The Ataxia telangiectasia Gene Product Is Required for Oxidative Stress-induced G1 and G2Checkpoint Function in Human Fibroblasts
2001; Elsevier BV; Volume: 276; Issue: 24 Linguagem: Inglês
10.1074/jbc.m011303200
ISSN1083-351X
AutoresRodney E. Shackelford, Cynthia L. Innes, Stella O. Sieber, Alexandra N. Heinloth, Steven A. Leadon, Richard S. Paules,
Tópico(s)Carcinogens and Genotoxicity Assessment
ResumoAtaxia telangiectasia (AT) is an autosomal recessive disorder characterized by neuronal degeneration accompanied by ataxia, telangiectasias, acute cancer predisposition, and sensitivity to ionizing radiation (IR). Cells from individuals with AT show unusual sensitivity to IR, severely attenuated cell cycle checkpoint functions, and poor p53 induction in response to IR compared with normal human fibroblasts (NHFs). The gene mutated in AT (ATM) has been cloned, and its product, pATM, has IR-inducible kinase activity. The AT phenotype has been suggested to be a consequence, at least in part, of an inability to respond appropriately to oxidative damage. To test this hypothesis, we examined the ability of NHFs and AT dermal fibroblasts to respond tot-butyl hydroperoxide and IR treatment. AT fibroblasts exhibit, in comparison to NHFs, increased sensitivity to the toxicity of t-butyl hydroperoxide, as measured by colony-forming efficiency assays. Unlike NHFs, AT fibroblasts fail to show G1 and G2 phase checkpoint functions or to induce p53 in response to t-butyl hydroperoxide. Treatment of NHFs with t-butyl hydroperoxide activates pATM-associated kinase activity. Our results indicate that pATM is involved in responding to certain aspects of oxidative damage and in signaling this information to downstream effectors of the cell cycle checkpoint functions. Our data further suggest that some of the pathologies seen in AT could arise as a consequence of an inability to respond normally to oxidative damage. Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by neuronal degeneration accompanied by ataxia, telangiectasias, acute cancer predisposition, and sensitivity to ionizing radiation (IR). Cells from individuals with AT show unusual sensitivity to IR, severely attenuated cell cycle checkpoint functions, and poor p53 induction in response to IR compared with normal human fibroblasts (NHFs). The gene mutated in AT (ATM) has been cloned, and its product, pATM, has IR-inducible kinase activity. The AT phenotype has been suggested to be a consequence, at least in part, of an inability to respond appropriately to oxidative damage. To test this hypothesis, we examined the ability of NHFs and AT dermal fibroblasts to respond tot-butyl hydroperoxide and IR treatment. AT fibroblasts exhibit, in comparison to NHFs, increased sensitivity to the toxicity of t-butyl hydroperoxide, as measured by colony-forming efficiency assays. Unlike NHFs, AT fibroblasts fail to show G1 and G2 phase checkpoint functions or to induce p53 in response to t-butyl hydroperoxide. Treatment of NHFs with t-butyl hydroperoxide activates pATM-associated kinase activity. Our results indicate that pATM is involved in responding to certain aspects of oxidative damage and in signaling this information to downstream effectors of the cell cycle checkpoint functions. Our data further suggest that some of the pathologies seen in AT could arise as a consequence of an inability to respond normally to oxidative damage. ataxia telangiectasia affinity-purified αATM 7 antibody ataxia telangiectasia-mutated gene product 5-bromo-2′-deoxyuridine bovine serum albumin 4′,6-diamidino-2-phenylindole heme oxygenase-1 ionizing radiation normal human fibroblast phosphate buffered saline gray phosphate-buffered saline polyacrylamide gel electrophoresis Ataxia telangiectasia (AT)1 is an autosomal recessive disorder characterized by immune disorders, acute cancer predisposition, telangiectasias, sensitivity to ionizing radiation (IR), and neuronal degeneration (1Kastan M.B. Onyekere O. Sidransky D. Volgelstein B. Craig R.W. Cancer Res. 1991; 51: 6304-6311PubMed Google Scholar). The number of individuals who carry one defective copy of the AT gene has been estimated to be around 0.5–1% of the general population (2Broeks A. Urbanus J.H. Floore A.N. Dahler E.C. Klijn J.G. Rutgers E.J. Devilee P. Russell N.S. van Leeuwen F.E. van't Veer L.J. Am. J. Hum. Genet. 