Antioxidant Enzymes and Cell Cycle Checkpoint Pathways

描述（由申请人提供）：作为氧化能量代谢副产物产生的活性氧（ROS：超氧化物和过氧化氢）已被证明是生理信号传导过程的重要介质。该提议旨在检验ROS（即，超氧化物和过氧化氢）通过氧化还原调节G1细胞周期调节蛋白来调节从G1到S期的进程。这一假设是基于我们的观察，即在正常成纤维细胞周期的G1期结束时的促氧化信号似乎是进入S期的刺激所必需的。此外，我们已经表明过表达的抗氧化酶（磷脂氢谷胱甘肽过氧化物酶和锰超氧化物歧化酶）诱导G1期延迟在人类乳腺癌和前列腺癌细胞。这些观察结果表明，细胞周期的氧化还原调节可以提供细胞周期调控蛋白和氧化代谢过程之间的机械联系，成功完成每个细胞周期阶段。提出了三个具体的目标，严格测试所述的假设：目的1：确定是否细胞内的抗氧化酶代谢超氧化物和过氧化氢（MnSOD，CuZnSOD，过氧化氢酶和/或谷胱甘肽过氧化物酶）调节细胞周期的进展，从GI到S在非恶性小鼠和人类成纤维细胞。目标二：确定超氧化物和过氧化氢的稳态水平是否随着细胞在细胞周期的各个阶段的进展而波动，以及细胞周期不同阶段之间ROS的波动是否由抗氧化酶表达的变化介导。目标3：确定抗氧化酶介导的从GI到S进展的改变是否是由细胞周期蛋白D1表达的氧化还原调节的变化引起的。对细胞内氧化还原环境和细胞周期进程的机制评估可以更好地理解正常和异常的细胞增殖。由于增殖性疾病是各种人类病理生理条件的核心，包括正常组织损伤以及癌症治疗期间的肿瘤细胞反应，因此完成本提案中的研究获得的结果可以为操纵细胞增殖以改善结局提供生化依据。

DESCRIPTION (provided by applicant): Reactive oxygen species (ROS: superoxide and hydrogen peroxide) produced as by products of oxidative energy metabolism have been shown to be important mediators of physiologic signaling processes. This proposal is designed to test the hypothesis that ROS (i.e., superoxide and hydrogen peroxide) regulate progression from G1 to S phase via redox regulation of G1 cell cycle regulatory proteins. This hypothesis is based on our observations that a pro-oxidant signal at the end of the G1 phase of the normal fibroblast cell cycle appears to be necessary for stimulation of entry into S-phase. Furthermore, we have shown over expression of antioxidant enzymes (phospholipid hydroperoxide glutathione peroxidase and Mn-superoxide dismutase) induce a G1-delay in human breast and prostate cancer cells. These observations suggest that redox regulation of the cell cycle could provide a mechanistic link between the cell cycle regulatory proteins and the oxidative metabolic processes necessary for the successful completion of each cell cycle phase. Three specific aims are proposed to rigorously test the stated hypothesis: Aim 1: Determine if intracellular antioxidant enzymes that metabolize superoxide and hydrogen peroxide (MnSOD, CuZnSOD, catalase and/or glutathione peroxidase) modulate cell cycle progression from GI to S in nonmalignant mouse and human fibroblasts. Aim 2: Determine if steady-state levels of superoxide and hydrogen peroxide fluctuate as cells progress through the various phases of the cell cycle and if fluctuations in ROS between different phases of the cell cycle are mediated by changes in antioxidant enzyme expression. Aim 3: Determine if antioxidant enzyme mediated alterations in progression from GI to S are caused by changes in the redox regulation of cyclin D1 expression. A mechanistic evaluation of intracellular redox environment and cell cycle progression could lead to a better understanding of normal and aberrant cellular proliferation. Since proliferative disorders are central to a variety of human pathophysiological conditions including normal tissue injury as well as tumor cell response during cancer therapy, results obtained from completion of the studies in this proposal could provide a biochemical rationale for manipulating cell proliferation to improve outcome.