Radiation-Induced Mutagenesis and Apoptotic Regulation

描述(由申请人提供)：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在限制人类细胞突变方面的新作用。过表达bcl2或bclX基因的L增加了X射线诱发的TK6细胞常染色体TK1基因突变。我们还看到杂合性缺失(LOH)突变的频率增加，伴随着非活性等位基因的复制--这表明更多的细胞通过同源重组修复(HRR)发生突变。我们的目标是了解常染色体突变增加的基础：由bcl2家族成员介导的S。提出四个目标：1)我们将检验这样一种假设，即在过表达bcl2或bclX L的TK6细胞中，TK1基因座LOH突变水平的升高与DNA双链断裂的同源定向修复(HDR)增加有关。我们将使用集成的DR-GFP报告程序来测量单个位点特定的DSB之后的基因转换。我们最近发现在TK6细胞中高表达bc1-X L促进hdr。我们计划将这一发现推广到其他bcl2家族成员和已经很好地研究了bcl2家族成员对PCD调控的FL5.12细胞。2)我们将验证这样一种假设，即Tk6-bclXL细胞中Tk1突变的频率升高是由于bclx L的一种不同于其抗凋亡功能的新活性所致。我们将建立表达不能阻断PCD的突变的bclX L的等基因TK6细胞，以评估X射线诱导的TK1突变是否受到调控。3)我们将通过维持IR后TK6细胞中较高水平的HsRAD51来验证BCL-X L促进X射线诱导的TK1突变的假说。我们将使用表达突变的HsRAD51(HsRAD51D-A)的等基因TK6细胞，该突变的HsRAD51(HsRAD51D-A)对促进HRR的caspase切割不敏感，野生型HsRAD51或不能促进HRR的HsRAD51的切割片段。如果HsRAD51D-A提高了X射线对TK1的诱变作用，我们将通过在TK6细胞中共表达这些蛋白来确定它是否与bc1-X L具有相同的作用途径。在HsRAD51水平升高是有毒的情况下，包括了新的策略。4)我们将验证PCD抑制本身可以增强X射线诱导的TK1突变的假设。我们将使用表达突变的proaspase-9(Cys287ala)的TK6细胞作为显性阴性来抑制PCD。阐明bcl2和bclX L在体外介导突变的机制将有助于阐明高表达bcl2的B细胞滤泡性淋巴瘤的突变机制。正是这种次级突变导致了人类的进展性、侵袭性疾病。

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.