot analysis shows that the PRO41-EGFP fusion protein is present in the membrane fraction. A fusion of the predicted N-terminal signal sequence of PRO41 with EGFP is secreted out of the cell, indicating that the signal sequence is functional. pro41 transcript levels are upregulated during sexual development. This increase in transcript levels was not observed in the sterile mutant pro1 that lacks a transcription factor gene. Moreover, microarray analysis of gene expression in the mutants pro1, pro41 and the pro1/41 double mutant showed that pro41 is partly epistatic to pro1. Taken together, these data show that PRO41 is a novel ER membrane protein essential for fruiting body formation in filamentous fungi.

IL-6-induced Bcl6 variant 2 supports IL-6-dependent myeloma cell proliferation and survival through STAT3

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Tsuyama, Naohiro; Danjoh, Inaho; Otsuyama, Ken-ichiro; Obata, Masanori; Tahara, Hidetoshi; Ohta, Tsutomu; Ishikawa, Hideaki

2005-01-01

IL-6 is a growth and survival factor for myeloma cells, although the mechanism by which it induces myeloma cell proliferation through gene expression is largely unknown. Microarray analysis showed that some B-cell lymphoma-associated oncogenes such as Bcl6, which is absent in normal plasma cells, were upregulated by IL-6 in IL-6-dependent myeloma cell lines. We found that Bcl6 variant 2 was upregulated by STAT3. ChIP assay and EMSA showed that STAT3 bound to the upstream region of variant 2 DNA. Expression of p53, a direct target gene of Bcl6, was downregulated in the IL-6-stimulated cells, and this process was impaired by an HDAC inhibitor. Bcl6 was knocked down by introducing small hairpin RNA, resulting in decreased proliferation and increased sensitivity to a DNA damaging agent. Thus, STAT3-inducible Bcl6 variant 2 appears to generate an important IL-6 signal that supports proliferation and survival of IL-6-dependent myeloma cells

Tax Protein-induced Expression of Antiapoptotic Bfl-1 Protein Contributes to Survival of Human T-cell Leukemia Virus Type 1 (HTLV-1)-infected T-cells*♦

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Macaire, Héloïse; Riquet, Aurélien; Moncollin, Vincent; Biémont-Trescol, Marie-Claude; Duc Dodon, Madeleine; Hermine, Olivier; Debaud, Anne-Laure; Mahieux, Renaud; Mesnard, Jean-Michel; Pierre, Marlène; Gazzolo, Louis; Bonnefoy, Nathalie; Valentin, Hélène

2012-01-01

Human T lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma (ATLL). ATLL is a severe malignancy with no effective treatment. HTLV-1 regulatory proteins Tax and HTLV-1 basic leucine zipper factor (HBZ) play a major role in ATLL development, by interfering with cellular functions such as CD4+ T-cell survival. In this study, we observed that the expression of Bfl-1, an antiapoptotic protein of the Bcl-2 family, is restricted to HTLV-1-infected T-cell lines and to T-cells expressing both Tax and HBZ proteins. We showed that Tax-induced bfl-1 transcription through the canonical NF-κB pathway. Moreover, we demonstrated that Tax cooperated with c-Jun or JunD, but not JunB, transcription factors of the AP-1 family to stimulate bfl-1 gene activation. By contrast, HBZ inhibited c-Jun-induced bfl-1 gene activation, whereas it increased JunD-induced bfl-1 gene activation. We identified one NF-κB, targeted by RelA, c-Rel, RelB, p105/p50, and p100/p52, and two AP-1, targeted by both c-Jun and JunD, binding sites in the bfl-1 promoter of T-cells expressing both Tax and HBZ. Analyzing the potential role of antiapoptotic Bcl-2 proteins in HTLV-1-infected T-cell survival, we demonstrated that these cells are differentially sensitive to silencing of Bfl-1, Bcl-xL, and Bcl-2. Indeed, both Bfl-1 and Bcl-xL knockdowns decreased the survival of HTLV-1-infected T-cell lines, although no cell death was observed after Bcl-2 knockdown. Furthermore, we demonstrated that Bfl-1 knockdown sensitizes HTLV-1-infected T-cells to ABT-737 or etoposide treatment. Our results directly implicate Bfl-1 and Bcl-xL in HTLV-1-infected T-cell survival and suggest that both Bfl-1 and Bcl-xL represent potential therapeutic targets for ATLL treatment. PMID:22553204

ONC201 kills solid tumor cells by triggering an integrated stress response dependent on ATF4 activation by specific eIF2α kinases

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Kline, C. Leah B.; Van den Heuvel, A. Pieter J.; Allen, Joshua E.; Prabhu, Varun V.; Dicker, David T.; El-Deiry, Wafik S.

2016-01-01

ONC201 (also called TIC10) is a small molecule that inactivates the cell proliferation- and cell survival-promoting kinases AKT and ERK and induces cell death through the pro-apoptotic protein TRAIL. ONC201 is currently in early phase clinical testing for various malignancies. Here, we found through gene expression and protein analyses that ONC201 triggered an increase in TRAIL abundance and cell death through an integrated stress response (ISR) involving the transcription factor ATF4, the tr...

