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口腔上皮细胞衰老和永生化的分子机制。细胞衰老是细胞分裂的不可逆停滞，在完成有限数量的细胞分裂后产生正常的体细胞。衰老的理论主要有DNA损伤说、线粒体说、端粒说等。这些衰老理论要么关注（1）外在环境因素，要么关注（2）内在遗传编程作为细胞衰老的原因。在这些外在因素中，氧化应激由于活性氧（ROS）的产生而对细胞和亚细胞大分子产生最有害的影响。相反，端粒假说认为端粒缩短在细胞衰老中起重要作用。这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，我们建立了体外衰老模型，其中细胞在衰老前经历22+/-3次细胞分裂。与成纤维细胞，复制NHOK表现出端粒酶活性，并保持恒定的端粒长度，这表明端粒长度无关的模式，在这些细胞的衰老。在我们最近的研究中，我们还发现NHOK衰老增强了线粒体蛋白的表达，增加了线粒体质量，这反映了细胞中的氧化应激反应。同时，衰老的NHOK中DNA修复基因的表达水平下调。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。目前建议的中心假设是：NHOK的衰老是由氧化应激的积累和细胞DNA修复能力受损导致的DNA损伤的积累引发的。为了验证这一假设，我们建议：（1）确定NHOK的表型和分子的变化，在氧化应激的反应，（2）调查在体外寿命的NHOK的DNA修复能力的变化，或没有氧化应激，和（3）确定的影响，破坏DNA修复途径对NHOK的表型和分子的变化，在氧化应激反应。有了这些目标，我们将解决的问题：什么是氧化应激的复制和NHOK衰老的影响？衰老是否与NHOK中DNA修复活性的降低和DNA损伤的积累有关？线粒体在这些细胞衰老中的作用是什么？我们能破坏NHOK的DNA修复能力并引发细胞的过早衰老吗？抗氧化剂能将DNA修复活性受损的细胞从过早衰老中拯救出来吗？

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?