p53 in Cellular Response to ROS-Mediated DNA Damage

p53 在细胞对 ROS 介导的 DNA 损伤的反应中的作用

• 批准号: 6891840
• 负责人: Peng Huang
• 金额: $ 30.24万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6891840
• 关键词: DNA damage; DNA repair; antineoplastics; apoptosis; cell line; cellular respiration; clinical research; confocal scanning microscopy; cytotoxicity; electron transport; enzyme activity; free radical oxygen; functional /structural genomics; gel mobility shift assay; high performance liquid chromatography; immunoprecipitation; mitochondria; neoplasm /cancer pharmacology; oxidative stress; p53 gene /protein; phosphorylation; protein kinase; protein protein interaction; protein structure function; terminal nick end labeling

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.

描述(由申请人提供)：肿瘤抑制基因P53在调节基因表达、细胞周期进展和DNA损伤后的细胞凋亡中发挥重要作用。我们在NIH R29拨款的支持下，对P53在药物诱导的DNA损伤修复中的作用进行了研究，得出了几个重要的发现。我们证明，P53的3‘-5’外切酶活性可以优先去除DNA中的错配核苷酸，提高DNA体外复制的保真度，抑制整个细胞中的错配突变，并可能作为药物诱导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，导致活性氧物种(ROS)的积累，与修复酶APE/Ref-1结合在ROS损伤的DNA上，并引发细胞凋亡。这种P53的激活受到线粒体呼吸活性的显著影响。这些观察，再加上线粒体在ROS的产生和氧化还原调节中发挥主要作用的事实，表明线粒体呼吸和P53激活在感知ROS介导的DNA损伤和导致细胞死亡之间存在逻辑联系。本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。我们将使用生物化学和分子生物学方法来研究以下特定目的：(1)检验假设，即P53作为ROS损伤的DNA传感器复合体的组成部分，在与损伤的DNA相互作用时被激活，并在DNA损伤持续时触发细胞凋亡。使用分离的蛋白质组分和含有明确的氧化损伤的DNA的体外测试将被用来测试P53和碱基切除修复分子如8-氧鸟嘌呤糖基酶和AP核酸内切酶之间的物理和功能相互作用。(2)表征线粒体呼吸在细胞对ROS介导的DNA损伤反应中对P53激活的新作用，并探讨其可能的机制。我们实验室已经建立了具有不同P53基因类型和线粒体呼吸缺陷的基因改变的细胞系，作为这些研究的独特工具。(3)评价ROS及相关抗癌药物引起的DNA氧化损伤对53基因激活的生物学影响。同基因的p53细胞株将被测试对ROS生成剂的差异敏感性。预计这些研究将进一步加深我们对P53影响细胞对DNA氧化损伤的反应以及对产生ROS的抗癌药物的敏感性的机制的理解。