Repair models of cell survival and corresponding computer program for survival curve fitting

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Shen Xun; Hu Yiwei

1992-01-01

Some basic concepts and formulations of two repair models of survival, the incomplete repair (IR) model and the lethal-potentially lethal (LPL) model, are introduced. An IBM-PC computer program for survival curve fitting with these models was developed and applied to fit the survivals of human melanoma cells HX118 irradiated at different dose rates. Comparison was made between the repair models and two non-repair models, the multitar get-single hit model and the linear-quadratic model, in the fitting and analysis of the survival-dose curves. It was shown that either IR model or LPL model can fit a set of survival curves of different dose rates with same parameters and provide information on the repair capacity of cells. These two mathematical models could be very useful in quantitative study on the radiosensitivity and repair capacity of cells

Role of Ku80-dependent end-joining in delayed genomic instability in mammalian cells surviving ionizing radiation

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Suzuki, Keiji; Kodama, Seiji; Watanabe, Masami

2010-01-01

Ionizing radiation induces delayed destabilization of the genome in the progenies of surviving cells. This phenomenon, which is called radiation-induced genomic instability, is manifested by delayed induction of radiation effects, such as cell death, chromosome aberration, and mutation in the progeny of cells surviving radiation exposure. Previously, there was a report showing that delayed cell death was absent in Ku80-deficient Chinese hamster ovary (CHO) cells, however, the mechanism of their defect has not been determined. We found that delayed induction of DNA double strand breaks and chromosomal breaks were intact in Ku80-deficient cells surviving X-irradiation, whereas there was no sign for the production of chromosome bridges between divided daughter cells. Moreover, delayed induction of dicentric chromosomes was significantly compromised in those cells compared to the wild-type CHO cells. Reintroduction of the human Ku86 gene complimented the defective DNA repair and recovered delayed induction of dicentric chromosomes and delayed cell death, indicating that defective Ku80-dependent dicentric induction was the cause of the absence of delayed cell death. Since DNA-PKcs-defective cells showed delayed phenotypes, Ku80-dependent illegitimate rejoining is involved in delayed impairment of the integrity of the genome in radiation-survived cells.

Role of Ku80-dependent end-joining in delayed genomic instability in mammalian cells surviving ionizing radiation

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Suzuki, Keiji, E-mail: kzsuzuki@nagasaki-u.ac.jp [Course of Life Sciences and Radiation Research, Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan); Kodama, Seiji [Research Institute for Advanced Science and Technology, Osaka Prefecture University, 1-2 Gakuen-machi, Sakai 599-8570 (Japan); Watanabe, Masami [Kyoto University Research Reactor Institute, Kumatori-cho Sennan-gun, Osaka 590-0494 (Japan)

2010-01-05

Ionizing radiation induces delayed destabilization of the genome in the progenies of surviving cells. This phenomenon, which is called radiation-induced genomic instability, is manifested by delayed induction of radiation effects, such as cell death, chromosome aberration, and mutation in the progeny of cells surviving radiation exposure. Previously, there was a report showing that delayed cell death was absent in Ku80-deficient Chinese hamster ovary (CHO) cells, however, the mechanism of their defect has not been determined. We found that delayed induction of DNA double strand breaks and chromosomal breaks were intact in Ku80-deficient cells surviving X-irradiation, whereas there was no sign for the production of chromosome bridges between divided daughter cells. Moreover, delayed induction of dicentric chromosomes was significantly compromised in those cells compared to the wild-type CHO cells. Reintroduction of the human Ku86 gene complimented the defective DNA repair and recovered delayed induction of dicentric chromosomes and delayed cell death, indicating that defective Ku80-dependent dicentric induction was the cause of the absence of delayed cell death. Since DNA-PKcs-defective cells showed delayed phenotypes, Ku80-dependent illegitimate rejoining is involved in delayed impairment of the integrity of the genome in radiation-survived cells.

Expression profiling of genes regulated by TGF-beta: Differential regulation in normal and tumour cells

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Takahashi Takashi

2007-04-01

Full Text Available Abstract Background TGF-beta is one of the key cytokines implicated in various disease processes including cancer. TGF-beta inhibits growth and promotes apoptosis in normal epithelial cells and in contrast, acts as a pro-tumour cytokine by promoting tumour angiogenesis, immune-escape and metastasis. It is not clear if various actions of TGF-beta on normal and tumour cells are due to differential gene regulations. Hence we studied the regulation of gene expression by TGF-beta in normal and cancer cells. Results Using human 19 K cDNA microarrays, we show that 1757 genes are exclusively regulated by TGF-beta in A549 cells in contrast to 733 genes exclusively regulated in HPL1D cells. In addition, 267 genes are commonly regulated in both the cell-lines. Semi-quantitative and real-time qRT-PCR analysis of some genes agrees with the microarray data. In order to identify the signalling pathways that influence TGF-beta mediated gene regulation, we used specific inhibitors of p38 MAP kinase, ERK kinase, JNK kinase and integrin signalling pathways. The data suggest that regulation of majority of the selected genes is dependent on at least one of these pathways and this dependence is cell-type specific. Interestingly, an integrin pathway inhibitor, RGD peptide, significantly affected TGF-beta regulation of Thrombospondin 1 in A549 cells. Conclusion These data suggest major differences with respect to TGF-beta mediated gene regulation in normal and transformed cells and significant role of non-canonical TGF-beta pathways in the regulation of many genes by TGF-beta.

