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• W2049477961 abstract "Mitochondria-targeted human 8-oxoguanine DNA glycosylase (mt-hOgg1) and aconitase-2 (Aco-2) each reduce oxidant-induced alveolar epithelial cell (AEC) apoptosis, but it is unclear whether protection occurs by preventing AEC mitochondrial DNA (mtDNA) damage. Using quantitative PCR-based measurements of mitochondrial and nuclear DNA damage, mtDNA damage was preferentially noted in AEC after exposure to oxidative stress (e.g. amosite asbestos (5–25 μg/cm2) or H2O2 (100–250 μm)) for 24 h. Overexpression of wild-type mt-hOgg1 or mt-long α/β 317–323 hOgg1 mutant incapable of DNA repair (mt-hOgg1-Mut) each blocked A549 cell oxidant-induced mtDNA damage, mitochondrial p53 translocation, and intrinsic apoptosis as assessed by DNA fragmentation and cleaved caspase-9. In contrast, compared with controls, knockdown of Ogg1 (using Ogg1 shRNA in A549 cells or primary alveolar type 2 cells from ogg1−/− mice) augmented mtDNA lesions and intrinsic apoptosis at base line, and these effects were increased further after exposure to oxidative stress. Notably, overexpression of Aco-2 reduced oxidant-induced mtDNA lesions, mitochondrial p53 translocation, and apoptosis, whereas siRNA for Aco-2 (siAco-2) enhanced mtDNA damage, mitochondrial p53 translocation, and apoptosis. Finally, siAco-2 attenuated the protective effects of mt-hOgg1-Mut but not wild-type mt-hOgg1 against oxidant-induced mtDNA damage and apoptosis. Collectively, these data demonstrate a novel role for mt-hOgg1 and Aco-2 in preserving AEC mtDNA integrity, thereby preventing oxidant-induced mitochondrial dysfunction, p53 mitochondrial translocation, and intrinsic apoptosis. Furthermore, mt-hOgg1 chaperoning of Aco-2 in preventing oxidant-mediated mtDNA damage and apoptosis may afford an innovative target for the molecular events underlying oxidant-induced toxicity. Mitochondria-targeted human 8-oxoguanine DNA glycosylase (mt-hOgg1) and aconitase-2 (Aco-2) each reduce oxidant-induced alveolar epithelial cell (AEC) apoptosis, but it is unclear whether protection occurs by preventing AEC mitochondrial DNA (mtDNA) damage. Using quantitative PCR-based measurements of mitochondrial and nuclear DNA damage, mtDNA damage was preferentially noted in AEC after exposure to oxidative stress (e.g. amosite asbestos (5–25 μg/cm2) or H2O2 (100–250 μm)) for 24 h. Overexpression of wild-type mt-hOgg1 or mt-long α/β 317–323 hOgg1 mutant incapable of DNA repair (mt-hOgg1-Mut) each blocked A549 cell oxidant-induced mtDNA damage, mitochondrial p53 translocation, and intrinsic apoptosis as assessed by DNA fragmentation and cleaved caspase-9. In contrast, compared with controls, knockdown of Ogg1 (using Ogg1 shRNA in A549 cells or primary alveolar type 2 cells from ogg1−/− mice) augmented mtDNA lesions and intrinsic apoptosis at base line, and these effects were increased further after exposure to oxidative stress. Notably, overexpression of Aco-2 reduced oxidant-induced mtDNA lesions, mitochondrial p53 translocation, and apoptosis, whereas siRNA for Aco-2 (siAco-2) enhanced mtDNA damage, mitochondrial p53 translocation, and apoptosis. Finally, siAco-2 attenuated the protective effects of mt-hOgg1-Mut but not wild-type mt-hOgg1 against oxidant-induced mtDNA damage and apoptosis. Collectively, these data demonstrate a novel role for mt-hOgg1 and Aco-2 in preserving AEC mtDNA integrity, thereby preventing oxidant-induced mitochondrial dysfunction, p53 mitochondrial translocation, and intrinsic apoptosis. Furthermore, mt-hOgg1 chaperoning of Aco-2 in preventing oxidant-mediated mtDNA damage and apoptosis may afford an innovative target for the molecular events underlying oxidant-induced toxicity. Reactive oxygen species (ROS) 2The abbreviations used are:ROSreactive oxygen species8-oxoG8-oxo-7,8-dihydroxyguanineOgg18-oxoguanine DNA glycosylasehOgg1human Ogg1AECalveolar epithelial cell(s)AT2alveolar type IIAco-2aconitase-2Q-PCRquantitative PCR. generated under physiologic conditions from the mitochondrial electron transport chain and other sources activate cellular signaling important for survival; however, higher levels of ROS cause oxidative DNA damage and apoptosis that promote aging, tumorigenesis, and degenerative diseases (1.Bohr V.A. Stevnsner T. de Souza-Pinto N.C. Mitochondrial DNA repair of oxidative damage in mammalian cells.Gene. 2002; 286: 127-134Crossref PubMed Scopus (156) Google Scholar, 2.Van Houten B. Woshner V. Santos J.H. Role of mitochondrial DNA in toxic responses to oxidative stress.DNA Repair. 2006; 5: 145-152Crossref PubMed Scopus (337) Google Scholar, 3.Figueira T.R. Barros M.H. Camargo A.A. Castilho R.F. Ferreira J.C. Kowaltowski A.J. Sluse F.E. Souza-Pinto N.C. Vercesi A.E. Mitochondria as a source of reactive oxygen and nitrogen species. From molecular mechanisms to human health.Antioxid. Redox. Signal. 2013; 18: 2029-2074Crossref PubMed Scopus (300) Google Scholar). Mitochondrial DNA (mtDNA) damage is more abundant and longer lasting than nuclear DNA damage after exposure to oxidative stress because of the proximity of mtDNA to the electron transport chain and because mtDNA lacks histones that serve as a barrier against ROS (2.Van Houten B. Woshner V. Santos J.H. Role of mitochondrial DNA in toxic responses to oxidative stress.DNA Repair. 2006; 5: 145-152Crossref PubMed Scopus (337) Google Scholar, 3.Figueira T.R. Barros M.H. Camargo A.A. Castilho R.F. Ferreira J.C. Kowaltowski A.J. Sluse F.E. Souza-Pinto N.C. Vercesi A.E. Mitochondria as a source of reactive oxygen and nitrogen species. From molecular mechanisms to human health.Antioxid. Redox. Signal. 2013; 18: 2029-2074Crossref PubMed Scopus (300) Google Scholar, 4.Yakes F.M. Van Houten B. Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress.Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 514-519Crossref PubMed Scopus (1447) Google Scholar). 8-Oxo-7,8-dihydroxyguanine (8-oxoG), which is one of the most abundant DNA adducts caused by oxidative stress, is causally associated with several cancers and neurodegenerative diseases because it induces mutations by preferentially mispairing with adenosine during replication (1.Bohr V.A. Stevnsner T. de Souza-Pinto N.C. Mitochondrial DNA repair of oxidative damage in mammalian cells.Gene. 2002; 286: 127-134Crossref PubMed Scopus (156) Google Scholar, 2.Van Houten B. Woshner V. Santos J.H. Role of mitochondrial DNA in toxic responses to oxidative stress.DNA Repair. 2006; 5: 145-152Crossref PubMed Scopus (337) Google Scholar). 8-Oxoguanine DNA glycosylase (Ogg1), a key enzyme in base excision repair, excises and repairs 8-oxoG in the mitochondria and nucleus (1.Bohr V.A. Stevnsner T. de Souza-Pinto N.C. Mitochondrial DNA repair of oxidative damage in mammalian cells.Gene. 2002; 286: 127-134Crossref PubMed Scopus (156) Google Scholar, 2.Van Houten B. Woshner V. Santos J.H. Role of mitochondrial DNA in toxic responses to oxidative stress.DNA Repair. 2006; 5: 145-152Crossref PubMed Scopus (337) Google Scholar, 3.Figueira T.R. Barros M.H. Camargo A.A. Castilho R.F. Ferreira J.C. Kowaltowski A.J. Sluse F.E. Souza-Pinto N.C. Vercesi A.E. Mitochondria as a source of reactive oxygen and nitrogen species. From molecular mechanisms to human health.Antioxid. Redox. Signal. 2013; 18: 2029-2074Crossref PubMed Scopus (300) Google Scholar, 4.Yakes F.M. Van Houten B. Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress.Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 514-519Crossref PubMed Scopus (1447) Google Scholar, 5.Richter C. Park J.W. Ames B.N. Normal oxidative damage to mitochondrial and nuclear DNA is extensive.Proc. Natl. Acad. Sci. U.S.A. 1988; 85: 6465-6467Crossref PubMed Scopus (1487) Google Scholar, 6.Dizdaroglu M. Jaruga P. Birincioglu M. Rodriguez H. Free radical-induced damage to DNA. Mechanisms and measurement.Free Radic. Biol. Med. 2002; 32: 1102-1115Crossref PubMed Scopus (804) Google Scholar). Mitochondrial base excision repair enzymes, which are all nuclear encoded and imported into the mitochondria, are primarily responsible for removing abnormal DNA adducts, including 8-oxoG from mtDNA (1.Bohr V.A. Stevnsner T. de Souza-Pinto N.C. Mitochondrial DNA repair of oxidative damage in mammalian cells.Gene. 2002; 286: 127-134Crossref PubMed Scopus (156) Google Scholar, 6.Dizdaroglu M. Jaruga P. Birincioglu M. Rodriguez H. Free radical-induced damage to DNA. Mechanisms and measurement.Free Radic. Biol. Med. 2002; 32: 1102-1115Crossref PubMed Scopus (804) Google Scholar). Accumulating evidence implicates a direct association between mtDNA damage and mitochondria-regulated (intrinsic) apoptosis; most notably, overexpression of mitochondria-targeted Ogg1 (mt-Ogg1) prevents mtDNA damage and intrinsic apoptosis in response to a variety of oxidative stresses in various cell types (7.Santos J.H. Hunakova L. Chen Y. Bortner C. Van Houten B. Cell sorting experiments link persistent mitochondrial DNA damage with loss of mitochondrial membrane potential and apoptotic cell death.J. Biol. Chem. 2003; 278: 1728-1734Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 8.Dobson A.W. Grishko V. LeDoux S.P. Kelley M.R. Wilson G.L. Gillespie M.N. Enhanced mtDNA repair capacity protects pulmonary artery endothelial cells from oxidant-mediated death.Am. J. Physiol. Lung Cell Mol. Physiol. 2002; 283: L205-L210Crossref PubMed Scopus (69) Google Scholar, 9.Shukla A. Jung M. Stern M. Fukagawa N.K. Taatjes D.J. Sawyer D. Van Houten B. Mossman B.T. Asbestos induces mitochondrial DNA damage and dysfunction linked to the development of apoptosis.Am. J. Physiol. Lung Cell Mol. Physiol. 2003; 285: L1018-L1025Crossref PubMed Scopus (84) Google Scholar, 10.Ruchko M. Gorodnya O. LeDoux S.P. Alexeyev M.F. Al-Mehdi A.B. Gillespie M.N. Mitochondrial DNA damage triggers mitochondrial dysfunction and apoptosis in oxidant-challenged lung endothelial cells.Am. J. Physiol. Lung Cell Mol. Physiol. 2005; 288: L530-L535Crossref PubMed Scopus (82) Google Scholar, 11.Panduri V. Liu G. Surapureddi S. Kondapalli J. Soberanes S. de Souza-Pinto N.C. Bohr V.A. Budinger G.R. Schumacker P.T. Weitzman S.A. Kamp D.W. Role of mitochondrial hOGG1 and aconitase in oxidant-induced lung epithelial cell apoptosis.Free Radic. Biol. Med. 2009; 47: 750-759Crossref PubMed Scopus (65) Google Scholar, 12.Ruchko M.V. Gorodnya O.M. Zuleta A. Pastukh V.M. Gillespie M.N. The DNA glycosylase Ogg1 defends against oxidant-induced mtDNA damage and apoptosis in pulmonary artery endothelial cells.Free Radic. Biol. Med. 2011; 50: 1107-1113Crossref PubMed Scopus (49) Google Scholar). However, whether alternative mechanisms modulate mtDNA damage that affect intrinsic apoptosis are not fully established. reactive oxygen species 8-oxo-7,8-dihydroxyguanine 8-oxoguanine DNA glycosylase human Ogg1 alveolar epithelial cell(s) alveolar type II aconitase-2 quantitative PCR. Ineffective repair of damaged alveolar epithelial cells (AEC) and AEC apoptosis are implicated in mediating pulmonary fibrosis in humans with idiopathic pulmonary fibrosis and in animal models of fibrotic lung disease, including asbestosis (13.King Jr., T.E. Pardo A. Selman M. Idiopathic pulmonary fibrosis.Lancet. 2011; 378: 1949-1961Abstract Full Text Full Text PDF PubMed Scopus (1334) Google Scholar, 14.Noble P.W. Barkauskas C.E. Jiang D. Pulmonary fibrosis. Patterns and perpetrators.J. Clin. Invest. 