BRCA1 modulates sensitivity to 5F-203 by regulating xenobiotic stress-inducible protein levels and EROD activity

Abstract

Purpose : We have investigated the effects of BRCA1 over-expression and knockdown on 5F-203-induced gene expression and cytotoxicity in human breast cancer cells. 5F-203 is a chemotherapeutic prodrug that both induces a p450 enzyme, CYP1A1, and is metabolically activated by CYP1A1. Methods : We used several molecular biological techniques to confirm our findings. BRCA1 regulates sensitivity to 5F-203 by regulating the expression of CYP1A1 mRNA and its EROD activity. XRE-Luc reporter assays, semi-quantitative RT-PCR, Western blot analysis, EROD activity measurements, gene knockdown and MTT cell survival assays were used for this study. Results : Our results show that the ability of 5F-203 treatments to increase CYP1A1 mRNA level and CYP1A1 enzymatic activity (EROD activity) are affected by BRCA1 protein levels. In addition, the ability of 5F-203 treatments to induce proteins, P53 and P53 target genes such as P21, is significantly decreased in BRCA1 knockdown cells, suggesting that BRCA1-related effects could at least partially explain why BRCA1 knockdown increases resistance to 5F-203-mediated cytotoxicity. We also observed altered expression of the two major transcription factors (AhR and ARNT) that affect CYP1A1 expression when BRCA1 protein levels are altered. Conclusion : BRCA1 is an important protein, which affects 5F-203-mediated cytotoxicity. Our findings are potentially clinically significant; they suggest that those patients most likely to respond to this new prodrug will have tumors containing normal amounts of BRCA1. Keywords : BRCA1 – 5F-203 – AhR – CYP1A1 – P53 – EROD activity