Combinatorial gene therapy renders increased survival in cirrhotic rats

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Armendáriz-Borunda Juan S

2010-05-01

Full Text Available Abstract Background Liver fibrosis ranks as the second cause of death in México's productive-age population. This pathology is characterized by acummulation of fibrillar proteins in hepatic parenchyma causing synthetic and metabolic disfunction. Remotion of excessive fibrous proteins might result in benefit for subjects increasing survival index. The goal of this work was to find whether the already known therapeutical effect of human urokinase Plasminogen Activator and human Matrix Metalloprotease 8 extends survival index in cirrhotic animals. Methods Wistar rats (80 g underwent chronic intoxication with CCl4: mineral oil for 8 weeks. Cirrhotic animals were injected with a combined dose of Ad-delta-huPA plus Ad-MMP8 (3 × 1011 and 1.5 × 1011 vp/Kg, respectively or with Ad-beta-Gal (4.5 × 1011 and were killed after 2, 4, 6, 8 and 10 days. Then, liver and serum were collected. An additional set of cirrhotic animals injected with combined gene therapy was also monitored for their probability of survival. Results Only the cirrhotic animals treated with therapeutical genes (Ad-delta-huPA+Ad-MMP-8 showed improvement in liver fibrosis. These results correlated with hydroxyproline determinations. A significant decrement in alpha-SMA and TGF-beta1 gene expression was also observed. Cirrhotic rats treated with Ad-delta-huPA plus Ad-MMP8 had a higher probability of survival at 60 days with respect to Ad-beta-Gal-injected animals. Conclusion A single administration of Ad-delta-huPA plus Ad-MMP-8 is efficient to induce fibrosis regression and increase survival in experimental liver fibrosis.

Cell Survival Signaling in Neuroblastoma

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Megison, Michael L.; Gillory, Lauren A.; Beierle, Elizabeth A.

2013-01-01

Neuroblastoma is the most common extracranial solid tumor of childhood and is responsible for over 15% of pediatric cancer deaths. Neuroblastoma tumorigenesis and malignant transformation is driven by overexpression and dominance of cell survival pathways and a lack of normal cellular senescence or apoptosis. Therefore, manipulation of cell survival pathways may decrease the malignant potential of these tumors and provide avenues for the development of novel therapeutics. This review focuses on several facets of cell survival pathways including protein kinases (PI3K, AKT, ALK, and FAK), transcription factors (NF-κB, MYCN and p53), and growth factors (IGF, EGF, PDGF, and VEGF). Modulation of each of these factors decreases the growth or otherwise hinders the malignant potential of neuroblastoma, and many therapeutics targeting these pathways are already in the clinical trial phase of development. Continued research and discovery of effective modulators of these pathways will revolutionize the treatment of neuroblastoma. PMID:22934706

Inhibition of Bcl-2 or IAP proteins does not provoke mutations in surviving cells

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Shekhar, Tanmay M. [Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3083 (Australia); Green, Maja M. [Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3083 (Australia); Department of Anatomy & Neuroscience, The University of Melbourne, Parkville 3010 (Australia); Rayner, David M.; Miles, Mark A.; Cutts, Suzanne M. [Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3083 (Australia); Hawkins, Christine J., E-mail: c.hawkins@latrobe.edu.au [Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3083 (Australia)

2015-07-15

Graphical abstract: - Highlights: • Mutagenicities of anti-cancer drugs were tested using HPRT, γH2AX and comet assays. • TRAIL, doxorubicin and etoposide were more mutagenic than BH3- or Smac-mimetics. • Physiologically achievable levels of the BH3-mimetic ABT-737 were not mutagenic. • High concentrations of ABT-737 provoked mutations via an off-target mechanism. • Even very high concentrations of IAP antagonists were not mutagenic. - Abstract: Chemotherapy and radiotherapy can cause permanent damage to the genomes of surviving cells, provoking severe side effects such as second malignancies in some cancer survivors. Drugs that mimic the activity of death ligands, or antagonise pro-survival proteins of the Bcl-2 or IAP families have yielded encouraging results in animal experiments and early phase clinical trials. Because these agents directly engage apoptosis pathways, rather than damaging DNA to indirectly provoke tumour cell death, we reasoned that they may offer another important advantage over conventional therapies: minimisation or elimination of side effects such as second cancers that result from mutation of surviving normal cells. Disappointingly, however, we previously found that concentrations of death receptor agonists like TRAIL that would be present in vivo in clinical settings provoked DNA damage in surviving cells. In this study, we used cell line model systems to investigate the mutagenic capacity of drugs from two other classes of direct apoptosis-inducing agents: the BH3-mimetic ABT-737 and the IAP antagonists LCL161 and AT-406. Encouragingly, our data suggest that IAP antagonists possess negligible genotoxic activity. Doses of ABT-737 that were required to damage DNA stimulated Bax/Bak-independent signalling and exceeded concentrations detected in the plasma of animals treated with this drug. These findings provide hope that cancer patients treated by BH3-mimetics or IAP antagonists may avoid mutation-related illnesses that afflict