2012; 122: 2756-2762Crossref PubMed Scopus (338) Google Scholar, 15.Cheresh P. Kim S.J. Tulasiram S. Kamp D.W. Oxidative stress and pulmonary fibrosis.Biochim. Biophys. Acta. 2013; 1832: 1028-1040Crossref PubMed Scopus (300) Google Scholar, 16.Liu G. Cheresh P. Kamp D.W. Molecular basis of asbestos-induced lung disease.Annu. Rev. Pathol. 2013; 8: 161-187Crossref PubMed Scopus (146) Google Scholar). Notably, targeted injury of alveolar type II (AT2) cells appears necessary for triggering pulmonary fibrosis (17.Sisson T.H. Mendez M. Choi K. Subbotina N. Courey A. Cunningham A. Dave A. Engelhardt J.F. Liu X. White E.S. Thannickal V.J. Moore B.B. Christensen P.J. Simon R.H. Targeted injury of type II alveolar epithelial cells induces pulmonary fibrosis.Am. J. Respir. Crit. Care Med. 2010; 181: 254-263Crossref PubMed Scopus (341) Google Scholar). We previously reported that oxidative stress caused by exposure to asbestos fibers (exogenous) or H2O2 (endogenous) induces AEC mitochondrial dysfunction, mitochondrial ROS production, p53 activation, and intrinsic apoptosis (11.Panduri V. Liu G. Surapureddi S. Kondapalli J. Soberanes S. de Souza-Pinto N.C. Bohr V.A. Budinger G.R. Schumacker P.T. Weitzman S.A. Kamp D.W. Role of mitochondrial hOGG1 and aconitase in oxidant-induced lung epithelial cell apoptosis.Free Radic. Biol. Med. 2009; 47: 750-759Crossref PubMed Scopus (65) Google Scholar, 16.Liu G. Cheresh P. Kamp D.W. Molecular basis of asbestos-induced lung disease.Annu. Rev. Pathol. 2013; 8: 161-187Crossref PubMed Scopus (146) Google Scholar, 18.Panduri V. Weitzman S.A. Chandel N. Kamp D.W. The mitochondria-regulated death pathway mediates asbestos-induced alveolar epithelial cell apoptosis.Am. J. Respir. Cell Mol. Biol. 2003; 28: 241-248Crossref PubMed Scopus (68) Google Scholar, 19.Panduri V. Surapureddi S. Soberanes S. Weitzman S.A. Chandel N. Kamp D.W. P53 mediates amosite asbestos-induced alveolar epithelial cell mitochondria-regulated apoptosis.Am. J. Respir. Cell Mol. Biol. 2006; 34: 443-452Crossref PubMed Scopus (56) Google Scholar). Furthermore, we showed that overexpression of wild-type mt-hOgg1 (mt-hOgg1-WT) and a mt-long α/β 317–323 hOgg1 mutant incapable of DNA repair (mt-hOgg1-Mut) each prevent intrinsic AEC apoptosis despite high levels of mitochondrial ROS stress in part because a novel function of hOgg1 chaperoning mitochondrial aconitase (Aco-2) from oxidative degradation (11.Panduri V. Liu G. Surapureddi S. Kondapalli J. Soberanes S. de Souza-Pinto N.C. Bohr V.A. Budinger G.R. Schumacker P.T. Weitzman S.A. Kamp D.W. Role of mitochondrial hOGG1 and aconitase in oxidant-induced lung epithelial cell apoptosis.Free Radic. Biol. Med. 2009; 47: 750-759Crossref PubMed Scopus (65) Google Scholar). Aco-2, one of the enzymes participating in the tricarboxylic acid cycle (TCA), acts as a biosensor for oxidative stress and preserves mtDNA in yeast independent of Aco-2 activity (20.Bulteau A.L. Ikeda-Saito M. Szweda L.I. Redox-dependent modulation of aconitase activity in intact mitochondria.Biochemistry. 2003; 42: 14846-14855Crossref PubMed Scopus (194) Google Scholar, 21.Chen X.J. Wang X. Kaufman B.A. Butow R.A. Aconitase couples metabolic regulation to mitochondrial DNA maintenance.Science. 