Abstract 2063: Oxidized ATM functions in intracellular redox regulation

Abstract

Abstract Background: Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in the signaling response to DNA double strand breaks (DSBs) by phosphorylating multiple targets involved in DNA repair and cell cycle control. In response to DSBs, ATM is activated through interactions with the Mre11-Rad50-Nbs1 (MRN) complex. Recent work from our group identified a second mechanism of ATM activation through direct oxidation that is independent of both MRN and DNA DSBs. ATM signaling through the p53 tumor suppressor in response to elevated ROS is known to prevent aneuploidy driven tumorigenesis and ATM deficiency is linked to oxidative stress. While ATM is best known for its role within the nuclear DNA damage response, recent studies implicate ATM in cytoplasmic signaling pathways. ATM activation through direct oxidation may function as a primary tumor suppressor mechanism and may serve to regulate the intracellular redox balance, yet the biological role of oxidized ATM remains to be elucidated. Methods: To investigate the role of ATM kinase activity in redox regulation, the levels of reactive oxygen species (ROS) were analyzed and compared in normal fibroblasts and various cancer cell lines with different genetic backgrounds following incubation with a specific inhibitor of the ATM kinase and mock treatment. To analyze the role of ATM activation through direct oxidation in redox regulation, ROS levels were measured and compared in human cell lines overexpressing wild-type ATM and a mutant form of ATM (C2991L) that is unresponsive to activation through direct oxidation. Cellular fractionation experiments were used to analyze the intracellular distribution of ATM following treatment with an oxidizing agent. Findings: Inhibition of ATM kinase activity for 24 hours resulted in increased ROS levels in normal human fibroblasts, WT and p53 -/- HCT116 colon cancer cell lines and hela cells, which are deficient in LKB1. Stable expression of the C2991L mutant form of ATM in 293 cells resulted in elevated levels of ROS when compared to 293 cells overexpressing wild-type ATM. Induced expression of the C2991L ATM mutant in cells lacking native ATM resulted in elevated ROS levels 24 hours post-induction when compared to the induced expression of wild-type ATM. Using cellular fractionation studies, we show that ATM levels in the cytoplasmic fraction increased at 30 minutes post-treatment with hydrogen peroxide and remained elevated up to 2 hours post-treatment without an accompanying increase in γ-H2AX, a widely utilized marker of DSBs. Conclusions: These findings indicate that oxidized ATM functions in regulating intracellular ROS levels and that this function is independent of ATM signaling through the p53 and LKB1 signaling pathways. These findings further show that ATM signaling in response to oxidative stress may not be restricted to known nuclear signaling pathways activated by DNA damage. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2063. doi:1538-7445.AM2012-2063