2000; 66: 494-500Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Those AT heterozygotes have been reported to exhibit elevated cancer risk, particularly for breast cancer and lymphoproliferative disease (3Swift M. Morrell D. Massey R.B. Chase C.L. N. Engl. J. Med. 1991; 325: 1831-1836Crossref PubMed Scopus (816) Google Scholar, 4Meyn M.S. Science. 1993; 260: 1327-1330Crossref PubMed Scopus (215) Google Scholar, 5Easton D.F. Int. J. Radiat. Biol. 1994; 66: 177-182Crossref PubMed Scopus (262) Google Scholar). Cultured AT dermal fibroblasts show increased chromosomal instability and acute sensitivity to IR in comparison to age-matched normal human fibroblasts (NHFs) (6Painter R.B. Young B.R. Biochim. Biophys. Acta. 1976; 418: 146-153Crossref PubMed Scopus (122) Google Scholar, 7Shiloh Y. Tabor E. Becker Y. Exp. Cell Res. 1982; 140: 191-199Crossref PubMed Scopus (46) Google Scholar, 8Scott D. Spreadborough A.R. Roberts S.A. Int. J. Radiat. Biol. 1994; 66: 157-163Crossref PubMed Scopus (57) Google Scholar, 9Gatti R.A. Berkel I. Boder E. Braedt G. Charmley P. Concannon P. Ersoy F. Foroud T. Jaspers N.G. Lange K. Lathrop G.M. Leppert M. Nakamura Y. O'Connell P. Paterson M. Salser W. Sanal O. Silver J. Sparkes R.S. Susi E. Weeks D.E. Wei S. White R. Yoder F. Nature. 1988; 336: 577-580Crossref PubMed Scopus (567) Google Scholar, 10McKinnon P.J. Hum. Genet. 1987; 75: 197-208Crossref PubMed Scopus (178) Google Scholar). Cells from individuals with AT exhibit poor p53 induction and severely impaired G1, S, and G2phase checkpoint functions in response to IR (11Painter R.B. Young B.R. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7315-7317Crossref PubMed Scopus (722) Google Scholar, 12Zampetti-Bosseler F. Scott D. Int. J. Radiat. Biol. 1981; 39: 547-558Crossref Scopus (198) Google Scholar, 13Beamish H. Lavin M.F. Int. J. Radiat. Biol. 1994; 65: 175-184Crossref PubMed Scopus (186) Google Scholar, 14Paules R.S. Levedakou E.N. Wilson S.J. Innes C.L. Rhodes N. Tlsty T.D. Galloway D.A. Donehower L.A. Tainsky M.A. Kaufmann W.K. Cancer Res. 1995; 55: 1763-1773PubMed Google Scholar). The gene mutated in AT, ATM, has been identified (15Savitsky K. Barshira A. Gilad S. Rotman G. Ziv Y. Vanagaite L. Tagle D.A. Smith S. Uziel T. Sfez S. Ashkenazi M. Pecker I. Frydman M. Harnik R. Patanjali S.R. Simmons A. Clines G.A. Sartiel A. Gatti R.A. Chessa L. Sanal O. Lavin M.F. Jaspers N.G.J. Malcolm A. Taylor R. Arlett C.F. Miki T. Weissman S.M. Lovett M. Collins F.S. Shiloh Y. Science. 1995; 268: 1749-1753Crossref PubMed Scopus (2377) Google Scholar), and the gene product, pATM, has been shown to have IR-inducible protein kinase activity (16Banin S. Moyal L. Shieh S.Y. Taya Y. Anderson C.W. Chessa L. Smorodinsky N.I. Prives C. Reiss Y. Shiloh Y. Ziv Y. Science. 1998; 281: 1674-1677Crossref PubMed Scopus (1711) Google Scholar,17Canman C.E. Lim D.S. Cimprich K.A. Taya Y. Tamai K. Sakaguchi K. Appella E. Kastan M.B. Siliciano J.D. Science. 1998; 281: 1677-1679Crossref PubMed Scopus (1712) Google Scholar). ATM shares homology with the family of phosphatidylinositol 3′-kinases (15Savitsky K. Barshira A. Gilad S. Rotman G. Ziv Y. Vanagaite L. Tagle D.A. Smith S. Uziel T. Sfez S. Ashkenazi M. Pecker I. Frydman M. Harnik R. Patanjali S.R. Simmons A. Clines G.A. Sartiel A. Gatti R.A. Chessa L. Sanal O. Lavin M.F. Jaspers N.G.J. Malcolm A. Taylor R. Arlett C.F. Miki T. Weissman S.M. Lovett M. Collins F.S. Shiloh Y. Science. 