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
Molecular Profiling Laboratory, Massachusetts General Hospital, Boston, Massachusetts, United States Cancer Research
(Impact Factor: 9.33).
11/2006; 66(20):10153-61. DOI: 10.1158/0008-5472.CAN-05-3696
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.
Available from: Ching Wooen Sze
- "As expected, the level of apoptosis induced in the form of caspase-3/7 activation and DNA fragmentation (TUNEL positive cells) was significantly higher in the HCV-infected control cells than in the HCV-infected BIK-depleted cells (Fig. 3B and Fig. S3). Our observation is in accordance with studies from other groups examining the effect of BIK knockdown and apoptosis, where BIKdepleted cells were more resistant to apoptosis induced by various agents (Fu et al., 2007; Hur et al., 2006; Li et al., 2008; Mebratu et al., 2008; Shimazu et al., 2007; Viedma-Rodriguez et al., 2013). In terms of viral induced apoptosis and BIK expression, our observation is in accordance with these studies. "
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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 · 3.32 Impact Factor
Available from: plosone.org
- "Our prior studies revealed the critical importance of BIK (a BH3-only family pro-apoptotic protein) and TP53 (a tumor suppressor transcription factor necessary for transcriptional induction of the BIK mRNA transcripts) in fulvestrant-induced apoptosis of MCF-7 cells , . To obtain further insights into the mechanism of fulvestrant actions, we performed RNAi knockdown screenings to identify additional molecules required for fulvestrant-induced MCF-7 cell apoptosis. "
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ABSTRACT: Fulvestrant is a representative pure antiestrogen and a Selective Estrogen Receptor Down-regulator (SERD). In contrast to the Selective Estrogen Receptor Modulators (SERMs) such as 4-hydroxytamoxifen that bind to estrogen receptor α (ERα) as antagonists or partial agonists, fulvestrant causes proteasomal degradation of ERα protein, shutting down the estrogen signaling to induce proliferation arrest and apoptosis of estrogen-dependent breast cancer cells. We performed genome-wide RNAi knockdown screenings for protein kinases required for fulvestrant-induced apoptosis of the MCF-7 estrogen-dependent human breast caner cells and identified the c-Src tyrosine kinase (CSK), a negative regulator of the oncoprotein c-Src and related protein tyrosine kinases, as one of the necessary molecules. Whereas RNAi knockdown of CSK in MCF-7 cells by shRNA-expressing lentiviruses strongly suppressed fulvestrant-induced cell death, CSK knockdown did not affect cytocidal actions of 4-hydroxytamoxifen or paclitaxel, a chemotherapeutic agent. In the absence of CSK, fulvestrant-induced proteasomal degradation of ERα protein was suppressed in both MCF-7 and T47D estrogen-dependent breast cancer cells whereas the TP53-mutated T47D cells were resistant to the cytocidal action of fulvestrant in the presence or absence of CSK. MCF-7 cell sensitivities to fulvestrant-induced cell death or ERα protein degradation was not affected by small-molecular-weight inhibitors of the tyrosine kinase activity of c-Src, suggesting possible involvement of other signaling molecules in CSK-dependent MCF-7 cell death induced by fulvestrant. Our observations suggest the importance of CSK in the determination of cellular sensitivity to the cytocidal action of fulvestrant.
PLoS ONE 04/2013; 8(4):e60889. DOI:10.1371/journal.pone.0060889 · 3.23 Impact Factor
Available from: sciencedirect.com
- "MCF-7 human breast cancer cells were obtained from the American Type Culture Collection (Rockville, MA, USA) and maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco-BRL, Rockville , IN, USA) with 100 U/ml penicillin, 100 lg/ml streptomycin and 10% fetal bovine serum (FBS, Haoyang Biological Manufacture Co. Ltd., Tianjin, China). The cells were grown as monolayers at 37 °C in a humidified 5% CO 2 incubator with a phenol red-free DMEM medium using 5% fetal bovine serum (HyClone, defined grade, Logan, UT), which supported proliferation of MCF7 cells with an efficacy similar to 30–60 p mol/L E2  . Cells were harvested using 0.5 g/l trypsin ( "
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ABSTRACT: The optimal sequencing for hormonal therapy and radiation are yet to be determined. We utilized fulvestrant, which is showing promise as an alternative to other agents in the clinical setting of hormonal therapy, to assess the cellular effects of concomitant anti-estrogen therapy (fulvestrant) with radiation (F+RT). This study was conducted to assess the effects of fulvestrant alone versus F+RT on hormone-receptor positive breast cancer to determine if any positive or negative combined effects exist. The effects of F+RT on human breast cancer cells were assessed using MCF-7 clonogenic and tetrazolium salt colorimetric (MTT) assays. The assays were irradiated with a dose of 0, 2, 4, 6 Gy +/- fulvestrant. The effects of F+RT vs. single adjuvant treatment alone on cell-cycle distribution were assessed using flow cytometry; relative expression of repair proteins (Ku70, Ku80, DNA-PKcs, Rad51) was assessed using Western Blot analysis. Cell growth for radiation alone vs. F+RT was 0.885±0.013 vs. 0.622±0.029 @2Gy, 0.599±0.045 vs. 0.475±0.054 @4 Gy, and 0.472±0.021 vs. 0.380±0.018 @6 Gy RT (p=0.003). While irradiation alone induced G2/M cell cycle arrest, the combination of F+RT induced cell redistribution in the G1 phase and produced a significant decrease in the proportion of cells in G2 phase arrest and in the S phase in breast cancer cells (p<0.01). Furthermore, levels of repair proteins DNA-PKcs and Rad51 were significantly decreased in the cells treated with F+RT compared with irradiation alone. F+RT leads to a decrease in the surviving fraction, increased cell cycle arrest, downregulating of nonhomologous repair protein DNA-PKcs and homologous recombination repair protein RAD51. Thus, our findings suggest that F+RT increases breast cancer cell radiosensitivity compared with radiation alone. These findings have salient implications for designing clinical trials using fulvestrant and radiation therapy.
Biochemical and Biophysical Research Communications 01/2013; 431(2). DOI:10.1016/j.bbrc.2013.01.006 · 2.30 Impact Factor
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