Radiation-Induced Mutagenesis and Apoptotic Regulation

描述（由申请方提供）：程序性细胞死亡（PCD）是消除外来或潜在危险细胞的基本过程。许多人类肿瘤已经找到了逃避PCD的方法。我们发现了PCD在限制人类细胞诱变中的新作用。BCL-2或BCL-XL的过表达增加了X射线诱导的TK 6细胞中常染色体TK 1位点的突变。我们还看到杂合性缺失（洛）突变频率增加，同时重复失活等位基因-这表明更多的细胞通过同源重组修复（HRR）发生突变。我们的目标是了解BCL-2家族成员介导的常染色体诱变剂增加的基础。提出了四个目标：1）我们将检验这样的假设：过表达BCL-2或BCL-XL的TK 6细胞中TK 1基因座的洛杂合性缺失突变水平升高与DNA双链断裂（DSB）的同源定向修复（HDR）增加有关。我们将使用整合的DR-GFP报告基因来测量单个位点特异性DSB后的基因转换。我们最近发现，高BCL-XL表达促进TK 6细胞中的HDR。我们计划将这一发现推广到其他BCL-2家族成员和FL 5.12细胞，其中BCL-2家族成员对PCD的调控已经得到了很好的研究。2)我们将测试TK 6-bclXL细胞中TK 1突变频率升高是由BCL-XL不同于其抗凋亡功能的新活性引起的假设。我们将开发表达不能阻断PCD的突变BCL-XL的同基因TK 6细胞，以评估X射线诱导的TK 1诱变是否受到调节。3)我们将检验BCL-XL通过在IR后的TK 6细胞中维持较高水平的HsRAD 51来促进X射线诱导的TK 1突变的假设。我们将使用表达突变体HsRAD 51（HsRAD 51 D-A）、野生型HsRAD 51或不能促进HRR的HsRAD 51的切割片段的同基因TK 6细胞，所述突变体对促进HRR的半胱天冬酶切割不敏感。如果HsRAD 51 D-A增强X射线诱导的TK 1诱变，我们将通过在TK 6细胞中共表达这些蛋白质来确定它是否在与BCL-XL相同的途径中起作用。新的策略包括在HsRAD 51水平升高是有毒的情况下。4)我们将检验PCD抑制本身可以增强X射线诱导的TK 1诱变的假设。我们将使用TK 6细胞表达突变体半胱氨酸天冬氨酸蛋白酶原-9（cys 287 ala），其作为显性阴性抑制PCD。阐明BCL-2和BCL-XL介导体外诱变的机制应阐明表达高水平BCL-2的B细胞滤泡性淋巴瘤的突变机制。正是继发性突变导致了人类疾病的进展和侵袭性。

DESCRIPTION (provided by applicant): Programmed cell death (PCD) is a fundamental process that eliminates extraneous or potentially dangerous cells. Many human tumors have found ways to evade PCD. We discovered a novel role for PCD in limiting mutagenesis in human cells. Over-expression of BCL-2 or BCL-X L increased x-ray-induced mutation at the autosomal TK1 locus in TK6 cells. We also saw an increased frequency of loss of heterozygosity (LOH) mutations with duplications of the inactive allele - suggesting that more cells were mutated via homologous recombinational repair (HRR). Our goal is to understand the basis for increased autosomal mutagenes:s mediated by BCL-2 family members. Four aims are proposed: 1) We will test the hypothesis that the elevated level of LOH mutations at the TK1 locus in TK6 cells that over-express BCL-2 or BCL-X L is associated with an increase in homology directed repair (HDR) of DNA double-strand breaks (DSBs). We will use an integrated DR-GFP reporter to measure gene conversion following a single, site-specific DSB. We recently showed that high BCL-X L expression promotes HDR in TK6 cells. We plan to generalize this finding to other BCL-2 family members and to FL5.12 cells in which PCD regulation by BCL-2 family members has been well studied. 2) We will test the hypothesis that the elevated frequencies of TK1 mutations in TK6-bclXL cells result from a novel activity of BCL-X L distinct from its anti-apoptotic function. We will develop isogenic TK6 cells that express mutated BCL-X L that can't block PCD to assess if x-ray-induced TK1 mutagenesis is modulated. 3) We will test the hypothesis that BCL-X L promotes x-ray-induced TK1 mutations by maintaining higher levels of HsRAD51 in TK6 cells post-IR. We will use isogenic TK6 cells that express a mutant HsRAD51 (HsRAD51D-A)'insensitive to caspase cleavage that promotes HRR, wildtype HsRAD51, or a cleavage fragment of HsRAD51 that can't promote HRR. If HsRAD51D-A elevates x-ray- induced TK1 mutagenesis, we will determine if it acts in the same pathway as BCL-X L by co-expressing these proteins in TK6 cells. New strategies are included in the event that an elevated level of HsRAD51 is toxic. 4) We will test the hypothesis that PCD suppression per se can enhance x-ray-induced TK1 mutagenesis. We will use TK6 cells expressing mutant procaspase-9 (cys287ala) that acts as a dominant negative to suppress PCD. Elucidating the mechanisms through which BCL-2 and BCL-X L mediate mutagenesis in vitro should illuminate mechanisms of mutation in B-cell follicular lymphomas that express high levels of BCL-2. It is the secondary mutations that lead to advance, aggressive disease in people.