2005; 307: 714-717Crossref PubMed Scopus (8) Google Scholar). However, the role of mtDNA damage in triggering AEC apoptosis as well as whether Aco-2 modulates mtDNA damage in eukaryotic cells (including AEC) are unknown. In this study we reasoned that preservation of mtDNA by mitochondria-targeted hOgg1 and/or Aco-2 is necessary for preventing oxidant (amosite asbestos or H2O2)-induced AEC mitochondrial dysfunction, p53 translocation to the mitochondria, and intrinsic apoptosis. Using a quantitative-PCR (Q-PCR)-based mitochondrial and nuclear DNA damage assay, we show that oxidative stress induces preferential mtDNA damage in AEC. Furthermore, using hOgg1 and Aco-2 over- and underexpression conditions, we establish that both hOgg1 and Aco-2 are important in limiting oxidant-induced mtDNA damage and subsequent p53 mitochondrial translocation and intrinsic apoptosis. Intriguingly, primary AT2 cells from ogg1−/− mice have reduced Aco-2 expression and increased levels of mtDNA lesions and intrinsic apoptosis under untreated conditions as compared with wild-type AT2 cells, and these deleterious effects are further enhanced after exposure to oxidative stress. Finally, we show that siRNA against Aco-2 alters the protective effects of mt-hOgg1-Mut but not mt-hOgg1-WT against oxidant-induced mtDNA damage and apoptosis. Collectively, these findings establish a novel role for mt-hOgg1 and Aco-2 in preserving AEC mtDNA integrity that prevents downstream mitochondrial dysfunction, p53 activation, and intrinsic apoptosis. Amosite asbestos fibers utilized herein were Union International Centere le Cancer reference standard samples kindly supplied by Drs. V. Timbrell and Andy Ghio (Environmental Protection Agency) as characterized and handled as described previously (18.Panduri V. Weitzman S.A. Chandel N. Kamp D.W. The mitochondria-regulated death pathway mediates asbestos-induced alveolar epithelial cell apoptosis.Am. J. Respir. Cell Mol. Biol. 2003; 28: 241-248Crossref PubMed Scopus (68) Google Scholar). All other reagents were purchased from Sigma unless otherwise stated. The A549 human lung adenocarcinoma cell line, MLE-12 mouse lung epithelial cell line, and RLE-6TN rat AT2 cell line were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Primary isolated AT2 cells were isolated from the lungs of wild-type C57/BL6 and ogg−/− mice using techniques previously described (22.Corti M. Brody A.R. Harrison J.H. Isolation and primary culture of murine alveolar type II cells.Am. J. Respir. Cell Mol. Biol. 1996; 14: 309-315Crossref PubMed Scopus (278) Google Scholar) and approved by the Animal Care and Use Committee. A549 cells, primary mice AT2 cells, and MLE-12 cells were maintained in DMEM (Invitrogen) with 2 mm l-glutamine supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. RLE-6TN cells were maintained at 37 °C in Ham-F12 (Invitrogen) with 2 mm l-glutamine supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were plated in 6-well plates or 100-mm dishes and grown to confluence before adding asbestos or H2O2 for 24 h as described (11.Panduri V. Liu G. Surapureddi S. Kondapalli J. Soberanes S. de Souza-Pinto N.C. Bohr V.A. Budinger G.R. Schumacker P.T. Weitzman S.A. Kamp D.W. Role of mitochondrial hOGG1 and aconitase in oxidant-induced lung epithelial cell apoptosis.Free Radic. Biol. Med. 2009; 47: 750-759Crossref PubMed Scopus (65) Google Scholar). Aco-2 (Invitrogen), wild-type, and mutant α-Ogg1 plasmid constructs were previously described (11.Panduri V. Liu G. Surapureddi S. Kondapalli J. Soberanes S. de Souza-Pinto N.C. Bohr V.A. Budinger G.R. Schumacker P.T. Weitzman S.A. Kamp D.W. Role of mitochondrial hOGG1 and aconitase in oxidant-induced lung epithelial cell apoptosis.Free Radic. Biol. Med. 2009; 47: 750-759Crossref PubMed Scopus (65) Google Scholar). Exogenous expression of hOgg1-WT and hOgg1-Mut was identified by immunostaining using c-Myc antibody (Santa Cruz Biotechnology, Dallas, TX). The plasmids were transiently transfected into A549 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. The transfection efficiency is >95%. After transfection for 48 h, the cells were tested for Western blot and DNA fragmentation as previously