1995; 268: 1749-1753Crossref PubMed Scopus (2377) Google Scholar), which include DNA-PK, ATR, MEC1, TEL1, TOR, and FRAP among others (18Keith C.T. Schreiber S.L. Science. 1995; 270: 50-51Crossref PubMed Scopus (451) Google Scholar). Members of this family of proteins have been reported to be involved in various aspects of the detection of DNA damage and control of cell cycle progression (18Keith C.T. Schreiber S.L. Science. 1995; 270: 50-51Crossref PubMed Scopus (451) Google Scholar,19Savitsky K. Sfez S. Tagle D.A. Ziv Y. Sartiel A. Collins F.S. Shiloh Y. Rotman G. Hum. Mol. Genet. 1995; 4: 2025-2032Crossref PubMed Scopus (478) Google Scholar). pATM has been suggested to function, at least in part, in the cellular response to oxidative damage (for review see Ref. 20Rotman G. Shiloh Y. BioEssays. 1997; 19: 911-917Crossref PubMed Scopus (144) Google Scholar). Support for this hypothesis comes from observations that pATM-deficient cells are unusually sensitive to the toxic effects of hydrogen peroxide, nitric oxide, and superoxide treatment as determined by colony-forming efficiency assays. Additionally, they resynthesize glutathione unusually slowly after depletion with diethyl maleate (21Vuillaume M. Mutat. Res. 1987; 186: 43-72Crossref PubMed Scopus (260) Google Scholar, 22Meredith M.J. Dodson M.L. Cancer Res. 1987; 47: 4576-4581PubMed Google Scholar, 23Vuillaume M. Best-Belpomme M. Lafont R. Hubert M. Decroix Y. Sarasin A. Carcinogenesis. 1989; 10: 1375-1381Crossref PubMed Scopus (12) Google Scholar, 24Ward A.J. Olive P.L. Burr A.H. Rosin M.P. Environ. Mol. Mutagen. 1994; 24: 103-111Crossref PubMed Scopus (56) Google Scholar, 25Green M.H.L. Marcovitch A.J. Harcourt S.A. Lowe J.E. Green I.C. Arlett C.F. Free Radic. Biol. Med. 1997; 22: 343-347Crossref PubMed Scopus (24) Google Scholar). Furthermore, Barlow and colleagues (26Barlow C. Dennery P.A. Shigenaga M.K. Smith M.A. Morrow J.D. Roberts L.J. Wynshaw-Boris A. Levine R.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9915-9919Crossref PubMed Scopus (222) Google Scholar) have shown thatATM-deficient mice have elevated markers of oxidative stress, particularly in organs such as the cerebellum, which are consistently affected in individuals with AT. Therefore, we hypothesized that pATM-deficient fibroblasts would lack normal cell cycle checkpoint function in response to oxidative stress. To test this hypothesis, we compared the effect of IR, and reactive oxygen species produced by treatment with t-butyl hydroperoxide, on normal and ATM-deficient human fibroblast strains. NHF1 is a normal human fibroblast culture derived from foreskins of apparently healthy neonates and was used at passages 13–19 (27Boyer J.C. Kaufmann W.K. Brylawski B.P. Cordeiro-Stone M. Cancer Res. 1990; 50: 2593-2598PubMed Google Scholar). GM03349, a normal dermal fibroblast strain from a 10-year-old male, was obtained from NIGMS (National Institutes of Health) Human Genetic Mutant Cell Repository (Camden, NJ) and used at passages 15–19. ATM-deficient dermal fibroblasts were obtained from the NIGMS Human Genetic Mutant Cell Repository (strain designations GM02052 and GM03395 (Camden, NJ)) and the NIA Aging Cell Repository (strain AG03058 (Camden, NJ)). The donor for the cells of strain GM02052 was a 15-year-old Moroccan female. Cells of this strain contain a mutation at nucleotide 103 that causes a change in coding from cysteine to thymidine, resulting in a stop codon at position 35 (28). The cells of strain GM03395 are dermal fibroblasts cultured from a skin biopsy of a 13-year-old