Inhibition of Bcl-2 or IAP proteins does not provoke mutations in surviving cells

International Nuclear Information System (INIS)

Shekhar, Tanmay M.; Green, Maja M.; Rayner, David M.; Miles, Mark A.; Cutts, Suzanne M.; Hawkins, Christine J.

2015-01-01

Graphical abstract: - Highlights: • Mutagenicities of anti-cancer drugs were tested using HPRT, γH2AX and comet assays. • TRAIL, doxorubicin and etoposide were more mutagenic than BH3- or Smac-mimetics. • Physiologically achievable levels of the BH3-mimetic ABT-737 were not mutagenic. • High concentrations of ABT-737 provoked mutations via an off-target mechanism. • Even very high concentrations of IAP antagonists were not mutagenic. - Abstract: Chemotherapy and radiotherapy can cause permanent damage to the genomes of surviving cells, provoking severe side effects such as second malignancies in some cancer survivors. Drugs that mimic the activity of death ligands, or antagonise pro-survival proteins of the Bcl-2 or IAP families have yielded encouraging results in animal experiments and early phase clinical trials. Because these agents directly engage apoptosis pathways, rather than damaging DNA to indirectly provoke tumour cell death, we reasoned that they may offer another important advantage over conventional therapies: minimisation or elimination of side effects such as second cancers that result from mutation of surviving normal cells. Disappointingly, however, we previously found that concentrations of death receptor agonists like TRAIL that would be present in vivo in clinical settings provoked DNA damage in surviving cells. In this study, we used cell line model systems to investigate the mutagenic capacity of drugs from two other classes of direct apoptosis-inducing agents: the BH3-mimetic ABT-737 and the IAP antagonists LCL161 and AT-406. Encouragingly, our data suggest that IAP antagonists possess negligible genotoxic activity. Doses of ABT-737 that were required to damage DNA stimulated Bax/Bak-independent signalling and exceeded concentrations detected in the plasma of animals treated with this drug. These findings provide hope that cancer patients treated by BH3-mimetics or IAP antagonists may avoid mutation-related illnesses that afflict

β3 integrin promotes chemoresistance to epirubicin in MDA-MB-231 through repression of the pro-apoptotic protein, BAD

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Nair, Madhumathy G.; Desai, Krisha; Prabhu, Jyothi S.; Hari, P.S.; Remacle, Jose; Sridhar, T.S., E-mail: tssridhar@sjri.res.in

2016-08-01

Resistance to anthracycline based chemotherapy is a major limitation in the treatment of breast cancer, particularly of the triple negative sub-type that lacks targeted therapies. Resistance that arises from tumor-stromal interaction facilitated by integrins provides the possibility of targeted disruption. In the present study, we demonstrate that integrin β3 signaling inhibits apoptosis induced by a DNA-damaging chemotherapeutic agent, epirubicin, in MDA-MB-231 breast cancer cells. Drug efflux based mechanisms do not contribute to this effect. We show that integrin β3 employs the PI3K-Akt and the MAPK pathway for enabling cell survival and proliferation. Further, our results indicate that integrin β3 helps inhibit epirubicin induced cytotoxicity by repression of the pro-apoptotic protein BAD, thus promoting an anti-apoptotic response. Myristoylated RGT peptide and a monoclonal antibody against integrin β3 brought about a reversal of this effect and chemosensitized the cells. These results identify β3 integrin signaling via repression of BAD as an important survival pathway used by breast cancer cells to evade chemotherapy induced stress. - Highlights: • Integrin β3 signaling promotes chemoresistance to epirubicin in breast cancer cells. • Integrin β3 promotes cell survival and proliferation in drug treated cells through the PI3K and MAPK pathways. • Integrin signaling helps evade drug induced cytotoxicity by repression of pro-apoptotic molecule; BAD.

p16(INK4A) inhibits the pro-metastatic potentials of osteosarcoma cells through targeting the ERK pathway and TGF-β1.

