title: Mitochondria are required for ATM activation by extranuclear oxidative stress in cultured human hepatoblastoma cell line Hep G2 cells.
abstract: Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in DNA damage response (DDR). A recent study reported that oxidized ATM can be active in the absence of DDR. However, the issue of where ATM is activated by oxidative stress remains unclear. Regarding the localization of ATM, two possible locations, namely, mitochondria and peroxisomes are possible. We report herein that ATM can be activated when exposed to hydrogen peroxide without inducing nuclear DDR in Hep G2 cells, and the oxidized cells could be subjected to subcellular fractionation. The first detergent-based fractionation experiment revealed that active, phosphorylated ATM was located in the second fraction, which also contained both mitochondria and peroxisomes. An alternative fractionation method involving homogenization and differential centrifugation, which permits the light membrane fraction containing peroxisomes to be produced, but not mitochondria, revealed that the light membrane fraction contained only traces of ATM. In contrast, the heavy membrane fraction, which mainly contained mitochondrial components, was enriched in ATM and active ATM, suggesting that the oxidative activation of ATM occurs in mitochondria and not in peroxisomes. In Rho 0-Hep G2 cells, which lack mitochondrial DNA and functional mitochondria, ATM failed to respond to hydrogen peroxide, indicating that mitochondria are required for the oxidative activation of ATM. These findings strongly suggest that ATM can be activated in response to oxidative stress in mitochondria and that this occurs in a DDR-independent manner.
authors.affiliation: Department of Radiation Medicine, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan; Department of Radiological Science, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8509, Japan. Electronic address: morita@tokushima-u.ac.jp.
authors.forename: Akinori
authors.initials: A
authors.lastname: Morita
authors.affiliation: Department of Radiation Medicine, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan.
authors.forename: Keiji
authors.initials: K
authors.lastname: Tanimoto
authors.affiliation: Department of Radiation Medicine, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan.
authors.forename: Tomoki
authors.initials: T
authors.lastname: Murakami
authors.affiliation: Department of Radiation Medicine, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Morinaga
authors.affiliation: Department of Radiation Medicine, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan; Department of Radiation Biology, Graduate School of Medicine, Tohoku University, Sendai 980-8575, Japan. Electronic address: hosoi@med.tohoku.ac.jp.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Hosoi
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins / Q000378:metabolism*; D004249:DNA Damage; D004272:DNA, Mitochondrial / Q000235:genetics / Q000378:metabolism; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D056945:Hep G2 Cells; D018197:Hepatoblastoma / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000378:metabolism*; D018384:Oxidative Stress; D020675:Peroxisomes / Q000378:metabolism
chemical_list: D004272:DNA, Mitochondrial; D004273:DNA, Neoplasm; D006861:Hydrogen Peroxide; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins
keywords: ATM; Hydrogen peroxide; Mitochondria; Oxidative stress; Peroxisome