black male, and the cells of strain AG03058 are dermal fibroblasts cultured from a skin biopsy from a 14-year-old black female. The exact mutations ofATM in strains GM03395 and AG03058 are not known. However, no pATM could be detected by immunoblotting in protein extracts from any of these strains, and the donors expressed the typical phenotype of the AT disease. These cells were used at passages 14–20. Normal human fibroblasts (NHF1 and GM03349 strains) were grown at 37 °C in a humidified 5% CO2 atmosphere in minimum Eagle's medium, supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and 2 mm glutamine (Life Technologies, Inc.) (NHF medium).ATM-deficient fibroblasts (GM02052, GM03395, and AG03058) were grown under the same conditions in minimum Eagle's medium supplemented with 20% fetal bovine serum, 2 mm glutamine, 0.4 mm serine, 0.2 mm aspartic acid, and 2 mm pyruvic acid (AT medium) (14Paules R.S. Levedakou E.N. Wilson S.J. Innes C.L. Rhodes N. Tlsty T.D. Galloway D.A. Donehower L.A. Tainsky M.A. Kaufmann W.K. Cancer Res. 1995; 55: 1763-1773PubMed Google Scholar). HeLa cells, obtained from the American Type Culture Collection (Manassas, VA), were cultured as above in minimum Eagle's medium with 5% fetal bovine serum and 2 mm glutamine. Cells were tested and found to be mycoplasma free. Logarithmically growing cell populations in 100-mm plastic dishes (Becton Dickinson Labware, Franklin Lakes, NJ) were exposed to γ-rays at room temperature using a 137Cs source at a rate of 2.6 Gy/min. Mock-treated control cells were subjected to the same movements both in and out of the incubators as treated cells, for both IR andt-butyl hydroperoxide treatments. When cells were treated with t-butyl hydroperoxide, the t-butyl hydroperoxide was added to cell cultures for 15 min. The cultures were then washed 2× with warm media, and the media were replaced. In experiments where some cell cultures were treated witht-butyl hydroperoxide, all plates within the same experiment were washed as described above. After treatment, the cells were incubated for the times indicated and harvested. Logarithmically growing fibroblast populations were harvested by typsinization, counted in a cell counter (Coulter Counter ZM, Coulter Corp., Miami, Fl), and replated at a density of 103 fibroblasts/100-mm tissue culture dish. After allowing cells to adhere for 12 h, the fibroblasts were exposed for 15 min to various concentrations oft-butyl hydroperoxide, washed, and incubated for 8–12 days in appropriate media. For assays employing mannitol, fibroblasts were treated 1 h with 1 mm mannitol, followed by treatment with t-butyl hydroperoxide in the continued presence of 1 mm mannitol, washed, and incubated as described above. Media were removed and colonies fixed by the addition of water/methanol (1:1, v/v) containing crystal violet (1 g/liter) and counted using a dissecting microscope. For each fibroblast strain, a minimum of two colony-forming assay experiments was performed, with each data point done in triplicate. G1 checkpoint function was assayed by flow cytometry using a modification of the cell cycle analysis protocol in Kastan et al. (1Kastan M.B. Onyekere O. Sidransky D. Volgelstein B. Craig R.W. Cancer Res. 1991; 51: 6304-6311PubMed Google Scholar) for simultaneous analysis of DNA synthesis and cell cycle. Logarithmically growing cells were either mock-treated, exposed to 3.0 Gy γ-IR, or exposed to various concentrations oft-butyl hydroperoxide as described above. 