Regulation of expression of BIK proapoptotic protein in human breast cancer cells: p53-dependent induction of BIK mRNA by fulvestrant and proteasomal degradation of BIK protein
ABSTRACT Induction of mRNA for BIK proapoptotic protein by doxorubicin or gamma-irradiation requires the DNA-binding transcription factor activity of p53. In MCF7 cells, pure antiestrogen fulvestrant also induces BIK mRNA and apoptosis. Here, we provide evidence that, in contrast to doxorubicin or gamma-irradiation, fulvestrant induction of BIK mRNA is not a direct effect of the transcriptional activity of p53, although p53 is necessary for this induction. It is known that p53 up-regulated modulator of apoptosis (PUMA) mRNA is induced directly by the transcriptional activity of p53. Whereas gamma-irradiation induced both BIK and PUMA mRNA, only BIK mRNA was induced by fulvestrant. Whereas both fulvestrant and doxorubicin induced BIK mRNA, only doxorubicin enhanced the DNA-binding activity of p53 and induced PUMA mRNA. Small interfering RNA (siRNA) suppression of p53 expression as well as overexpression of dominant-negative p53 effectively inhibited the fulvestrant induction of BIK mRNA, protein, and apoptosis. Transcriptional activity of a 2-kb BIK promoter, which contained an incomplete p53-binding sequence, was not affected by fulvestrant when tested by reporter assay. Fulvestrant neither affected the stability of the BIK mRNA transcripts. Interestingly, other human breast cancer cells, such as ZR75-1, constitutively expressed BIK mRNA even without fulvestrant. In these cells, however, BIK protein seemed to be rapidly degraded by proteasome, and siRNA suppression of BIK in ZR75-1 cells inhibited apoptosis induced by MG132 proteasome inhibitor. These results suggest that expression of BIK in human breast cancer cells is regulated at the mRNA level by a mechanism involving a nontranscriptional activity of p53 and by proteasomal degradation of BIK protein.
SourceAvailable from: Senthilvelan Manohar[Show abstract] [Hide abstract]
ABSTRACT: The hippocampus, which is critical for memory and spatial navigation, contains a proliferating stem cell niche that is especially vulnerable to antineoplastic drugs such as cisplatin. Although the damaging effects of cisplatin have recently been recognized, the molecular mechanisms underlying its toxic effects on this vital region are largely unknown. Using a focused apoptosis gene array, we analyzed the early cisplatin-induced changes in gene expression in the hippocampus of adult Sprague-Dawley rats and compared the results to those from the inferior colliculus, a non-mitotic auditory region resistant to cisplatin-induced cell death. Two days after a 12 mg/kg dose of cisplatin, significant increases were observed in five proapoptotic genes: Bik, Bid, Bok, Trp53p2, and Card6 and a significant decrease in one antiapoptotic gene Bcl2a1. In contrast, Nol3, an antiapoptotic gene, showed a significant increase in expression. The cisplatin-induced increase in Bid mRNA and decrease in Bcl2a1 mRNA were accompanied by a corresponding increase and decrease of their respective proteins in the hippocampus. In contrast, the cisplatin-induced changes in Bcl2a1, Bid, Bik, and Bok gene expression in the inferior colliculus were strikingly different from those in the hippocampus consistent with the greater susceptibility of the hippocampus to cisplatin toxicity. Cisplatin also significantly reduced immunolabeling of the cell proliferation marker Ki67 in the subgranular zone of the hippocampus 2 days post-treatment. These results indicate that cisplatin-induced hippocampal cell death is mediated by increased expression of proapoptotic and decreased antiapoptotic genes and proteins that likely inhibit hippocampal cell proliferation.Neurotoxicity Research 11/2013; 25(4). DOI:10.1007/s12640-013-9443-y · 3.15 Impact Factor
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ABSTRACT: Hepatitis C virus (HCV) induces cytopathic effects in the form of hepatocytes apoptosis thought to be resulted from the interaction between viral proteins and host factors. Using pathway specific PCR array, we identified 9 apoptosis-related genes that are dysregulated during HCV infection, of which the BH3-only pro-apoptotic Bcl-2 family protein, BIK, was consistently up-regulated at the mRNA and protein levels. Depletion of BIK protected host cells from HCV-induced caspase-3/7 activation but not the inhibitory effect of HCV on cell viability. Furthermore, viral RNA replication and release were significantly suppressed in BIK-depleted cells and over-expression of the RNA-dependent RNA polymerase, NS5B, was able to induce BIK expression. Immunofluorescence and co-immunoprecipitation assays showed co-localization and interaction of BIK and NS5B, suggesting that BIK may be interacting with the HCV replication complex through NS5B. These results imply that BIK is essential for HCV replication and that NS5B is able to induce BIK expression.Virology 11/2014; 474. DOI:10.1016/j.virol.2014.10.027 · 3.28 Impact Factor
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ABSTRACT: The Epstein-Barr virus (EBV) establishes a lifelong latent infection in humans. EBV infection of primary B-cells causes cell activation and proliferation, a process driven by the viral latency III gene expression program which includes EBV nuclear proteins (EBNAs), latent membrane proteins and untranslated RNAs including microRNAs. Some latently infected cells enter the long-lived memory B-cell compartment and express only EBNA1 transiently (Lat I) or no EBV protein at all (Lat 0). Targeting the molecular machinery that controls B-cell fate decisions, including the Bcl-2 family of apoptosis-regulating proteins, is crucial to the EBV cycle of infection. Here, we show that BIK (also known as NBK), which encodes a pro-apoptotic 'sensitiser' protein, is repressed by the EBNA2-driven Lat III program but not the Lat I program. BIK repression occurred soon after infection of primary B-cells by EBV but not by a recombinant EBV in which the EBNA2 gene had been knocked out. Ectopic BIK induced apoptosis in Lat III cells by a mechanism dependent on its BH3 domain and the activation of caspases. We show that EBNA2 represses BIK in EBV-negative B lymphoma-derived cell lines and that this host-virus interaction can inhibit the pro-apoptotic effect of TGF-β1, a key physiological mediator of B-cell homeostasis. Reduced levels of TGF-β1-associated regulatory SMAD proteins were bound to the BIK promoter in response to EBV Lat III or ectopic EBNA2. This data is evidence of an additional mechanism used by EBV to promote B-cell survival, namely the transcriptional repression of the BH3-only sensitiser BIK. Over 90% of adult humans are infected with the Epstein-Barr virus (EBV). EBV establishes a lifelong silent infection, its DNA residing in small numbers of blood B-cells that are a reservoir from which low-level virus re-activation and shedding in saliva intermittently occurs. Importantly, EBV DNA is found in some B-cell derived tumors in which viral genes play a key role in tumor cell emergence and progression. Here, we report for the first time that EBV can shut-off a B-cell gene called BIK. When activated by a molecular signal called TGF-β1, BIK plays an important role in killing unwanted B-cells, including those infected by viruses. We describe the key EBV-B-cell molecular interactions that lead to BIK shut-off. These findings further our knowledge of how EBV prevents the death of its host cell during infection. They are also relevant to certain post-transplant lymphomas where unregulated cell growth is caused by EBV genes.Journal of Virology 02/2014; DOI:10.1128/JVI.03642-13 · 4.65 Impact Factor