DNA damage signaling in oxygen toxicity in lung cells

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DESCRIPTION (provided by applicant): Hyperoxia is known to produce elevated levels of reactive oxygen species (ROS) that can damage DNA. The cellular response to DNA damage is complex and involves gene-products that recognize DNA damage and transduce the signal to various sensors, which, in turn, inhibit proliferation, stimulate repair or induce apoptosis. Although the DNA damage response induced by oxidants such as hydrogen peroxide (H202) or other peroxides have been studied, less is known about how cells respond to DNA damage in hyperoxia. Elucidating how lung cells respond to hyperoxic DNA damage is critical for understanding pulmonary oxygen toxicity. ATM (ataxia telangiecea) and ATR (ATM-Rad3-related) are members of the phosphatidylinositol 3-kinase-related kinases (PIKK) family of proteins that have been shown to transduce DNA damage signals in response to exposure to genotoxic agents. The experiments proposed here test the hypothesis that ATR transduces hyperoxia-mediated DNA damage signals by activating checkpoint proteins p53 and/or checkpoint kinase1 (Chk1). This results in inactivation of cdc25C, which inhibits cdc2 kinase activity, thereby preventing cell cycle progression. First, we will determine whether ATR is activated in hyperoxia in lung cells. This will be achieved using inhibitors for PIKKs, and by genetic approaches involving dominant-negative constructs of ATR or ATM, and the use of ATM+/+ or ATM-/- cells (Aim 1). Next we will determine the nature of DNA damage induced in hyperoxia and how that differs from H202 or UV (Aim 2). In our next specific aim we will determine whether hyperoxia specifically activates Chk1 directly or in an ATR-dependent manner resulting in cdc25C inactivation. We will use specific inhibitors of Chk1 and genetic approaches to delineate the role of Chk1 and cdc25C in DNA damage signaling in hyperoxia (Aim 3). In our next specific aim (Aim 4) we will determine the mechanisms of inactivation of cdc2 kinase activity. We will use various kinase and phosphatase assays and genetic and inhibitor studies to define the mechanisms of inhibition of cdc2 in hyperoxia. Using these molecular approaches, we will define the mechanisms of DNA damage signaling n hyperoxia, which will significantly improve our understanding of pulmonary oxygen toxicity in lung cells