4 h following treatment, BrdUrd (Roche Molecular Biochemicals) was added to the media to a final concentration of 10 μm, and cells were incubated for an additional 2 h. Cells were harvested, fixed in phosphate-buffered saline (PBS)/methanol at a 1:2 (v/v) ratio, and stored at −20 °C. 5 × 105 cells from each sample were stained for BrdUrd incorporation in a solution of Tween 20/BSA plus anti-BrdUrd antibody (Becton Dickinson catalog number 347580) as recommended by the manufacturer. Fluorescein isothiocyanate-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) was used as secondary antibody. Cells were stained with PBS containing 5 μg/ml propidium iodide (Roche Molecular Biochemicals) and analyzed using a Becton Dickinson FACSort. Twenty thousand cells were counted for each analysis. Flow cytometric experiments were done twice with each treatment point done in duplicate. After treatment, cells were harvested and solubilized on ice in kinase lysis buffer with inhibitors (10 mm sodium phosphate (pH 7.2), 150 mm NaCl, 1% Nonidet P-40, 1 mm EDTA, 5 mm EGTA, 5 mmβ-glycerophosphate, 1 mm dithiothreitol, 120 kallikrein IU/ml aprotinin, and 10 μg/ml leupeptin). Protein concentration was determined using a detergent-compatible protein assay kit (Bio-Rad) with bovine serum albumin (BSA) as a standard. Although the amount of protein used in different kinase assay experiments varied, within each experiment the protein concentrations employed were the same for all samples. In p34CDC2/cyclin B histone H1 in vitrokinase activity assays 50–100 μg of protein was used per kinase reaction, whereas 500–700 μg of protein was used per p33CDK2/cyclin E histone H1 in vitro kinase assay. The desired amount of protein from solubilized extracts was aliquoted into 1.5-ml microcentrifuge tubes, and volumes were adjusted to 500 μl with kinase lysis buffer. The immunoprecipitations were done with either 0.5 μl of anti-human cyclin B Powerclonal antibody (catalog number 05-373, Upstate Biotechnology, Inc., Lake Placid, NY) or 2.0 μl of anti-human cyclin E Powerclonal antibody (catalog number 05-371, Upstate Biotechnology, Inc., Lake Placid, NY). Samples were precleared with protein G-agarose beads (Life Technologies, Inc.), then incubated with the primary antibody for 2 h, followed by the addition of protein G-agarose beads. Kinase reactions were carried out in histone H1 kinase buffer (20 mm HEPES (pH 7.3), 80 mm β-glycerophosphate, 20 mm EGTA, 50 mm MgCl2, 5 mm MnCl2, 1 mm dithiothreitol, 60 kallikrein IU/ml aprotinin, 10 μg/ml leupeptin, 10 μmcyclic AMP-dependent protein kinase-inhibitory peptide), with 8 μg of histone H1 and 10 μCi of [32γ-P]ATP (3,000 Ci/mmol, Amersham Pharmacia Biotech) for 30 min at 37 °C. The kinase reactions were stopped by addition of 2× SDS sample buffer (4% SDS, 150 mm Tris (pH 6.8), 20% glycerol, 1 mmβ-mercaptoethanol, 0.02% bromphenol blue), and proteins were resolved by 12% SDS-PAGE. Gels were stained with Coomassie Blue to verify equal histone protein loading, dried, and subjected to autoradiography with Hyperfilm MP (Amersham Pharmacia Biotech). The radiolabeled protein substrates in the dried gels were then quantified using a Molecular Dynamics PhosphorImager and ImageQuant software. All kinase assays were performed at least in triplicate. After appropriate treatment, fibroblasts were fixed on 100-mm dishes by the gentle addition of cold methanol. After 10 min the plates were air-dried and stored at 4 °C until staining with 0.2 μg/ml 4′,6-diamidino-2-phenylindole (DAPI). DAPI-stained cells then were examined by fluorescence microscopy. The percentage of mitotic cells (the mitotic index) was determined from counts of a minimum of 5,000 cells. All mitotic delay treatments were performed in duplicate, and all experiments were done in duplicate. Immunoprecipitations of p53 were performed with whole cell extracts from five 100-mm tissue culture dishes (∼800 μg protein/IP) as described above for protein kinase assays, using 2.5 μl of anti-p53 murine monoclonal antibody (catalog number OP03, Oncogene Research