Antioxidant Enzymes and Cell Cycle Checkpoint Pathways

描述（由申请人提供）：活性氧（ROS：超氧化物和过氧化氢）作为氧化能量代谢的副产物已被证明是生理信号传导过程的重要介质。该提案旨在验证ROS（即超氧化物和过氧化氢）通过G1细胞周期调节蛋白的氧化还原调节G1期到S期的进程的假设。这一假设是基于我们的观察，即正常成纤维细胞周期G1期结束时的促氧化信号似乎是刺激进入s期所必需的。此外，我们已经证明抗氧化酶（磷脂氢过氧化物谷胱甘肽过氧化物酶和锰超氧化物歧化酶）的过度表达诱导了人类乳腺癌和前列腺癌细胞的g1延迟。这些观察结果表明，细胞周期的氧化还原调节可以在细胞周期调节蛋白和成功完成每个细胞周期阶段所必需的氧化代谢过程之间提供机制联系。目的1：确定细胞内代谢超氧化物和过氧化氢的抗氧化酶（MnSOD、CuZnSOD、过氧化氢酶和谷胱甘肽过氧化物酶）是否调节非恶性小鼠和人类成纤维细胞从GI到S的细胞周期进程。目的2：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活性氧的波动是否由抗氧化酶表达的变化介导。目的3：确定抗氧化酶介导的从GI到S的变化是否由细胞周期蛋白D1表达的氧化还原调节变化引起。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。由于增殖性疾病是多种人类病理生理状况的核心，包括正常组织损伤以及癌症治疗期间的肿瘤细胞反应，因此本提案中完成的研究结果可以为操纵细胞增殖以改善结果提供生化基础。

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.