title: DNA double-strand breaks activate ATM independent of mitochondrial dysfunction in A549 cells.
abstract: Excessive nuclear or mitochondrial DNA damage can lead to mitochondrial dysfunction, decreased energy production, and increased generation of reactive oxygen species (ROS). Although numerous cell signaling pathways are activated when cells are injured, the ataxia telangiectasia mutant (ATM) protein has emerged as a major regulator of the response to both mitochondrial dysfunction and nuclear DNA double-strand breaks (DSBs). Because mitochondrial dysfunction is often a response to excessive DNA damage, it has been difficult to determine whether nuclear and/or mitochondrial DNA DSBs activate ATM independent of mitochondrial dysfunction. In this study, mitochondrial and nuclear DNA DSBs were generated in the A549 human lung adenocarcinoma cell line by infecting with retroviruses expressing the restriction endonuclease PstI fused to a mitochondrial targeting sequence (MTS) or nuclear localization sequence (NLS) and a hemagglutinin antigen epitope tag (HA). Expression of MTS-PstI-HA or NLS-PstI-HA activated the DNA damage response defined by phosphorylation of ATM, the tumor suppressor protein p53 (TP53), KRAB-associated protein (KAP)-1, and structural maintenance of chromosomes (SMC)-1. Phosphorylated ATM and SMC1 were detected in nuclear fractions, whereas phosphorylated TP53 and KAP1 were detected in both mitochondrial and nuclear fractions. PstI also enhanced expression of the cyclin-dependent kinase inhibitor p21 and inhibited cell growth. This response to DNA damage occurred in the absence of detectable mitochondrial dysfunction and excess production of ROS. These findings reveal that DNA DSBs are sufficient to activate ATM independent of mitochondrial dysfunction and suggest that the activated form of ATM and some of its substrates are restricted to the nuclear compartment, regardless of the site of DNA damage.
authors.affiliation: Department of Environmental Medicine, The University of Rochester, Rochester, NY 14642, USA.
authors.forename: Lidza
authors.initials: L
authors.lastname: Kalifa
authors.affiliation: Department of Anesthesiology, The University of Rochester, Rochester, NY 14642, USA.
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Gewandter
authors.affiliation: Department of Pediatrics, The University of Rochester, Rochester, NY 14642, USA.
authors.forename: Rhonda J
authors.initials: RJ
authors.lastname: Staversky
authors.affiliation: Department of Biology, The University of Rochester, Rochester, NY 14642, USA.
authors.forename: Elaine A
authors.initials: EA
authors.lastname: Sia
authors.affiliation: Department of Anesthesiology, The University of Rochester, Rochester, NY 14642, USA.
authors.forename: Paul S
authors.initials: PS
authors.lastname: Brookes
authors.affiliation: Department of Pediatrics, The University of Rochester, Rochester, NY 14642, USA. Electronic address: michael_oreilly@urmc.rochester.edu.
authors.forename: Michael A
authors.initials: MA
authors.lastname: O'Reilly
mesh_terms: D017209:Apoptosis / Q000235:genetics; D064007:Ataxia Telangiectasia Mutated Proteins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000235:genetics*; D049109:Cell Proliferation / Q000235:genetics; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000096:biosynthesis; D004247:DNA / Q000737:chemistry; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000235:genetics; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D010766:Phosphorylation; D017382:Reactive Oxygen Species / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D014162:Transfection; D000076183:Tripartite Motif-Containing Protein 28; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D000098550:Structural Maintenance of Chromosome Protein 1
chemical_list: D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D050759:Cyclin-Dependent Kinase Inhibitor p21; D017382:Reactive Oxygen Species; D012097:Repressor Proteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D000098550:Structural Maintenance of Chromosome Protein 1; D004247:DNA; C101116:TRIM28 protein, human; D000076183:Tripartite Motif-Containing Protein 28; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C024388:CTGCAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific
keywords: ATM; DNA damage; Free radicals; Mitochondrial dysfunction; Oxidative stress