described (11.Panduri V. Liu G. Surapureddi S. Kondapalli J. Soberanes S. de Souza-Pinto N.C. Bohr V.A. Budinger G.R. Schumacker P.T. Weitzman S.A. Kamp D.W. Role of mitochondrial hOGG1 and aconitase in oxidant-induced lung epithelial cell apoptosis.Free Radic. Biol. Med. 2009; 47: 750-759Crossref PubMed Scopus (65) Google Scholar, 18.Panduri V. Weitzman S.A. Chandel N. Kamp D.W. The mitochondria-regulated death pathway mediates asbestos-induced alveolar epithelial cell apoptosis.Am. J. Respir. Cell Mol. Biol. 2003; 28: 241-248Crossref PubMed Scopus (68) Google Scholar, 19.Panduri V. Surapureddi S. Soberanes S. Weitzman S.A. Chandel N. Kamp D.W. P53 mediates amosite asbestos-induced alveolar epithelial cell mitochondria-regulated apoptosis.Am. J. Respir. Cell Mol. Biol. 2006; 34: 443-452Crossref PubMed Scopus (56) Google Scholar) as well as mtDNA damage (see below). hOgg1-specific shRNA with the target sequences of 5′- UCCAAGGUGUGCGACUGCUGCGACA-3′ (Integrated DNA Technologies, Coralville, IA), RNA interference for Aco-2 (Invitrogen), and scrambled shRNA controls (Invitrogen) were transiently transfected into A549 cells using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer's recommendations. After transfection for 48 h, the cells were tested for Western blot and DNA fragmentation as previously described (11.Panduri V. Liu G. Surapureddi S. Kondapalli J. Soberanes S. de Souza-Pinto N.C. Bohr V.A. Budinger G.R. Schumacker P.T. Weitzman S.A. Kamp D.W. Role of mitochondrial hOGG1 and aconitase in oxidant-induced lung epithelial cell apoptosis.Free Radic. Biol. Med. 2009; 47: 750-759Crossref PubMed Scopus (65) Google Scholar) as well as mtDNA damage (see below). Genomic DNA, including both nuclear and mtDNA, was assessed by Q-PCR as described elsewhere (23.Santos J.H. Mandavilli B.S. Van Houten B. Measuring oxidative mtDNA damage and repair using quantitative PCR.Methods Mol. Biol. 2002; 197: 159-176PubMed Google Scholar). Briefly, genomic DNA was extracted using the Qiagen Genomic-tip 20/G and Qiagen DNA Buffer Set (Qiagen, Gaithersburg, MD) per the manufacturer's protocol. The cells were incubated in a lysis buffer containing RNase A (Invitrogen) and proteinase K (Qiagen) for 2 h. Total DNA contained in the extracts is bound to Qiagen Genomic-tip 20/G columns, washed, and eluted. Eluted DNA was incubated with isopropyl alcohol at −80 °C overnight and centrifuged 12,000 × g for 60 min. To wash DNA, we evenly added 0.7 ml (70%) of ethanol to each aliquot and centrifuged samples at 9500 − g for 30 min. After removing the supernatant, Tris-EDTA buffer (pH 7.5) was added the each tube, and the DNA concentration was measured. PCR was performed using Ex-taq (Clontech, Mountain View, CA) with specific primers (Table 1) to amplify a fragment of the mitochondrial genome, both a short and long fragment and nuclear DNA (β-globulin) as described (23.Santos J.H. Mandavilli B.S. Van Houten B. Measuring oxidative mtDNA damage and repair using quantitative PCR.Methods Mol. Biol. 2002; 197: 159-176PubMed Google Scholar). Each DNA was quantified by Pico-green (Invitrogen) using the FL600 Microplate Fluorescence Reader parameters excitation and emission wavelengths 485 and 530 nm. Then the data were obtained from the small fragment were subsequently used to normalize the results of the mitochondrial long fragment (23.Santos J.H. Mandavilli B.S. Van Houten B. Measuring oxidative mtDNA damage and repair using quantitative PCR.Methods Mol. Biol. 2002; 197: 159-176PubMed Google Scholar). The number of mitochondrial lesions was calculated by the equation, D = (1–2−(Δlong−Δshort)) × 10,000 (bp)/size of the long fragment (bp).TABLE 1The sequences of primer pairs to amplify human, mouse, and rat target genes for Q-PCR-based DNA