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Silva, Gabriela; Aboussekhra, Abdelilah

2016-05-01

Extracellular signal-regulated kinase (ERK) is a downstream component of the evolutionarily conserved mitogen-activated protein kinase-signaling pathway, which controls the expression of a plethora of genes implicated in various physiological processes. This pathway is often hyper-activated by mutations or abnormal extracellular signaling in different types of human cancer, including the most common primary malignant bone tumor osteosarcomas. p16(INK4A) is an important tumor suppressor gene frequently lost in osteosarcomas, and is associated with the progression of these malignancies. We have shown, here, that the ERK1/2 protein kinase is also activated by p16(INK4A) down-regulation in osteosarcoma cells and normal human as well as mouse cells. This inhibitory effect is associated with the suppression of the upstream kinase MEK1/2, and is mediated via the repression of miR-21-5p and the consequent up-regulation of the MEK/ERK antagonist SPRY2 in osteosarcoma cells. Furthermore, we have shown that p16(INK4) inhibits the migration/invasion abilities of these cells through miR-21-5p-dependent inhibition of ERK1/2. In addition, we present clear evidence that p16(INK4) represses the paracrine pro-migratory effect of osteosarcoma cells on stromal fibroblasts through the inhibition of the TGF-β1 expression/secretion. This effect is also ERK1/2-dependent, indicating that in addition to their cell-autonomous actions, p16(INK4) and ERK1/2 have also non-cell-autonomous cancer-related functions. Together, these results indicate that the tumor suppressor p16(INK4) protein represses the carcinogenic process of osteosarcoma cells not only as a cell cycle regulator, but also as a negative regulator of pro-carcinogenic/-metastatic pathways. This indicates that targeting the ERK pathway is of utmost therapeutic value. © 2015 Wiley Periodicals, Inc.

Emerging paradigms and questions on pro-angiogenic bone marrow-derived myelomonocytic cells.

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Laurent, Julien; Touvrey, Cédric; Botta, Francesca; Kuonen, François; Ruegg, Curzio

2011-01-01

Cancer-related inflammation has emerged in recent years as a major event contributing to tumor angiogenesis, tumor progression and metastasis formation. Bone marrow-derived and inflammatory cells promote tumor angiogenesis by providing endothelial progenitor cells that differentiate into mature endothelial cells, and by secreting pro-angiogenic factors and remodeling the extracellular matrix to stimulate angiogenesis though paracrine mechanisms. Several bone marrow-derived myelonomocytic cells, including monocytes and macrophages, have been identified and characterized by several laboratories in recent years. While the central role of these cells in promoting tumor angiogenesis, tumor progression and metastasis is nowadays well established, many questions remain open and new ones are emerging. These include the relationship between their phenotype and function, the mechanisms of pro-angiogenic programming, their contribution to resistance to anti-angiogenic treatments and to metastasis and their potential clinical use as biomarkers of angiogenesis and anti-angiogenic therapies. Here, we will review phenotypical and functional aspects of bone marrow-derived myelonomocytic cells and discuss some of the current outstanding questions.

Description and physical localization of the bovine survival of motor neuron gene (SMN).

Science.gov (United States)

Pietrowski, D; Goldammer, T; Meinert, S; Schwerin, M; Förster, M

1998-01-01

Proximal spinal muscular atrophy (SMA) is an autosomal recessive disease in humans and other mammals, characterized by degeneration of anterior horn cells of the spinal cord. In humans, the survival of motor neuron gene (SMN) has been recognized as the SMA-determining gene and has been mapped to 5q13. In cattle, SMA is a recurrent, inherited disease that plays an important economic role in breeding programs of Brown Swiss stock. Now we have identified the full- length cDNA sequence of the bovine SMN gene. Molecular analysis and characterization of the sequence documents 85% identity to its human counterpart and three evolutionarily conserved domains in different species. Physical mapping data reveals that bovine SMN is localized to chromosome region 20q12-->q13, supporting the conserved synteny of this chromosomal region between humans and cattle.

Transcriptional profiling of MEF2-regulated genes in human neural progenitor cells derived from embryonic stem cells

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Shing Fai Chan

2015-03-01

Full Text Available The myocyte enhancer factor 2 (MEF2 family of transcription factors is highly expressed in the brain and constitutes a key determinant of neuronal survival, differentiation, and synaptic plasticity. However, genome-wide transcriptional profiling of MEF2-regulated genes has not yet been fully elucidated, particularly at the neural stem cell stage. Here we report the results of microarray analysis comparing mRNAs isolated from human neural progenitor/stem cells (hNPCs derived from embryonic stem cells expressing a control vector versus progenitors expressing a constitutively-active form of MEF2 (MEF2CA, which increases MEF2 activity. Microarray experiments were performed using the Illumina Human HT-12 V4.0 expression beadchip (GEO#: GSE57184. By comparing vector-control cells to MEF2CA cells, microarray analysis identified 1880 unique genes that were differentially expressed. Among these genes, 1121 genes were up-regulated and 759 genes were down-regulated. Our results provide a valuable resource for identifying transcriptional targets of MEF2 in hNPCs.

Adult rat bone marrow stromal cells express genes associated with dopamine neurons

International Nuclear Information System (INIS)

Kramer, Brian C.; Woodbury, Dale; Black, Ira B.

