Tumor suppressor p53 transcriptionally regulates Bak expression in pancreatic cancer

4976 Introduction: The threshold for induction of the mitochondrial apoptosis pathway is determined by the relative balance of pro- and anti-apoptotic members of the Bcl-2 family. Underexpression of pro-apoptotic proteins or relative overexpression of anti-apoptotic proteins may result in apoptotic resistance in cancer cells. We have previously shown that Bak, a pro-apoptotic member of the Bcl-2 family, is underexpressed in pancreatic cancer. The mechanisms of Bak gene regulation are not clearly defined. Tumor suppressor p53 has been show to upregulate Bax gene expression through direct transcriptional transactivation. We identified two putative p53 transcriptional sites in the Bak promoter with homology to consensus p53-binding motifs. We hypothesize that p53 regulates the transcription of Bak through binding at these motifs. Methods: The human pancreatic cancer cell lines BxPC-3 (wildtype p53) and MIA-PaCa-2 (mutant p53) were used for all experiments. Bak mRNA and protein levels were determined by RT-PCR and Western blotting, respectively. To demonstrate binding of p53 to the putative sites in the Bak promoter, electromobility shift assays were performed. A Bak promoter/luciferase reporter was also constructed to functionally evaluate Bak gene promoter function. The effect of site-directed mutagenesis of the putative p53 sites was performed and the effect examined in both cell lines. Finally, the effect of restoration of wildtype-p53 in the MIA-PaCa-2 cell line (mutant p53) on Bak transcription was determined. Results: Bak RNA and protein levels were higher in BxPC-3 (wildtype p53) than MIA-PaCa-2 (mutant p53). Gel-shifted products to the putative p53 sites in the Bak promoter was demonstrated in BxPC-3, but not MIA-PaCa-2. Mutation of the putative p53 sites eliminated the gel shift product and reduced promoter activity in the promoter/luciferase construct in BxPC-3, but had no effect in MIA-PaCa-2. Conclusions: Tumor suppressor p53 transcriptionally regulates the pro-apoptotic protein Bak in pancreatic cancer. Loss of functional p53, a common event in pancreatic cancer, may result in suppression of Bak transcription. Therapy targeted at transcriptionally increasing Bak may be a novel method of therapy to increase pro-apoptotic protein content and therefore restore apoptotic sensitivity in pancreatic cancer.