p53 in Cellular Response to ROS-Mediated DNA Damage

描述（申请人提供）：肿瘤抑制因子p53在DNA损伤时调控基因表达、细胞周期进程和细胞凋亡中发挥重要作用。在NIH R29资助下，我们对p53在药物诱导DNA损伤修复中的作用进行了研究，并取得了一些重要发现。我们证明了p53的3‘- 5’外切酶活性优先从DNA中去除错配核苷酸，提高了体外DNA复制保真度，抑制了全细胞中的错配突变，并可能作为药物诱导DNA损伤的传感器成分。重要的是，我们观察到p53被引起活性氧（ROS）积累的抗癌药物激活，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。这种p53的激活受到线粒体呼吸活动的显著影响。这些观察结果，以及线粒体在ROS生成和氧化还原调控中发挥主要作用的事实，表明线粒体呼吸和p53激活之间存在逻辑联系，以感知ROS介导的DNA损伤并导致细胞死亡。本研究项目的长期目标是研究p53和线粒体在ros介导的DNA损伤和药物诱导的细胞凋亡中的作用，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)验证p53作为ros损伤DNA传感器复合物的组成部分，在与受损DNA相互作用时被激活，并在DNA损伤持续时触发细胞凋亡的假设。体外实验使用分离的蛋白质成分和含有明确氧化损伤的DNA，将用于测试p53与碱基切除修复分子（如8-氧鸟嘌呤糖基化酶和AP内切酶）之间的物理和功能相互作用。(2)在细胞应答ros介导的DNA损伤过程中，鉴定线粒体呼吸对p53激活的新作用，并探讨其潜在机制。我们的实验室已经建立了具有各种p53基因型和基因改变线粒体呼吸缺陷的细胞系，作为这些研究的独特工具。(3)评估53激活对活性氧和相关抗癌药物引起的DNA氧化损伤的生物学影响。等基因p53细胞系将被测试对ros生成剂的不同敏感性。预计这些研究将进一步加深我们对p53影响细胞对氧化DNA损伤的反应和对产生ROS的抗癌药物的敏感性的机制的理解。

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.