2006-01-01

An intensive search is underway to identify candidates to replace the cells that degenerate in Parkinson's disease (PD). To date, no suitable substitute has been found. We have recently found that adult rat bone marrow stromal cells (MSCs) can be induced to assume a neuronal phenotype in vitro. These findings may have particular relevance to the treatment of PD. We now report that adult MSCs express multiple dopaminergic genes, suggesting that they are potential candidates for cell therapy. Using RT-PCR, we have examined families of genes that are associated with the development and/or survival of dopaminergic neurons. MSCs transcribe a variety of dopaminergic genes including patched and smoothened (components of the Shh receptor), Gli-1 (downstream mediator of Shh), and Otx-1, a gene associated with formation of the mesencephalon during development. Furthermore, Shh treatment elicits a 1.5-fold increase in DNA synthesis in cultured MSCs, suggesting the presence of a functional Shh receptor complex. We have also found that MSCs transcribe and translate Nurr-1, a nuclear receptor essential for the development of dopamine neurons. In addition, MSCs express a variety of growth factor receptors including the glycosyl-phosphatidylinositol-anchored ligand-binding subunit of the GDNF receptor, GFRα1, as well as fibroblast growth factor receptors one and four. The expression of genes that are associated with the development and survival of dopamine neurons suggests a potential role for these cells in the treatment of Parkinson's disease

The pro-survival function of p53 in HeLa cells

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Kim, Jin Kyu; Kang, Mi Young; Jang, Eun Yeong; Kim, Jin Hong [Korea Atomic Energy Research Institute, Advanced Radiation Technology Institute, Jeongeup (Korea, Republic of)

2014-11-15

The rate of apoptosis and autophagy was variable with different p53 status after IR treatment of cells. The influence of p53 status on cell fate suggests a role of p53 in two fundamentally important cell biological pathways: autophagy and apoptosis. p53 coordinates cell cycle arrest and apoptosis to govern cell fate. This study was done to identify p53-mediated regulation of cell's fate. Autophagy induced by IR may prevent cells from undergoing apoptosis, implying an interlink modulation between autophagy and apoptosis. The rate of apoptosis and autophagy was determined with different p53 status after IR treatment of HeLa cells in this study. Our research on IR-induced cellular responses may provide new information about fate decision between the processes of apoptosis and autophagy.

Identification of an IL-1-induced gene expression pattern in AR+ PCa cells that mimics the molecular phenotype of AR- PCa cells.

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Thomas-Jardin, Shayna E; Kanchwala, Mohammed S; Jacob, Joan; Merchant, Sana; Meade, Rachel K; Gahnim, Nagham M; Nawas, Afshan F; Xing, Chao; Delk, Nikki A

2018-06-01

In immunosurveillance, bone-derived immune cells infiltrate the tumor and secrete inflammatory cytokines to destroy cancer cells. However, cancer cells have evolved mechanisms to usurp inflammatory cytokines to promote tumor progression. In particular, the inflammatory cytokine, interleukin-1 (IL-1), is elevated in prostate cancer (PCa) patient tissue and serum, and promotes PCa bone metastasis. IL-1 also represses androgen receptor (AR) accumulation and activity in PCa cells, yet the cells remain viable and tumorigenic; suggesting that IL-1 may also contribute to AR-targeted therapy resistance. Furthermore, IL-1 and AR protein levels negatively correlate in PCa tumor cells. Taken together, we hypothesize that IL-1 reprograms AR positive (AR + ) PCa cells into AR negative (AR - ) PCa cells that co-opt IL-1 signaling to ensure AR-independent survival and tumor progression in the inflammatory tumor microenvironment. LNCaP and PC3 PCa cells were treated with IL-1β or HS-5 bone marrow stromal cell (BMSC) conditioned medium and analyzed by RNA sequencing and RT-QPCR. To verify genes identified by RNA sequencing, LNCaP, MDA-PCa-2b, PC3, and DU145 PCa cell lines were treated with the IL-1 family members, IL-1α or IL-1β, or exposed to HS-5 BMSC in the presence or absence of Interleukin-1 Receptor Antagonist (IL-1RA). Treated cells were analyzed by western blot and/or RT-QPCR. Comparative analysis of sequencing data from the AR + LNCaP PCa cell line versus the AR - PC3 PCa cell line reveals an IL-1-conferred gene suite in LNCaP cells that is constitutive in PC3 cells. Bioinformatics analysis of the IL-1 regulated gene suite revealed that inflammatory and immune response pathways are primarily elicited; likely facilitating PCa cell survival and tumorigenicity in an inflammatory tumor microenvironment. Our data supports that IL-1 reprograms AR + PCa cells to mimic AR - PCa gene expression patterns that favor AR-targeted treatment resistance and cell survival. © 2018 Wiley

Assessment of the Pro12Ala Polymorphism in the PPAR-γ2 Gene among Type 2 Diabetes Patients in a Nigerian Population