Products, Cambridge, MA) per sample. Eluted proteins were resolved by 12% SDS-PAGE and transferred to 0.2-μm nitrocellulose membranes. The blots were probed with anti-p53 antibody (a generous gift from Dr. B. Alex Merrick, NIEHS), which had been raised in rabbits against immunopurified human r-p53 expressed in a baculovirus expression system. By using anti-rabbit IgG peroxidase-conjugated goat antibody (Roche Molecular Biochemicals), p53 protein was visualized by chemiluminescence (Pierce) followed by exposure to Hyperfilm MP (Amersham Pharmacia Biotech). p53 immunoprecipitations and Western analysis were performed in triplicate. To examine the effect of t-butyl hydroperoxide on HO-1 protein levels in normal and AT fibroblasts, we treated AT and NHF1 fibroblasts with 1–10 μm t-butyl hydroperoxide for 4 h. Cells were then harvested and lysed as described above; 100 μg of whole cell protein extract was loaded per lane; proteins were resolved by 12% SDS-PAGE and transferred to nitrocellulose membranes as described above. Nitrocellulose blots were probed with anti-HO-1 rabbit polyclonal antibody (catalog number PA3-019, Affinity Bioreagents, Golden, CO) and visualized using anti-rabbit IgG peroxidase-conjugated antibody (Roche Molecular Biochemicals) as described above. All HO-1 induction Western blots were done in triplicate. In order to isolate the highest affinity, highest specificity αATM 7 antibodies from the rabbit polyclonal antiserum, antibody was affinity-purified using the original peptide immunogen. Peptide corresponding to pATM residues 826–840 (ATM-N826 peptide) was immobilized to generate a peptide column in the following manner. Ten mg of ATM-N826 peptide were mixed with 1.0 ml of Affi-Gel 15 (Bio-Rad). The reaction mixture was mixed at room temperature for 2–3 h, and then the organic solvents were replaced with 10% ethanolamine in water for 1 h at room temperature to terminate the reaction and block any unreacted groups that remained. The derivatized Affi-Gel was poured into a column and cleaned by passing 4 m potassium thiocyanate through the packing. The column was washed with PBS and stored in PBS with 0.02% sodium azide at 4 °C. To affinity-purify antipeptide antibody, IgG was isolated from the whole polyclonal rabbit serum using an Econo-Pac Protein A kit (Bio-Rad). Twenty five to 30 mg of the protein A-purified IgG was added to the Affi-Gel peptide column and tumbled overnight at 4 °C. The antibody-bound peptide beads were washed with 5 column volumes of 1m potassium thiocyanate, and fractions were eluted with 4m potassium thiocyanate. BSA (0.1%) was added to peak fractions prior to dialysis in 1× PBS overnight at 4 °C. The peak fractions were verified by Western blotting of total protein extracts from normal and ATM-deficient fibroblasts that had been resolved by 6% SDS-PAGE (acrylamide/bisacrylamide ratio of 100:1). Affinity-purified αATM 7 (αATM 7 a.p.) antibody was stored in 20% glycerol and 0.02% sodium azide at −20 °C. The pATM in vitro PHAS-1 kinase assays were done as described previously (16Banin S. Moyal L. Shieh S.Y. Taya Y. Anderson C.W. Chessa L. Smorodinsky N.I. Prives C. Reiss Y. Shiloh Y. Ziv Y. Science. 1998; 281: 1674-1677Crossref PubMed Scopus (1711) Google Scholar,17Canman C.E. Lim D.S. Cimprich K.A. Taya Y. Tamai K. Sakaguchi K. Appella E. Kastan M.B. Siliciano J.D. Science. 