Environmental Regulation of Cellular Responses to DNA Damage

总结 该项目的首要目标是了解DNA损伤反应（DDR）如何激活 细胞凋亡DDR是协调多种细胞功能的激酶驱动的信号传导途径， 包括：监测DNA损伤、招募DNA修复酶和激活细胞周期 检查站逮捕。总的来说，这些事件促进了遗传毒性应激后的生存。或者，在一些实施例中， 例DDR激活细胞凋亡。尽管对这些功能的了解很多， 仍然不清楚为什么复员方案的启动在某些情况下导致生存，而在另一些情况下导致死亡。当前 该模型表明，p53控制着阻止和修复的决定，或者激活细胞死亡。这 模型不能解释常见的观察结果，即癌细胞-通常缺乏p53-可以强烈地 当暴露于DNA损伤时激活细胞凋亡。因此，还必须存在其他不明机制， 促进DDR诱导的细胞死亡。我们确定复员方案启动机制的战略 凋亡是在缺乏p53但保留高水平DNA的细胞中进行功能性遗传筛选 损伤敏感性我们的筛选已经确定了caspase-1的激活和caspase-1相关的 炎性细胞因子信号传导是强烈的DNA损伤诱导的细胞死亡所必需的。这是出乎意料 因为与caspase家族的其他成员不同，caspase-1被认为不参与细胞凋亡， 死亡在这个建议中，我们将使用活细胞显微镜实验来确定具有以下特征的细胞的命运： 激活caspase-1。此外，我们将使用生化和遗传实验来确定机制， caspase-1被DNA损伤激活。最后，我们将使用高通量靶向蛋白质组学和 基因组分析，以及数据驱动的统计建模，以确定caspase- 1信号传导与DDR整合以促进凋亡性细胞死亡。这项研究的主要成果将是 了解炎症和DDR信号之间的细胞内和细胞间串扰 有助于促进细胞凋亡的激活。这些信息可能有助于理解变量对 在组织和人群中观察到的DNA损伤，最终可能会提高我们的能力， 可靠地控制DNA损伤后的生死决定。

SUMMARY The overarching goal of this project is to understand how the DNA Damage Response (DDR) activates apoptotic cell death. The DDR is a kinase driven signaling pathway that coordinates multiple cellular functions, including: surveillance for DNA damage, recruitment of DNA repair enzymes, and activation of cell cycle checkpoint arrest. Collectively, these events promote survival following genotoxic stress. Alternatively, in some cases the DDR activates apoptosis. Although much is known about these each of these functions, it still remains unclear why activation of the DDR leads to survival in some cases and death in others. The current model suggests that p53 controls the decision to arrest and repair, or alternatively, to activate cell death. This model does not explain the common observation that cancer cells – which often lack p53 – can robustly activate apoptosis when exposed to DNA damage. Thus, other unidentified mechanisms must also exist to facilitate DDR-induced cell death. Our strategy for identifying mechanisms by which the DDR activates apoptosis was to perform functional genetic screens in cells that lack p53 but retain high levels of DNA damage sensitivity. Our screen has identified that activation of caspase-1 and caspase-1 associated inflammatory cytokine signaling is required for robust DNA damage induced cell death. This was unexpected because unlike other members of the caspase family, caspase-1 is not thought to contribute to apoptotic cell death. In this proposal, we will use live cell microscopy experiments to determine the fate of cells that have activated caspase-1. Additionally, we will use biochemical and genetic experiments to determine mechanisms by which caspase-1 is activated by DNA damage. Finally, we will use high-throughput targeted proteomic and genomic analyses, together with data driven statistical modeling, to determine mechanisms by which caspase- 1 signaling is integrated with the DDR to promote apoptotic cell death. A major outcome from this study will be an understanding of how intra-cellular and inter-cellular crosstalk between inflammatory and DDR signaling helps to facilitate activation of apoptosis. This information may help to understand the variable sensitivity to DNA damage that is observed across tissues and across people, and may ultimately improve our ability to reliably control life-death decisions following DNA damage.