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Godwill Azeh Engwa

2018-04-01

Full Text Available The association between the Pro12Ala polymorphism of the PPARγ2 gene, type 2 diabetes (T2D, and obesity in certain ethnic populations has been reported. However, this relationship has not yet been described among diabetes patients in Nigeria. This study investigated the relationship between the Pro12Ala polymorphism in the PPARγ2 gene, obesity, and lipid abnormalities characterizing T2D among patients in Nigeria. This case-control study recruited 73 T2D and 75 non-diabetic (ND patients. Demographic and clinical data were collected and blood glucose levels together with serum lipid profile for patients were measured. Pro12Ala polymorphism in the PPARγ2 gene was genotyped by restriction fragment length-Polymerase Chain Reaction (RFLP-PCR. The PPAR-γ2 gene (amplicon size = 270 base pair was successfully amplified for all samples. Following restriction enzyme digestion and analysis by agarose gel electrophoresis, amplicons from samples showed a band of size 270 bp and were of the wild homozygous Pro/Pro genotype. Ala12 variant was totally absent from the study population. Obesity, estimated using Body Mass Index (BMI and waist circumference (WC, was significantly higher (p < 0.05 in T2D patients compared to the non-diabetic patients. More so, the prevalence of lipid abnormalities; hypercholesterolaemia (TC > 200 mg/dL, hypertriglyceridaemia (TG > 150 mg/dL, high HDL (>100 mg/dL, and low HDL (<50 mg/dL was significantly greater (p < 0.001 in T2D patients compared to non-diabetic patients. Results obtained further indicated lack of significant association between PPAR-γ2 gene polymorphism, T2D, and obesity. However, obesity and dyslipidaemia were strongly associated in T2D patients.

Dynamics of the transcriptome response of cultured human embryonic stem cells to ionizing radiation exposure

International Nuclear Information System (INIS)

Sokolov, Mykyta V.; Panyutin, Irina V.; Panyutin, Igor G.; Neumann, Ronald D.

2011-01-01

One of the key consequences of exposure of human cells to genotoxic agents is the activation of DNA damage responses (DDR). While the mechanisms underpinning DDR in fully differentiated somatic human cells have been studied extensively, molecular signaling events and pathways involved in DDR in pluripotent human embryonic stem cells (hESC) remain largely unexplored. We studied changes in the human genome-wide transcriptome of H9 hESC line following exposures to 1 Gy of gamma-radiation at 2 h and 16 h post-irradiation. Quantitative real-time PCR was performed to verify the expression data for a subset of genes. In parallel, the cell growth, DDR kinetics, and expression of pluripotency markers in irradiated hESC were monitored. The changes in gene expression in hESC after exposure to ionizing radiation (IR) are substantially different from those observed in somatic human cell lines. Gene expression patterns at 2 h post-IR showed almost an exclusively p53-dependent, predominantly pro-apoptotic, signature with a total of only 30 up-regulated genes. In contrast, the gene expression patterns at 16 h post-IR showed 354 differentially expressed genes, mostly involved in pro-survival pathways, such as increased expression of metallothioneins, ubiquitin cycle, and general metabolism signaling. Cell growth data paralleled trends in gene expression changes. DDR in hESC followed the kinetics reported for human somatic differentiated cells. The expression of pluripotency markers characteristic of undifferentiated hESC was not affected by exposure to IR during the time course of our analysis. Our data on dynamics of transcriptome response of irradiated hESCs may provide a valuable tool to screen for markers of IR exposure of human cells in their most naive state; thus unmasking the key elements of DDR; at the same time, avoiding the complexity of interpreting distinct cell type-dependent genotoxic stress responses of terminally differentiated cells.

Dynamics of the transcriptome response of cultured human embryonic stem cells to ionizing radiation exposure

Energy Technology Data Exchange (ETDEWEB)

Sokolov, Mykyta V., E-mail: sokolovm@mail.nih.gov [Nuclear Medicine Division, Department of Radiology and Imaging Sciences, Clinical Center, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892 (United States); Panyutin, Irina V., E-mail: ipanyutinv@mail.nih.gov [Nuclear Medicine Division, Department of Radiology and Imaging Sciences, Clinical Center, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892 (United States); Panyutin, Igor G., E-mail: igorp@helix.nih.gov [Nuclear Medicine Division, Department of Radiology and Imaging Sciences, Clinical Center, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892 (United States); Neumann, Ronald D., E-mail: rneumann@mail.nih.gov [Nuclear Medicine Division, Department of Radiology and Imaging Sciences, Clinical Center, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892 (United States)

