Tissue factor/FVIIa activates Bcl-2 and prevents doxorubicin-induced apoptosis in neuroblastoma cells

<p>Abstract</p> <p>Background</p> <p>Tissue factor (TF) is a transmembrane protein that acts as a receptor for activated coagulation factor VII (FVIIa), initiating the coagulation cascade. Recent studies demonstrate that expression of tumor-derived TF also mediates intracellular signaling relevant to tumor growth and apoptosis. Our present study investigates the possible mechanism by which the interaction between TF and FVIIa regulates chemotherapy resistance in neuroblastoma cell lines.</p> <p>Methods</p> <p>Gene and siRNA transfection was used to enforce TF expression in a TF-negative neuroblastoma cell line and to silence endogenous TF expression in a TF-overexpressing neuroblastoma line, respectively. The expression of TF, Bcl-2, STAT5, and Akt as well as the phosphorylation of STAT5 and Akt in gene transfected cells or cells treated with JAK inhibitor and LY294002 were determined by Western blot assay. Tumor cell growth was determined by a clonogenic assay. Cytotoxic and apoptotic effect of doxorubicin on neuroblastoma cell lines was analyzed by WST assay and annexin-V staining (by flow cytometry) respectively.</p> <p>Results</p> <p>Enforced expression of TF in a TF-negative neuroblastoma cell line in the presence of FVIIa induced upregulation of Bcl-2, leading to resistance to doxorubicin. Conversely, inhibition of endogenous TF expression in a TF-overexpressing neuroblastoma cell line using siRNA resulted in down-regulation of Bcl-2 and sensitization to doxorubicin-induced apoptosis. Additionally, neuroblastoma cells expressing high levels of either endogenous or transfected TF treated with FVIIa readily phosphorylated STAT5 and Akt. Using selective pharmacologic inhibitors, we demonstrated that JAK inhibitor I, but not the PI3K inhibitor LY294002, blocked the TF/FVIIa-induced upregulation of Bcl-2.</p> <p>Conclusion</p> <p>This study shows that in neuroblastoma cell lines overexpressed TF ligated with FVIIa produced upregulation of Bcl-2 expression through the JAK/STAT5 signaling pathway, resulting in resistance to apoptosis. We surmise that this TF-FVIIa pathway may contribute, at least in part, to chemotherapy resistance in neuroblastoma.</p

A New Method for Determining the Connection Resistance of the Compression Connector in Cable Joint

The compression connector of a cable joint is one of the major components causing joint overheating. This paper proposed a new model to determine the connection resistance of the compression connector. It innovatively integrated electrical contacts model analysis (ECMA) with finite element analysis (FEA) in the modeling. The compacted stranded structure of the cable conductor was taken into account in the proposed model. The streamline distortion effect on the connection resistance was also established. To verify the applicability of the proposed model, the connection resistances of five compression connectors with different cross sections were measured. The modeling results and measurement results were in close agreement with each other

NFAT5 Restricts Bovine Herpesvirus 1 Productive Infection in MDBK Cell Cultures

ABSTRACT Bovine herpesvirus 1 (BoHV-1), an important bovine viral pathogen, causes severe disease in the upper respiratory tract and reproductive system. Tonicity-responsive enhancer-binding protein (TonEBP), also known as nuclear factor of activated T cells 5 (NFAT5), is a pleiotropic stress protein involved in a range of cellular processes. In this study, we showed that the knockdown of NFAT5 by siRNA increased BoHV-1 productive infection and overexpression of NFAT5 via plasmid transfection decreased virus production in bovine kidney (MDBK) cells. Virus productive infection at later stages significantly increased transcription of NFAT5 but not appreciably alter measurable NFAT5 protein levels. Virus infection relocalized NFAT5 protein and decreased the cytosol accumulation. Importantly, we found a subset of NFAT5 resides in mitochondria, and virus infection led to the depletion of mitochondrial NFAT5. In addition to full-length NFAT5, another two isoforms with distinct molecular weights were exclusively detected in the nucleus, where the accumulation was differentially affected following virus infection. In addition, virus infection differentially altered mRNA levels of PGK1, SMIT, and BGT-1, the canonical downstream targets regulated by NFAT5. Taken together, NFAT5 is a potential host factor that restricts BoHV-1 productive infection, and virus infection hijacks NFAT5 signaling transduction by relocalization of NFAT5 molecules in cytoplasm, nucleus, and mitochondria, as well as altered expression of its downstream targets. IMPORTANCE Accumulating studies have revealed that NFAT5 regulates disease development due to infection of numerous viruses, underlying the importance of the host factor in virus pathogenesis. Here, we report that NFAT5 has capacity to restrict BoHV-1 productive infection in vitro. And virus productive infection at later stages may alter NFAT5 signaling pathway as observed by relocalization of NFAT5 protein, reduced accumulation of NFAT5 in cytosol, and differential expression of NFAT5 downstream targets. Importantly, for the first time, we found that a subset of NFAT5 resides in mitochondria, implying that NFAT5 may regulate mitochondrial functions, which will extend our knowledge on NFAT5 biological activities. Moreover, we found two NFAT5 isoforms with distinct molecular weights were exclusively detected in the nucleus, where the accumulation was differentially affected following virus infection, representing a novel regulation mechanism on NFAT5 function in response to BoHV-1infection

