Mechanisms of Carcinogenesis in BRCA2 Mutant Cells

BRCA2基因是一种癌症易感基因，在乳腺癌、前列腺癌、卵巢癌、食道癌和胰腺癌中成熟。作为对这一批评的回应，我们明显修改了这一建议，研究小鼠BRCA2缺陷细胞(BRCA2-)，而不是人BRCA2缺陷Vance细胞，因为小鼠细胞中的遗传缺陷更简单。我们在小鼠BRCA2-细胞中的初步数据表明，在BRCA2缺陷细胞中，RAD51迅速被caspase-3切割，BRCA2或RAD51的过表达或caspase-3的抑制逆转了放射性和DNA修复缺陷。抗caspase-3的RAD51突变体(D-A187Rad51)在BRCA2中逆转辐射敏感性和DNA修复--当这两种蛋白都在接近生理水平表达时，其程度比野生型RAD51更大。我们最近已经纯化了RAD51和caspase-3蛋白，以便进行体外机制研究。我们提供的新数据表明，RAD51在BRCA2-中主要是细胞质的，野生型BRCA2的表达将RAD51重新定位到细胞核，而突变型BRCA2的表达不影响RAD51的定位。这些发现导致了以下假设：1.BRCA2在双链DNA断裂修复中正常发挥作用；2.BRCA2和Rda51在同一生化途径中介导DNA修复；3.BRCA2通过抑制caspase-3和其他蛋白酶的降解来稳定RAD51；4.BRCA2影响RAD51的核定位。为了检验假设，我们提出了这些目标。目的1：确定BRCA2蛋白介导双链断裂修复和肿瘤发生的功能结构域。目的2：了解BRCA2缺陷癌细胞中已知的额外突变是否会导致BRCA2细胞的辐射敏感性和DNA修复缺陷。目的：通过体外实验确定BRCA2-RAD51相互作用的机制及其体内意义。目的：分析BRCA2-RAD51基因定位的机制。目的5：建立和应用活体成像方法，实时研究RAD51在活细胞中的定位和DNA修复。

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.