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

非复制人类细胞和组织中的 DNA 损伤反应激酶信号传导

• 批准号: 10091482
• 负责人: Michael George Kemp
• 金额: $ 29.6万
• 依托单位: WRIGHT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10091482
• 关键词: ATM function; ATR gene; Adjuvant; Aging; Antineoplastic Agents; Biological Models; Cancer Etiology; Cell Cycle; Cell Cycle Progression; Cell Death; Cell Differentiation process; Cell Survival; Cell division; Cell physiology; Cells; Chemotherapy-Oncologic Procedure; Cultured Cells; DNA; DNA Damage; DNA biosynthesis; DNA lesion; DNA replication fork; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Data; Disease susceptibility; ERCC3 gene; Effectiveness; Epidermis; Eukaryota; Event; Exhibits; Exposure to; Foundations; Genetic Transcription; Genotoxic Stress; Goals; Health; Holoenzymes; Homeostasis; Human; Human body; In Vitro; KRP protein; Lead; Life; Link; Malignant Neoplasms; Mission; Mitotic; Modeling; Movement; Mus; Mutagenesis; Mutagens; Organ; Outcome; Pathway interactions; Phase; Phase Transition; Phosphotransferases; Physiological; Play; Population; Prevention; Process; Property; Protein Kinase; Proteins; Public Health; Publishing; RNA Polymerase II; Research; Risk; Role; Signal Transduction; Skin; Skin Tissue; Source; Stress; Testing; Therapeutic; Tissues; Toxic effect; Ultraviolet B Radiation; United States National Institutes of Health; Work; ataxia telangiectasia mutated protein; base; biological adaptation to stress; cancer therapy; carcinogenesis; cell type; chemotherapeutic agent; environmental agent; genotoxicity; homologous recombination; human tissue; improved; in vivo; inhibitor/antagonist; innovation; insight; irradiation; kinase inhibitor; novel; programs; replication stress; response; small molecule inhibitor; stem cell differentiation; stressor; transcription factor S-II; transcription factor TFIIH

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.

项目摘要/摘要 内源性DNA损伤对DNA聚合酶运动的干扰 环境和化疗药物既是人类癌症和衰老的原因，也是一种常见的 多种抗癌药物的作用机制。这种与遗传毒素相关的复制压力也是一种 公认的ATR(共济失调毛细血管扩张突变和RAD3相关)蛋白激酶的激活物，它发挥着 细胞DNA损伤过程中调控DNA复制和细胞周期相变的关键作用 回应(DDR)。ATR的小分子抑制剂已成为改善血管紧张素转换酶的潜在佐剂 常见癌症化疗方案的疗效分析。不幸的是，我们对ATR的作用的理解 可能偏向于涉及DNA合成和细胞分裂的细胞过程，因为 活跃复制细胞的模型系统，通常用于研究ATR功能。但是，使用非 在体外复制培养细胞和体外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亚单位 多功能蛋白TFIIH(转录因子II-H)。此外，我们发现，与之形成鲜明对比的是 对于ATR激酶抑制对复制细胞的DNA损伤增敏作用，ATR抑制在非 相反，复制细胞可以保护非复制细胞免受几种DNA损伤剂的致命影响。 因此，这项建议的目的是更清楚地定义ATR激酶激活和 在非复制的人类细胞中的功能，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。这一点的中心假设是 提出的观点是，在非复制细胞中ATR激酶激活的机制在 与复制细胞相比，转录相关的ATR激酶信号有很大的不同 DNA损伤对细胞和组织命运的影响。这项拟议的研究的基本原理是它将 更全面地了解ATR激酶如何影响细胞和组织对DNA的反应 对人体有害，并可能导致使用ATR激酶抑制剂来限制某些DNA的毒性 具有破坏性的化合物。我们的假设将通过实现以下三个具体目标来验证：1)定义 暴露于非复制型静止和分化的人细胞中ATR激酶激活的机制 基因毒性应激；2)表征ATR激酶抑制在非 在体外复制细胞；以及3)验证非复制细胞中ATR激酶的激活和功能模式 人和小鼠皮肤组织的体外和体内实验。我们的方法是创新的，因为它将调查 人类细胞和组织中DNA损伤反应的未知和生理相关方面。这个 所提出的研究具有重要意义，因为它将为ATR如何调制提供新的机制见解。 依赖的DNA损伤信号可能在人类细胞和组织中提供治疗益处。