DNA damage response kinase signaling in non-replicating human cells and tissues

项目总结/摘要 内源性DNA损伤对DNA聚合酶运动的干扰， 环境和化学治疗剂都是人类癌症和衰老的原因， 许多抗癌药物的作用机制。这种基因毒素相关的复制应激也是一种良好的- 公认的ATR（共济失调毛细血管扩张突变和rad 3相关）蛋白激酶激活剂， 在细胞DNA损伤过程中，在调节DNA复制和细胞周期时相转换中起关键作用 响应（DDR）。ATR的小分子抑制剂已经成为改善免疫应答的潜在佐剂。 常见癌症化疗方案的有效性。不幸的是，我们对ATR作用的理解 在DDR中，可能偏向于涉及DNA合成和细胞分裂的细胞过程， 典型地用于研究ATR功能的活跃复制细胞的模型系统。然而，使用非- 复制体外培养的细胞和离体人类皮肤组织外植体，我们的初步数据显示， ATR激活的新模式，与转录应激密切相关，特别是与XPB亚基 多功能蛋白质TFIIH（转录因子II-H）。此外，我们发现， ATR激酶抑制对复制细胞的DNA损伤增敏作用，ATR抑制在非复制细胞中的作用， 相反，复制细胞保护非复制细胞免受几种DNA损伤剂的致死作用。 因此，本提案的目的是更清楚地定义ATR激酶激活的机制， 在非复制型人类细胞中发挥作用，并确定ATR抑制剂是否对 暴露于DNA损伤剂的人体组织的重要细胞群。这个问题的核心假设是 这一建议是，ATR激酶在非复制细胞中的激活机制在细胞内表现出独特的性质， 与复制细胞相比，转录相关的ATR激酶信号传导具有深刻的不同， 对细胞和组织命运的影响。这项拟议研究的理由是，它将 更全面地了解ATR激酶如何影响细胞和组织对DNA的反应 并可能导致使用ATR激酶抑制剂来限制某些DNA的毒性 有害化合物我们的假设将通过以下三个具体目标进行测试：1）定义 ATR激酶在非复制性静止和分化的人类细胞中活化的机制 遗传毒性应激; 2）表征非遗传毒性应激中ATR激酶抑制的积极和消极后果。 体外复制细胞; 3）研究非复制细胞中ATR激酶的激活和功能模式 人和小鼠皮肤组织的离体和体内。我们的方法是创新的，因为它将调查一个 在人类细胞和组织中DNA损伤反应的未探索和生理学相关方面。的 拟议的研究是重要的，因为它将提供新的机制的见解如何调节ATR- 依赖性DNA损伤信号传导可以在人细胞和组织中提供治疗益处。

PROJECT SUMMARY/ABSTRACT The interference of DNA polymerase movement by DNA damage induced by endogenous, environmental, and chemotherapeutic agents is both a cause of cancer and aging in humans and a common mechanism of action of many anti-cancer drugs. This genotoxin-associated replication stress is also a well- recognized activator of the ATR (ataxia telangiectasia-mutated and rad3-related) protein kinase, which plays critical roles in regulating DNA replication and cell cycle phase transitions during the cellular DNA damage response (DDR). Small molecule inhibitors of ATR have emerged as potential adjuvants to improve the effectiveness of common cancer chemotherapy regimens. Unfortunately, our understanding of the role of ATR in the DDR may be biased towards cellular processes involving DNA synthesis and cell division because of the model systems of actively replicating cells that are typically used to study ATR function. However, using non- replicating cultured cells in vitro and human skin tissue explants ex vivo, our preliminary data have revealed a novel mode of ATR activation that is closely linked with transcription stress and specifically with the XPB subunit of the multi-functional protein TFIIH (transcription factor II-H). Moreover, we have found that in striking contrast to the DNA damage-sensitizing effects of ATR kinase inhibition on replicating cells, ATR inhibition in non- replicating cells instead protects non-replicating cells from the lethal effects of several DNA damaging agents. The objective of this proposal is to therefore more clearly define the mechanisms of ATR kinase activation and function in non-replicating human cells and to determine whether ATR inhibitors provide therapeutic benefit to important cell populations of human tissues exposed to DNA damaging agents. The central hypothesis of this proposal is that the mechanism of ATR kinase activation in non-replicating cells exhibits unique properties in comparison to replicating cells and that transcription-associated ATR kinase signaling has profoundly different effects on cell and tissue fate in response to DNA damage. The rationale for this proposed research is that it will provide a more complete understanding of how the ATR kinase impacts cellular and tissue responses to DNA damaging agents in humans and may lead to the use of ATR kinase inhibitors to limit the toxicity of certain DNA damaging compounds. Our hypothesis will be tested by carrying out the following three specific aims: 1) Define the mechanism of ATR kinase activation in non-replicating quiescent and differentiated human cells exposed to genotoxic stress; 2) Characterize the positive and negative consequences of ATR kinase inhibition in non- replicating cells in vitro; and 3) Validate the modes of ATR kinase activation and function in non-replicating cells of human and mouse skin tissue ex vivo and in vivo. Our approach is innovative because it will investigate an unexplored and physiologically relevant aspect of the DNA damage response in human cells and tissues. The proposed research is significant because it will provide novel mechanistic insights into how modulation of ATR- dependent DNA damage signaling may provide therapeutic benefits in human cells and tissues.