Mechanisms of Carcinogenesis in BRCA2 Mutant Cells

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The BRCA2 gene is a cancer predisposition gene, which is matured in breast cancer, prostate cancer, ovarian cancer, esophageal cancer , and pancreatic cancer. In response to the critique, we have markedly modified this proposal to study mouse BRCA2 defective cells (BRCA2--) instead of human BRCA2 defective Vance cells, since the genetic defect in the mouse cells is simpler. Our preliminary data in mouse BRCA2-- cells shows that Rad51 is rapidly cleaved by caspase-3 in BRCA2 defective cells and that overexpression of BRCA2 or Rad51, or inhibition of caspase-3 reverse radioactivity and the DNA repair defect. A caspase-3 resistant Rad51 mutant (D- A187Rad51) reverses radio sensitivity and the DNA repair in brca2-- to a greater extent than wildtype Rad51 when both proteins are expressed at near physiologic levels. We have recently purified Rad51 and caspase-3 proteins to allow in vitro mechanistic studies. We present new data showing Rad51 is mostly cytoplasmic in brca2--, and that wildtype BRCA2 expression relocates Rad51 to the nucleus whereas mutant BRCA2 expression does not effect Rad51 localization. These fundings lead to the following hypotheses: 1. BRCA2 normally functions in double strand DNA break repair; 2. BRCA2 and Rda51 function in the same biochemical pathway mediating DNA repair; 3. BRCA2 stabilizes Rad51 by inhabiting degradation by caspase-3 and other proteases; 4. BRCA2 influences nuclear localization of Rad51. To test hypothesis we propose these aims. Aim 1: Determine the functional domains of BRCA2 protein which mediate double strand break repair and tumorigenesis. Aim 2: See if known additional mutations in BRCA2 defective cancer cells contribute to radiation sensitivity & DNA repair defects in brca2-- cells. Aim 3: Determine the mechanism and in vivo significance off BRCA2- Rad51 interactions by in vitro assays. Aim 4: Analyze mechanism of BRCA2- Rad51 localization. Aim 5: Develop and use in Vivo imaging methods to study Rad51 localization and DNA repair in living cells, in real time.