p53 in Cellular Response to ROS-Mediated DNA Damage

描述（由申请人提供）：肿瘤抑制因子p53在调节基因表达、细胞周期进程和响应DNA损伤的细胞凋亡中发挥重要作用。我们对 p53 在修复药物引起的 DNA 损伤中的作用的研究得到了 NIH R29 资助的支持，取得了一些重要的发现。我们证明，p53 的 3'- 5' 核酸外切酶活性优先去除 DNA 中的错配核苷酸，增强体外 DNA 复制保真度，抑制全细胞中的错配突变，并可能作为药物诱导的 DNA 损伤的传感器组件。重要的是，我们观察到 p53 被抗癌药物激活，导致活性氧 (ROS) 积累，与修复酶 APE/Ref-1 相互作用，结合 ROS 损伤的 DNA，并引发细胞凋亡。 p53 的激活受到线粒体呼吸活动的显着影响。这些观察结果，加上线粒体在 ROS 生成和氧化还原调节中发挥重要作用的事实，表明线粒体呼吸和 p53 激活在感知 ROS 介导的 DNA 损伤并导致细胞死亡方面存在逻辑联系。该研究项目的长期目标是研究 p53 和线粒体在细胞对 ROS 介导的 DNA 损伤和药物诱导的细胞凋亡的反应中的作用，并评估它们在癌症治疗中的相关性。我们将使用生化和分子生物学方法来研究以下具体目标：（1）检验以下假设：p53 作为 ROS 损伤的 DNA 传感器复合物的一个组成部分，在与损伤的 DNA 相互作用时被激活，并在 DNA 损伤持续存在时触发细胞凋亡。使用分离的蛋白质成分和含有确定的氧化损伤的 DNA 的体外测定将用于测试 p53 和碱基切除修复分子（例如 8-氧代鸟嘌呤糖基化酶和 AP 核酸内切酶）之间的物理和功能相互作用。 (2) 表征细胞对 ROS 介导的 DNA 损伤做出反应期间线粒体呼吸对 p53 激活的新作用，并研究其潜在机制。我们的实验室已建立了具有各种 p53 基因型和呼吸缺陷的遗传改变线粒体的细胞系，作为这些研究的独特工具。 (3) 评估 53 激活响应 ROS 和相关抗癌药物引起的氧化 DNA 损伤的生物学后果。将测试同基因 p53 细胞系对 ROS 生成剂的不同敏感性。预计拟议的研究将进一步了解 p53 影响细胞对氧化 DNA 损伤的反应以及对产生 ROS 的抗癌药物的敏感性的机制。

DESCRIPTION (provided by applicant): The tumor suppressor p53 plays important roles in regulating gene expression, cell cycle progression, and apoptosis in response to DNA damage. Our studies on the role of p53 in repair of drug-induced DNA damage, supported by a NIH R29 grant, have led to several important findings. We demonstrated that the 3'- 5' exonuclease activity of p53 preferentially removed mismatched nucleotides from DNA and enhanced DNA replication fidelity in vitro, suppressed mismatch mutations in whole cells, and may function as a sensor component for drug-induced DNA damage. Importantly, we observed that p53 was activated by anticancer agents that cause accumulation of reactive oxygen species (ROS), interacted with a repair enzyme APE/Ref-1 in binding to ROS-damaged DNA, and triggered apoptosis. This p53 activation was significantly affected by the mitochondrial respiratory activity. These observations, together with the facts that mitochondria play a major role in ROS generation and redox regulation, suggest a logical link between the mitochondrial respiration and p53 activation in sensing ROS-mediated DNA damage and causing cell death. The long-term goals of this research project are to investigate the roles of p53 and mitochondria in cellular response to ROS-mediated DNA damage and drug-induced apoptosis, and to evaluate their relevance in cancer therapeutics. We will use biochemical and molecular biology methods to investigate the following specific aims: (1) Test the hypothesis that p53 functions as a component of a sensor complex for ROS-damaged DNA, is activated during interaction with the damaged DNA, and trigger apoptosis when DNA damage is persistent. In vitro assays using isolated protein components and DNA containing defined oxidative damage will be employed to test the physical and functional interactions between p53 and base excision repair molecules such as 8- oxoguanine glycosylase and AP endonuclease. (2) Characterize the novel role of mitochondrial respiration on p53 activation during cellular response to ROS-mediated DNA damage, and investigate the underlying mechanisms. Cell lines with various p53 genotypes and with genetically altered mitochondria deficient in respiration have been established in our laboratory as unique tools for these studies. (3) Evaluate the biological consequences of 53 activation in response to oxidative DNA damage caused by ROS and relevant anticancer agents. Isogenic p53 cell lines will be tested for differential sensitivity to ROS-generating agents. It is anticipated that the proposed studies will further our understanding of the mechanisms by which p53 affect cellular response to oxidative DNA damage and sensitivity to anticancer agents that generate ROS.