damage assayHumanβ-Globin gene (nucleus,13.5 kb)5′-TTG AGA CGC ATG AGA CGT GCA G-3′Sense5′-GCA CTG GCT TAG GAG TTG GAC T-3′AntisenseMitochondria long fragment (8.9 kb)5′- TCT AAG CCT CCT TAT TCG AGC CGA-3′Sense5′-TTT CAT CAT GCG GAG ATG TTG GAT GG-3′AntisenseMitochondria short fragment (222 bp)5′-CCC CAC AAA CCC CAT TAC TAA ACC CA-3′Sense5′-TTT CAT CAT GCG GAG ATG TTG GAT GG-3′Mouseβ-Globin gene (nucleus,8.7 kb)5′-TTG AGA CTG TGA TTG GCA ATG CCT-3′Sense5′-CCT TTA ATG CCC ATC CCG GAC T-3′AntisenseMitochondria long fragment (10 kb)5′-GCC AGC CTG ACC CAT AGC CAT AAT AT-3′Sense5′-GAG AGA TTT TAT GGG TGT AAT GCG G-3′AntisenseMitochondria short fragment (117 bp)5′-CCC AGC TAC TAC CAT CAT TCA AGT-3′Sense5′-GAT GGT TTG GGA GAT TGG TTG ATG T-3′RatTRPM-2 (nucleus,12.5 kb)5′-AGA CGG GTG AGA CAG CTG CAC CTT TTC-3′Sense5′-CGA GAG CAT CAA GTG CAG GCA TTA GAG-3′AntisenseMitochondria long fragment (13.4 kb)5′-AAA ATC CCC GCA AAC AAT GAC CAC CC-3′Sense5′-GGC AAT TAA GAG TGG GAT GGA GCC AA-3′AntisenseMitochondria short fragment (235 bp)5′-CCT CCC ATT CAT TAT CGC CGC CCT TGC-3′Sense5′-GTC TGG GTC TCC TAG TAG GTC TGG GAA-3′ Open table in a new tab DNA fragmentation for apoptosis was assessed using a histone-associated DNA fragmentation (mono and oligonucleosomes) Cell Death detection kit (Cell Signaling Technology, Beverly, MA) as previously described (11.Panduri V. Liu G. Surapureddi S. Kondapalli J. Soberanes S. de Souza-Pinto N.C. Bohr V.A. Budinger G.R. Schumacker P.T. Weitzman S.A. Kamp D.W. Role of mitochondrial hOGG1 and aconitase in oxidant-induced lung epithelial cell apoptosis.Free Radic. Biol. Med. 2009; 47: 750-759Crossref PubMed Scopus (65) Google Scholar, 18.Panduri V. Weitzman S.A. Chandel N. Kamp D.W. The mitochondria-regulated death pathway mediates asbestos-induced alveolar epithelial cell apoptosis.Am. J. Respir. Cell Mol. Biol. 2003; 28: 241-248Crossref PubMed Scopus (68) Google Scholar, 19.Panduri V. Surapureddi S. Soberanes S. Weitzman S.A. Chandel N. Kamp D.W. P53 mediates amosite asbestos-induced alveolar epithelial cell mitochondria-regulated apoptosis.Am. J. Respir. Cell Mol. Biol. 2006; 34: 443-452Crossref PubMed Scopus (56) Google Scholar). Apoptosis was also determined by flow cytometric analysis of Annexin V staining using an APC Annexin V kit (BD Pharmingen) according to the manufacturer's recommendations. Briefly, the cells were washed twice using cold PBS and then resuspended in 1× binding buffer (10 mm Hepes (pH 7.4), 140 mm NaCl, and 2.5 mm CaCl2) at a concentration of 1 × 106 cells/ml. 1 × 105 cells were transferred to a new test tube, and 5 μl of APC Annexin V was added for 15 min at room temperature in the dark. 400 μl of 1× binding buffer with 3 μm 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI, Invitrogen) were added to each tube. At the end of the incubation, the cells were analyzed by a FACSAria 4-Laser (BD Pharmingen). The values were determined with H2O2-induced A549 cells and are described under “Results” (n = 3). Cell lysates were collected, and immunoblotting was performed as described (11.Panduri V. Liu G. Surapureddi S. Kondapalli J. Soberanes S. de Souza-Pinto N.C. Bohr V.A. Budinger G.R. Schumacker P.T. Weitzman S.A. Kamp D.W. Role of mitochondrial hOGG1 and aconitase in oxidant-induced lung epithelial cell apoptosis.Free Radic. Biol. Med. 2009; 47: 750-759Crossref PubMed Scopus (65) Google Scholar, 19.Panduri V. Surapureddi S. Soberanes S. Weitzman S.A. Chandel N. Kamp D.W. P53 mediates amosite asbestos-induced alveolar epithelial cell mitochondria-regulated apoptosis.Am. J. Respir. Cell Mol. Biol. 2006; 34: 443-452Crossref PubMed Scopus (56) Google Scholar). For p53 localization studies, we separated the total cellular protein into the mitochondrial and the cytosolic