2011-05-10

One of the key consequences of exposure of human cells to genotoxic agents is the activation of DNA damage responses (DDR). While the mechanisms underpinning DDR in fully differentiated somatic human cells have been studied extensively, molecular signaling events and pathways involved in DDR in pluripotent human embryonic stem cells (hESC) remain largely unexplored. We studied changes in the human genome-wide transcriptome of H9 hESC line following exposures to 1 Gy of gamma-radiation at 2 h and 16 h post-irradiation. Quantitative real-time PCR was performed to verify the expression data for a subset of genes. In parallel, the cell growth, DDR kinetics, and expression of pluripotency markers in irradiated hESC were monitored. The changes in gene expression in hESC after exposure to ionizing radiation (IR) are substantially different from those observed in somatic human cell lines. Gene expression patterns at 2 h post-IR showed almost an exclusively p53-dependent, predominantly pro-apoptotic, signature with a total of only 30 up-regulated genes. In contrast, the gene expression patterns at 16 h post-IR showed 354 differentially expressed genes, mostly involved in pro-survival pathways, such as increased expression of metallothioneins, ubiquitin cycle, and general metabolism signaling. Cell growth data paralleled trends in gene expression changes. DDR in hESC followed the kinetics reported for human somatic differentiated cells. The expression of pluripotency markers characteristic of undifferentiated hESC was not affected by exposure to IR during the time course of our analysis. Our data on dynamics of transcriptome response of irradiated hESCs may provide a valuable tool to screen for markers of IR exposure of human cells in their most naive state; thus unmasking the key elements of DDR; at the same time, avoiding the complexity of interpreting distinct cell type-dependent genotoxic stress responses of terminally differentiated cells.

Low GILT expression is associated with poor patient survival in diffuse large B-cell lymphoma

Directory of Open Access Journals (Sweden)

Hannah ePhipps-Yonas

2013-12-01

Full Text Available The MHC class II-restricted antigen processing pathway presents antigenic peptides acquired in the endocytic route for the activation of CD4+ T cells. Multiple cancers express MHC class II, which may influence the anti-tumor immune response and patient outcome. Low MHC class II expression is associated with poor survival in diffuse large B-cell lymphoma (DLBCL, the most common form of aggressive non-Hodgkin lymphoma. Therefore, we investigated whether gamma-interferon-inducible lysosomal thiol reductase (GILT, an upstream component of the MHC class II-restricted antigen processing pathway that is not regulated by the transcription factor class II transactivator, may be important in DLBCL biology. GILT reduces protein disulfide bonds in the endocytic compartment, exposing additional epitopes for MHC class II binding and facilitating antigen presentation. In each of four independent gene expression profiling cohorts with a total of 585 DLBCL patients, low GILT expression was significantly associated with poor overall survival. In contrast, low expression of a classical MHC class II gene, HLA-DRA, was associated with poor survival in one of four cohorts. The association of low GILT expression with poor survival was independent of established clinical and molecular prognostic factors, the International Prognostic Index and the cell of origin classification, respectively. Immunohistochemical analysis of GILT expression in 96 DLBCL cases demonstrated variation in GILT protein expression within tumor cells which correlated strongly with GILT mRNA expression. These studies identify a novel association between GILT expression and clinical outcome in lymphoma. Our findings underscore the role of antigen processing in DLBCL and suggest that molecules targeting this pathway warrant investigation as potential therapeutics.

Expression of core clock genes in colorectal tumour cells compared with normal mucosa

DEFF Research Database (Denmark)

Fonnes, S; Donatsky, A M; Gögenur, I

2015-01-01

AIM: Experimental studies have shown that some circadian core clock genes may act as tumour suppressors and have an important role in the response to oncological treatment. This study investigated the evidence regarding modified expression of core clock genes in colorectal cancer and its...... expression of colorectal cancer cells compared with healthy mucosa cells from specimens analysed by real-time or quantitative real-time polymer chain reaction. The expression of the core clock genes Period, Cryptochrome, Bmal1 and Clock in colorectal tumours were compared with healthy mucosa and correlated...... with clinicopathological features and survival. RESULTS: Seventy-four articles were identified and 11 studies were included. Overall, gene expression of Period was significantly decreased in colorectal cancer cells compared with healthy mucosa cells. This tendency was also seen in the gene expression of Clock. Other core...

Integrative analysis of genome-wide gene copy number changes and gene expression in non-small cell lung cancer.

Directory of Open Access Journals (Sweden)

Verena Jabs

Full Text Available Non-small cell lung cancer (NSCLC represents a genomically unstable cancer type with extensive copy number aberrations. The relationship of gene copy number alterations and subsequent mRNA levels has only fragmentarily been described. The aim of this study was to conduct a genome-wide analysis of gene copy number gains and corresponding gene expression levels in a clinically well annotated NSCLC patient cohort (n = 190 and their association with survival. While more than half of all analyzed gene copy number-gene expression pairs showed statistically significant correlations (10,296 of 18,756 genes, high correlations, with a correlation coefficient >0.7, were obtained only in a subset of 301 genes (1.6%, including KRAS, EGFR and MDM2. Higher correlation coefficients were associated with higher copy number and expression levels. Strong correlations were frequently based on few tumors with high copy number gains and correspondingly increased mRNA expression. Among the highly correlating genes, GO groups associated with posttranslational protein modifications were particularly frequent, including ubiquitination and neddylation. In a meta-analysis including 1,779 patients we found that survival associated genes were overrepresented among highly correlating genes (61 of the 301 highly correlating genes, FDR adjusted p<0.05. Among them are the chaperone CCT2, the core complex protein NUP107 and the ubiquitination and neddylation associated protein CAND1. In conclusion, in a comprehensive analysis we described a distinct set of highly correlating genes. These genes were found to be overrepresented among survival-associated genes based on gene expression in a large collection of publicly available datasets.