Transfection of the TF gene and its effect on Bcl-2 expression and sensitivity to doxorubicin

, SK-N-SH cells lacking TF expression were transfected with the expression plasmid pcDNA3.1/TF and control plasmid (an empty neo pcDNA3.1 plasmid). Expression of transfected TF was detected by western blot assay. , the effect of interaction between TF and FVIIa on Bcl-2 and Bax expression. SK-N-SH transfected with TF and SH-EP1 expressing endogenous TF were both incubated with 10 nM FVIIa for the indicated time points. Cell extracts were then analyzed for Bcl-2 and Bax expression by western blot. Labels under Bcl-2 bands in the blot represent expression levels after normalization for actin, compared with untreated (0) samples (defined as 1 unit) , effect of enforced TF expression on cell survival of SK-N-SH cells in the presence or absence of FVIIa in response to doxorubicin. Cells transfected with TF and control cells (1 × 10) were treated with or without 10 nM FVIIa and different doses of doxorubicin. Cells were incubated for 48 h, then cell viability was determined by WST assay. , a similar WST assay of dose-dependent inhibition of FVIIa on doxorubicin-induced cell death. SK-N-SH cells transfected with TF were treated with 2 μM doxorubicin in the presence or absence of different concentrations of FVIIa as indicated.<p><b>Copyright information:</b></p><p>Taken from "Tissue factor/FVIIa activates Bcl-2 and prevents doxorubicin-induced apoptosis in neuroblastoma cells"</p><p>http://www.biomedcentral.com/1471-2407/8/69</p><p>BMC Cancer 2008;8():69-69.</p><p>Published online 6 Mar 2008</p><p>PMCID:PMC2275284.</p><p></p

The effects of TF silencing by siRNA on the expression of endogenous TF, Bcl-2 and Bcl-XL as well as cell growth and apoptosis induced by doxorubicin in SH-EP1

, cells cultured with 10 nM FVIIa were treated with different concentrations of either TF siRNA or control siRNA for 24 h. Expression of endogenous TF, Bcl-2 and Bcl-XL was detected by western blot assay. , clonogenic assay of SH-EP1 in culture with TF siRNA. Cells (1 × 10) were cultured in soft agarose in the presence of FVIIa for 2 weeks with different doses of TF siRNA and control siRNA, respectively, and resulting colonies counted. Data for the colony counts is shown (mean ± S.D. for triplicate cultures). western blot assay for activation of caspase-3 and cleavage of the death substrate PARP in SH-EP1 cells that were treated with 2 μM doxorubicin (Dox) and 200 nM of either TF siRNA or control siRNA for the indicated time points. quantitative detection of apoptotic cells induced by Dox when given in combination with either TF siRNA or control siRNA. SH-EP1 cells were treated with Dox (2 μM) plus siRNA (200 nM) for the indicated times, and apoptotic cells were detected by flow cytometry. Data represents the percentage of annexin-V positive cells.<p><b>Copyright information:</b></p><p>Taken from "Tissue factor/FVIIa activates Bcl-2 and prevents doxorubicin-induced apoptosis in neuroblastoma cells"</p><p>http://www.biomedcentral.com/1471-2407/8/69</p><p>BMC Cancer 2008;8():69-69.</p><p>Published online 6 Mar 2008</p><p>PMCID:PMC2275284.</p><p></p