Antioxidant Enzymes and Cell Cycle Checkpoint Pathways

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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 G! phase of the normal fibroblast cell cycle appears to be necessary for stimulation of entry into S-phase (Cancer Research 63:2109-2117, 2003). Furthermore, we have shown over expression of antioxidant enzymes (phospholipid hydroperoxide glutathione peroxidase and Mn-superoxide dismutase) induce a Grdelay in human breast and prostate cancer cells (Free Radical Research 37:621-630, 2003; Oncogene, 24:77-89, 2005). 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.