1998; 281: 1677-1679Crossref PubMed Scopus (1712) Google Scholar). Cells were incubated for 90 min following exposure with 6.0 Gy γ-IR or a 15-min exposure to 300 μm t-butyl hydroperoxide and then lysed in kinase lysis buffer with 10 mm β-glycerophosphate and 1 mmNaVO3 added. Cell lysates were clarified by centrifugation; the protein concentration was determined; the volume was adjusted to 0.9 ml with kinase lysis buffer plus inhibitors, and protein G-agarose beads were added to pre-clear the lysates as described above. After 30 min, the pre-cleared lysates were removed from the protein G-agarose beads and added to either 0.1 ml of kinase lysis buffer with inhibitors alone or containing αATM 7 a.p. antibody. In order to test antibody specificity, in some treatments the αATM 7 a.p. antibody was incubated for 30 min prior to its addition to the cell lysates with either 25 μg of the peptide that the antibody was raised against (ATM-N826) or with 25 μg of an irrelevant peptide sequence of the same length. All pATM kinase reactions were performed at least in triplicate using 1.5–3.0 mg protein lysate per immunoprecipitation. To quantify the damage induced in NHFs andATM-deficient fibroblasts by t-butyl hydroperoxide and IR, we measured thymine glycol formation in the DNA from ATM-normal NHF1 cells and ATM-deficient AG03058 fibroblasts following treatment by each agent. Six plates of logarithmically growing NHF1 or AG03058 fibroblasts, at ∼70% confluence, were treated with 100 or 300 μm t-butyl hydroperoxide or 6 Gy γ-IR, as described above. Cells were harvested from each plate on ice with 5 ml of cold PBS as soon as possible following treatment. The cells were pelleted, washed 1 time in cold PBS, re-pelleted in 1.5-ml microcentrifuge tubes, and quick-frozen in dry ice/ethanol until assayed. Determination of thymine glycol formation was performed as described previously (29Le X.C. Xing J.Z. Lee J. Leadon S.A. Weinfeld M. Science. 1998; 280: 1066-1069Crossref PubMed Scopus (187) Google Scholar). Each experimental point was performed in triplicate. To examine the relative toxicity of reactive oxygen species exposure between normal and pATM-deficient fibroblasts, we treated different fibroblast strains witht-butyl hydroperoxide. We employed t-butyl hydroperoxide as a source of oxidative stress as it is poorly hydrolyzed by catalase (30Winston G.W. Harvey W. Berl L. Cederbaum A.I. Biochem. J. 1983; 216: 415-421Crossref PubMed Scopus (61) Google Scholar). We reasoned that this is important since catalase activity has been reported to be low inATM-deficient cells (31Vuillaume M. Calvayrac R. Best Belpomme M. Tarroux P. Hubert M. Decroix Y. Sarasin A. Cancer Res. 1986; 46: 538-544PubMed Google Scholar, 32Watters D. Kedar P. Spring K. Bjorkman J. Chen P. Gatei M. Birrell G. Garrone B. Srinivasa P. Crane D.I. Lavin M.F. J. Biol. Chem. 1999; 274: 34277-34282Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) and thus, using a peroxide that can be hydrolyzed by catalase, would introduce experimental variation due to differences in cellular catalase activities. To initiate these studies, two normal and two ATM-deficient fibroblast strains were treated with increasing levels of IR, and toxicity was assayed by colony-forming efficiency. As reported previously (7Shiloh Y. Tabor E. Becker Y. Exp. Cell Res. 1982; 140: 191-199Crossref PubMed Scopus (46) Google Scholar), exposure to increasing amounts of IR inhibited colony formation in ATM-deficient fibroblast strains more effectively than in NHFs (Fig.1 A). To compare the effects oft-butyl hydroperoxide on normal and ATM-deficient fibroblasts, normal NHF1 and GM03349 cells and ATM-deficient GM02052 and GM03395 cells were treated with increasing concentrations of t-butyl hydroperoxide. As shown in Fig. 1 B, the ATM-deficient fibroblasts were more sensitive to the colony-forming inhibiting effects of t-butyl hydroperoxide than the normal fibroblasts. LC50 (lethal concentration for 50% of population) for both normal fibroblast strains was in the 40–50 μm t-butyl hydroperoxide range, whereas the LC50 for the ATM-deficient dermal fibroblast strains was in the 6–8 μm range (Fig. 1 B). The colony-inhibiting effect