Oxidative Stress, DNA Damage, and Cellular Aging

描述（由申请人提供）：我们研究的长期目标是了解人类口腔上皮细胞衰老和永生的分子机制。细胞衰老是一种不可逆的细胞分裂停滞，在完成有限数量的细胞分裂后导致正常体细胞的产生。人们提出了几种关于衰老的理论，包括DNA损伤理论、线粒体理论和端粒假说。这些衰老理论要么关注(1)外在环境因素，要么关注(2)细胞衰老的内在遗传程序。在外在因素中，氧化应激对细胞和亚细胞大分子的影响最大，因为它会产生活性氧（ROS）。相反，端粒假说维持了端粒缩短在细胞衰老中的重要作用。两种衰老机制的理论都在一定程度上得到了实验验证，尽管在某些细胞系统中确实存在例外。为了确定正常人口腔角质形成细胞（NHOK）的衰老模式，我们建立了衰老前细胞经历22+/- 3次细胞分裂的体外衰老模型。与成纤维细胞不同，复制的NHOK表现出端粒酶活性并保持端粒长度不变，表明这些细胞的衰老模式与端粒长度无关。在我们最近的研究中，我们还发现NHOK的衰老增加了线粒体蛋白的表达，增加了线粒体质量，这反映了细胞的氧化应激反应。同时，DNA修复基因的表达水平在衰老NHOK中下调。因此，我们的数据表明NHOK中氧化应激、DNA损伤和衰老之间存在联系。当前提案的中心假设是：NHOK的衰老是由氧化应激积累和细胞DNA修复能力受损导致的DNA损伤积累引发的。为了验证这一假设，我们提出：(1)确定NHOK在氧化应激下的表型和分子变化；(2)研究NHOK在体外生命周期内存在或不存在氧化应激时DNA修复能力的变化；(3)确定破坏DNA修复途径对NHOK在氧化应激下表型和分子变化的影响。基于这些目的，我们将探讨氧化应激对NHOK的复制和衰老有何影响？衰老是否与NHOK中DNA修复活性降低和DNA损伤积累有关？线粒体在这些细胞衰老中的作用是什么？我们能否破坏NHOK的DNA修复能力并引发细胞过早衰老？DNA修复能力受损的细胞能否通过抗氧化剂从过早衰老中拯救出来？

DESCRIPTION (provided by applicant): The long-term goal of our research is to understand the molecular mechanism of senescence and immortalization in human oral epithelial cells. Cellular senescence is an irreversible arrest of cell divisions that results in normal somatic cells after completing a finite number of cell divisions. Several theories of senescence have been proposed, including DNA damage theory, mitochondrial theory, and telomere hypothesis. These theories of aging focus either on (1) extrinsic environmental factors or (2) intrinsic genetic programming as the cause of cellular senescence. Among the extrinsic factors, oxidative stress imposes the most detrimental effect on cells and subcellular macromolecues due to generation of reactive oxygen species (ROS). In contrast, the telomere hypothesis maintains the important role of telomere shortening in senescence of cells. Both theories of senescence mechanism have been experimentally validated to some extent, although exceptions do exist in some cell systems. To determine the mode of senescence in normal human oral keratinocytes (NHOK), we established an in vitro model of senescence, in which the cells undergo 22+/- 3 cell divisions before senescence. Unlike fibroblasts, replicating NHOK demonstrated telomerase activity and maintained constant length of telomeres, suggesting telomere length-independent mode of senescence in these cells. In our recent study, we also found that senescence of NHOK enhanced expression of mitochondrial proteins and increased mitochondrial mass, which reflect oxidative stress response in cells. Simutaneously, the expression levels of DNA repair genes were down-regulated in senescing NHOK. Therefore, our data point to the linkage between oxidative stress, DNA damage, and senescence in NHOK. The central hypothesis of the current proposal is: Senescence of NHOK is triggered by accumulation of DNA damage that results from accumulation of oxidative stress and impaired DNA repair capacity of cells. To test this hypothesis, we propose to: (1) determine the phenotypic and molecular alterations of NHOK in response to oxidative stress, (2) investigate the changes in the DNA repair capacities of NHOK during in vitro life span with or without oxidative stress, and (3) determine the effect of disrupting the DNA repair pathways on phenotypic and molecular alterations of NHOK in response to oxidative stress. With these Aims, we will address the questions: What is the effect of oxidative stress on replication and senescence of NHOK? Is senescence associated with reduction in DNA repair activities and accumulation of DNA damage in NHOK? What is the role of mitochondria in senescence of these cells? Can we disrupt the DNA repair capacities in NHOK and trigger premature aging in cells? Can the cells with impaired DNA repair activities be rescued from premature senescence by antioxidants?