Role of ROS-Induced DNA Damage in Mitochondria-Regulated Apoptosis

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DESCRIPTION (provided by applicant): Human cells are constantly exposed to oxidative stress. Reactive oxygen species (ROS) are endogenously produced due to leakage of partially reduced O2- anion radical species from the mitochondrial respiration complexes. Environmental agents, such as heavy metals, and other genotoxicants also generate ROS due to mitochondrial dysfunction and various oxidases. Enzymatically or via metal catalyzed reactions, O2- is converted to other ROS, namely H2O2 and OH radical. In addition to oxidatively damaging proteins and lipids, ROS also cause DNA damage, such as mutagenic abasic sites (APS) and their oxidized derivatives (OAS), in nuclear and mitochondrial DNA, which have implications for the induction of cancers, the aging processes, and other pathophysiologies. Due to the lack of protective histones and mitochondrial genome's proximity to sites of ROS generation, it accumulates more oxidative damage than the nuclear genome. APE1 could be the central player in the DNA base excision repair pathway in the mitochondria and translocates to this site to repair APS and OAS in the mitochondrial genome after cleavage of its nuclear localization sequence present at the N-terminus. APE1 also functions as redox factor 1 due to its ability to reductively activate transcription factors, such as c-Jun and p53. My sponsor's laboratory has established a conditional APE1 null mouse embryonic fibroblast (MEF) system with 'floxed' human APE1 gene to examine the role of APE1 in the defense against apoptosis. Studies done in his laboratory have shown that expression of Cre recombinase, which cleaves at loxP sites to delete APE1 in these MEFs, elicits apoptosis. Also available for this project is a new tetracycline-regulated APE1 siRNA expressing HeLa cell line. Our fundamental hypothesis is that accumulation of APS and OAS mainly in the mitochondrial genome, caused by the absence of APE1's repair activity, would activate ROS-initiated apoptosis. We will test this hypothesis by pursuing the following three specific aims: Aim 1, we will generate repair-deficient and mitochondrial-targeted transductants of the conditional APE1 MEF and HeLa cell mutants in order to specifically examine the role of APE1's repair activity in the mitochondria in modulating apoptosis; Aim 2, we will examine the role of APS and OAS as initiators of apoptotic signaling by measuring kinetics of ROS, APS, and OAS production in MEFs lacking only the repair activity of APE1; Aim 3, we will investigate how exogenous modulation of oxidative state affects apoptosis. Thus, the proposed research should unravel the mechanism of progression from oxidative damage to mitochondria-mediated apoptosis. Since alterations of this mechanism seem to help cancer cells to evade apoptosis, our findings could help design new therapeutic approaches for preventing and treating cancer.