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 ;-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 ;-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 ;-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. (Cancer Res 2006; 66(20): 10153-61)

Antiestrogen-resistant subclones of MCF-7 human breast cancer cells are derived from a common monoclonal drug-resistant progenitor

“…tic protein (4,5,13,14). In all AE-sensitive sublines and in the polyclonal MCF-7 cells, Fv strongly induced BIK expression and suppressed ER␣ expression ( Fig.…”

“…ER␣ and BIK protein expression was determined by Western blotting (4,5). Determination of MCF-7 transcriptome using Affymetrix U133-plus 2.0 microarray and 2D hierarchical clustering were performed as described in our previous studies (13,14).…”

“…1E and S3). Because the cytocidal Fv action requires a longer period of drug exposure (4,5), cell viability was intact when cell cycle analysis was performed. The lack of correlation between the cytostatic and cytocidal effects of Fv suggests independent mechanisms for these 2 Fv effects.…”

“…Because proliferation and survival of MCF-7 cells in culture or as xenograft are strictly dependent on estrogen (3)(4)(5), this cell line has been widely used to study mechanisms of ET resistance. Reflecting the extensive intratumoral heterogeneity of malignant epithelial cells in human breast cancer (6), MCF-7 is a polyclonal cell line consisting of highly heterogeneous cancer cells that differ remarkably in their phenotypic and cytogenetic characteristics (7,8).…”

Gene Expression Profiling of Pulmonary Fibrosis Identifies Twist1 as an Antiapoptotic Molecular “Rectifier” of Growth Factor Signaling

“…This may have been because Bik was below the limit of detection of our antibodies or rapidly degraded by the proteasome. 43 Several potential Twist1 targets have been shown to up-regulate Bim and PUMA, including c-Jun N-terminal kinase, 44 FoxO, 45,46 and p53. 47,48 In preliminary experiments, we were unable to detect c-Jun Nterminal kinase activation following Twist1 knockdown, and we did not find evidence of increased accumulation of nuclear FoxO proteins or of the unphosphorylated transcriptionally active forms (data not shown).…”