Mechanisms of Carcinogenesis in BRCA2 Mutant Cells

BRCA2突变细胞的致癌机制

• 批准号: 6399060
• 负责人: Jeffrey T Holt
• 金额: $ 30.15万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2002-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6399060
• 关键词: DNA repair; antisense nucleic acid; athymic mouse; brca gene; breast neoplasms; carcinogenesis; clinical research; cysteine endopeptidases; enzyme inhibitors; fibroblasts; gene mutation; human subject; oligonucleotides; p53 gene /protein; radiation sensitivity; tumor suppressor proteins

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.

BRCA 2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，我们对这一提议进行了显著修改，以研究小鼠BRCA 2缺陷细胞（BRCA 2-）而不是人类BRCA 2缺陷万斯细胞，因为小鼠细胞中的遗传缺陷更简单。我们在小鼠BRCA 2--细胞中的初步数据显示，在BRCA 2缺陷细胞中，Rad 51被caspase-3快速切割，并且BRCA 2或Rad 51的过表达或caspase-3的抑制逆转放射性和DNA修复缺陷。抗半胱天冬酶-3的Rad 51突变体（D-A187 Rad 51）逆转放射敏感性和brca 2中的DNA修复--当两种蛋白质均以接近生理水平表达时，其逆转程度大于野生型Rad 51。我们最近纯化了Rad 51和caspase-3蛋白，以进行体外机制研究。我们目前的新数据显示，Rad 51主要是在brca 2细胞质-，野生型BRCA 2表达重新定位Rad 51的细胞核，而突变体BRCA 2表达不影响Rad 51的本地化。这些发现导致了以下假设：1。BRCA 2通常在双链DNA断裂修复中发挥作用; 2. BRCA 2和Rda 51在介导DNA修复的相同生化途径中起作用; 3. BRCA 2通过抑制caspase-3和其他蛋白酶的降解来稳定Rad 51; 4. BRCA 2影响Rad 51的核定位。为了检验假设，我们提出了这些目标。目的1：确定BRCA 2蛋白介导双链断裂修复和肿瘤发生的功能域。目的2：了解BRCA 2缺陷癌细胞中已知的其他突变是否有助于BRCA 2-细胞中的辐射敏感性和DNA修复缺陷。目的3：通过体外试验确定BRCA 2-Rad 51相互作用的机制和体内意义。目的4：分析BRCA 2-Rad 51的定位机制。目的5：开发和使用体内成像方法来研究Rad 51在活细胞中的定位和DNA修复，在真实的时间。