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中的新作用。BCL-2或BCL-X L的过表达增加了TK6细胞常染色体TK1位点的x射线诱导突变。我们还发现，随着非活性等位基因的重复，杂合性缺失（LOH）突变的频率增加，这表明更多的细胞是通过同源重组修复（HRR）发生突变的。我们的目标是了解由BCL-2家族成员介导的常染色体突变增加的基础。我们提出了四个目的：1)我们将验证在过度表达BCL-2或BCL-X L的TK6细胞中，TK1位点LOH突变水平升高与DNA双链断裂（DSBs）的同源定向修复（HDR）增加有关的假设。我们将使用集成DR-GFP报告器来测量单个位点特异性DSB后的基因转化。我们最近发现BCL-X - L的高表达促进了TK6细胞的HDR。我们计划将这一发现推广到其他BCL-2家族成员和FL5.12细胞，其中BCL-2家族成员对PCD的调节已经得到了很好的研究。2)我们将验证TK6-bclXL细胞中TK1突变频率升高的假设，这是由于BCL-X L具有不同于其抗凋亡功能的新活性。我们将开发表达不能阻断PCD的突变BCL-X L的等基因TK6细胞，以评估x射线诱导的TK1突变是否被调节。3)我们将验证BCL-X L通过在ir后维持较高水平的HsRAD51在TK6细胞中促进x射线诱导的TK1突变的假设。我们将使用表达突变型HsRAD51 （HsRAD51D-A）、野生型HsRAD51或不能促进HRR的HsRAD51切割片段的等基因TK6细胞。如果HsRAD51D-A提高x射线诱导的TK1突变，我们将通过在TK6细胞中共表达这些蛋白来确定它是否与BCL-X - L在相同的途径中起作用。新的策略包括在HsRAD51水平升高是有毒的情况下。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procaspase-9 （cys287ala）的TK6细胞作为显性阴性基因来抑制PCD。阐明BCL-2和BCL-X L在体外介导突变的机制，有助于阐明高水平表达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发展和侵袭。

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.