fractions using a Mitochondria Isolation kit (Thermo Fisher Scientific Inc., Rockford, IL) according to the manufacturer's recommendations as previously described (19.Panduri V. Surapureddi S. Soberanes S. Weitzman S.A. Chandel N. Kamp D.W. P53 mediates amosite asbestos-induced alveolar epithelial cell mitochondria-regulated apoptosis.Am. J. Respir. Cell Mol. Biol. 2006; 34: 443-452Crossref PubMed Scopus (56) Google Scholar). Protein concentration was quantified by BCA protein assay kit (Thermo Fisher Scientific). Proteins were resolved in 4∼20% acrylamide gel (Bio-Rad), transferred onto a nitrocellulose membrane, and incubated with specific antibodies. Membranes were developed with an ECL chemiluminescence detection kit (GE Healthcare Bio-Sciences, Pittsburgh, PA). The antibodies for Western blotting included polyclonal antibodies directed against hOgg1 (Novus Biological, Cambridge, UK), mitochondrial aconitase (Abcam, Cambridge, UK), cleaved caspase-9 (Cell Signaling Technology), and cytochrome oxidase IV (Cell Signaling Technology). Anti-GAPDH, c-Myc, and p53 were purchased from Santa Cruz Biotechnologies. The protein bands were visualized by enhanced chemiluminescence reaction (GE Healthcare Bio-Sciences) and quantified by densitometry using Eagle Eye software (Stratagene, La Jolla, CA). The results of each experimental condition were determined from the mean of duplicate-triplicate trials. Data was expressed as the means ± S.E. (n = 3 unless otherwise stated). A two-tailed Student's t test was used to assess the significance of differences between two groups. Analysis of variance was used when comparing more than two groups; differences between two groups within the set were analyzed by a Fisher's protected least significant differences test as well as Tukey tests. Probability values <0.05 were considered significant. To investigate the effect of oxidative stress (asbestos or H2O2) on AEC DNA damage, we used a Q-PCR-based measurement of mitochondrial and nuclear DNA damage. We found that mtDNA lesions in A549 cells are increased in a dose-dependent manner after exposure to asbestos (5–25 μg/cm2) or H2O2 (100–250 μm) over 24 h (Fig. 1A). Oxidative stress also induced mtDNA damage in primary isolated murine AT2, MLE-12, and RLE-6TN rat AT2 cells, although the levels of mtDNA lesions were less than that noted in A549 cells (Fig. 1A). Increased mtDNA lesions after exposure to asbestos or H2O2 corresponded with slightly reduced mtDNA copy number in all four AEC (Fig. 1B). Notably, negligible levels of nuclear DNA damage to β-globulin (human and mice) or clusterin (rat) were detected in these AEC after asbestos or H2O2 exposure over 24 h (Fig. 1). These data show that oxidative stress from either asbestos or H2O2 induces greater levels of mtDNA as compared with nuclear DNA damage in various types of AEC. Previous studies have established that mitochondrial-targeted mt-hOgg1-WT prevents oxidant-induced apoptosis in part by augmenting repair of mtDNA damage, but a similar role in AEC is unclear (8.Dobson A.W. Grishko V. LeDoux S.P. Kelley M.R. Wilson G.L. Gillespie M.N. Enhanced mtDNA repair capacity protects pulmonary artery endothelial cells from oxidant-mediated death.Am. J. Physiol. Lung Cell Mol. Physiol. 2002; 283: L205-L210Crossre" @default.
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• W2049477961 date "2014-02-01" @default.
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• W2049477961 title "Mitochondria-targeted Ogg1 and Aconitase-2 Prevent Oxidant-induced Mitochondrial DNA Damage in Alveolar Epithelial Cells" @default.
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• W2049477961 doi "https://doi.org/10.1074/jbc.m113.515130" @default.
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