of t-butyl hydroperoxide in both normal and ATM-deficient fibroblast strains was biphasic, with an initial high sensitivity to low concentrations oft-butyl hydroperoxide (1–10 μm), followed by less sensitivity at higher concentrations (>10 μm). This apparent biphasic response is pATM-independent and suggests to us that higher concentrations of t-butyl hydroperoxide induce a pATM-independent resistance or adaptive response to the effects oft-butyl hydroperoxide in both cell types (Fig.1 B, compare 1–10 μm to 10–300 μm t-butyl hydroperoxide). Peroxides are thought to exert some of their damaging effects through the production of reactive oxygen intermediates via events such as the Fenton reaction (for review see Ref. 33Shackelford R.E. Kaufmann W.K. Paules R.S. Environ. Health Perspect. 1999; 107: 5-24Crossref PubMed Scopus (228) Google Scholar). We pretreated fibroblasts with mannitol for 1 h prior to t-butyl hydroperoxide treatment in order to ascertain if the colony-inhibiting effects oft-butyl hydroperoxide could be reduced by co-treatment with an antioxidant. Mannitol, which is effective at scavenging hydroxyl radicals (34Regoli F. Winston G.W. Toxicol. Appl. Pharmacol. 1999; 156: 96-105Crossref PubMed Scopus (364) Google Scholar), was used as an antioxidant. As shown in Fig.1 C, pretreatment with mannitol partially inhibited the killing effect of t-butyl hydroperoxide on both normal andATM-deficient fibroblast strains. For most concentrations examined, the differences between treatment with and without mannitol are significant. However, pretreatment with mannitol of theATM-deficient cells showed less reduction of killing following treatment with the highest concentration oft-butyl hydroperoxide (100 μm) (Fig.1 C). This is likely to be due to the very low number ofATM-deficient cells that can still form colonies at this concentration of t-butyl hydroperoxide. Nevertheless, these data show that the toxic effect of t-butyl hydroperoxide can be partially reversed by pretreatment with an antioxidant. G1 checkpoint function, as reflected by delay of entry into S phase, was assayed following exposure to oxidative stress. Exposure of NHF1 fibroblasts in logarithmic growth phase to t-butyl hydroperoxide over a 10–100 μm range resulted in a concentration-dependent suppression of S phase entry, as measured by flow cytometry (Table I and Fig. 2). When theATM-deficient fibroblast strain AG03058 was subjected to the same treatment, comparatively little inhibition of S phase entry was observed over the same concentrations of t-butyl hydroperoxide (Table I and Fig. 2). As demonstrated previously (11Painter R.B. Young B.R. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7315-7317Crossref PubMed Scopus (722) Google Scholar,35Beamish H. Khanna K.K. Lavin M.F. Radiat. Res. 1994; 138: 5130-5133Crossref Scopus (48) Google Scholar), normal fibroblasts exhibit G1 checkpoint arrest in response to IR, whereas ATM-deficient fibroblasts did not (Table I and Fig. 2).Table IFlow cytometric analysis of ATM-normal NHF1 and ATM-deficient AG03058 fibroblasts following treatment with increasing concentrations of t-butyl hydroperoxide (A) and 3.0 Gy IR (B)Treatment/concentrationRelative percentage of cells in early S phase1-aPercentage of treated cells in early S phase relative to the percentage of mock-treated control cells in early S phase.NHF1 (ATM +/+)AG03058 (ATM −/−)A.t-Butyl-OOH0 μm100 (±8)1-b±, standard deviation.100 (±8)10 μm100 (±7)104 (±10)30 μm70 (±11)117 (±4)100 μm54 (±5)93 (±5)B. γ-IR0 Gy100 (±7)100 (±7)3.0 Gy19 (±9)96 (±6)1-a Percentage of treated cells in early S phase relative to the percentage of mock-treated control cells in early S phase.1-b ±, standard deviation. Open table in
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