The Ataxia telangiectasia Gene Product Is Required for Oxidative Stress-induced G 1 and G 2 Checkpoint Function in Human Fibroblasts*

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by neuronal degeneration ac-companied by ataxia, telangiectasias, acute cancer pre-disposition, 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 to t- 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 G 1 and G 2 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

the cells of strain AG03058 are dermal fibroblasts cultured from a skin biopsy from a 14-year-old black female. The exact mutations of ATM 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% CO 2 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) (14). 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 ␥-rays at room temperature using a 137 Cs 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 and t-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 2ϫ with warm media, and the media were replaced. In experiments where some cell cultures were treated with t-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.
Cell Colony-forming Efficiency Assay-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 10 3 fibroblasts/100-mm tissue culture dish. After allowing cells to adhere for 12 h, the fibroblasts were exposed for 15 min to various concentrations of t-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.
Flow Cytometry for G 1 Checkpoint Function-G 1 checkpoint function was assayed by flow cytometry using a modification of the cell cycle analysis protocol in Kastan et al. (1) for simultaneous analysis of DNA synthesis and cell cycle. Logarithmically growing cells were either mock-treated, exposed to 3.0 Gy ␥-IR, or exposed to various concentrations of t-butyl hydroperoxide as described above. 4 h following treatment, BrdUrd (Roche Molecular Biochemicals) was added to the media to a final concentration of 10 M, 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 Ϫ20°C. 5 ϫ 10 5 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 Immu-noResearch Laboratories, West Grove, PA) was used as secondary antibody. Cells were stained with PBS containing 5 g/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.
Protein Analysis and Histone H1 in Vitro Kinase Assays-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 mM ␤-glycerophosphate, 1 mM dithiothreitol, 120 kallikrein IU/ml aprotinin, and 10 g/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 p34 CDC2 /cyclin B histone H1 in vitro kinase activity assays 50 -100 g of protein was used per kinase reaction, whereas 500 -700 g of protein was used per p33 CDK2 /cyclin E histone H1 in vitro kinase assay. The desired amount of protein from solubilized extracts was aliquoted into 1.5-ml microcen-trifuge tubes, and volumes were adjusted to 500 l with kinase lysis buffer. The immunoprecipitations were done with either 0.5 l of antihuman cyclin B Powerclonal antibody (catalog number 05-373, Upstate Biotechnology, Inc., Lake Placid, NY) or 2.0 l 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 ␤-glycerophosphate, 20 mM EGTA, 50 mM MgCl 2 , 5 mM MnCl 2 , 1 mM dithiothreitol, 60 kallikrein IU/ml aprotinin, 10 g/ml leupeptin, 10 M cyclic AMP-dependent protein kinase-inhibitory peptide), with 8 g of histone H1 and 10 Ci of [ 32 ␥-P]ATP (3,000 Ci/mmol, Amersham Pharmacia Biotech) for 30 min at 37°C. The kinase reactions were stopped by addition of 2ϫ SDS sample buffer (4% SDS, 150 mM Tris (pH 6.8), 20% glycerol, 1 mM ␤-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 Phosphor-Imager and ImageQuant software. All kinase assays were performed at least in triplicate.
Quantification of Mitotic Delay Induced by t-Butyl Hydroperoxide and IR-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 g/ml 4Ј,6diamidino-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.
Protein Analyses-Immunoprecipitations of p53 were performed with whole cell extracts from five 100-mm tissue culture dishes (ϳ800 g protein/IP) as described above for protein kinase assays, using 2.5 l 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-m 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 M t-butyl hydroperoxide for 4 h. Cells were then harvested and lysed as described above; 100 g 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.
Affinity Purification of pATM Antibody, ␣ATM 7 a.p.-In order to isolate the highest affinity, highest specificity ␣ATM 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 1 M potassium thiocyanate, and fractions were eluted with 4 M potassium thiocyanate. BSA (0.1%) was added to peak fractions prior to dialysis in 1ϫ 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 ␣ATM 7 (␣ATM 7 a.p.) antibody was stored in 20% glycerol and 0.02% sodium azide at Ϫ20°C. pATM in Vitro PHAS-1 Kinase Assay-The pATM in vitro PHAS-1 kinase assays were done as described previously (16,17). Cells were incubated for 90 min following exposure with 6.0 Gy ␥-IR or a 15-min exposure to 300 M t-butyl hydroperoxide and then lysed in kinase lysis buffer with 10 mM ␤-glycerophosphate and 1 mM NaVO 3 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 ␣ATM 7 a.p. antibody. In order to test antibody specificity, in some treatments the ␣ATM 7 a.p. antibody was incubated for 30 min prior to its addition to the cell lysates with either 25 g of the peptide that the antibody was raised against (ATM-N826) or with 25 g 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.

Quantification of Thymine Glycol Formation in ATM-normal and ATM-deficient Fibroblasts after Treatment with t-Butyl
Hydroperoxide and IR-To quantify the damage induced in NHFs and ATM-deficient fibroblasts by t-butyl hydroperoxide and IR, we measured thymine glycol formation in the DNA from ATM-normal NHF1 cells and ATMdeficient AG03058 fibroblasts following treatment by each agent. Six plates of logarithmically growing NHF1 or AG03058 fibroblasts, at ϳ70% confluence, were treated with 100 or 300 M t-butyl hydroperox-ide or 6 Gy ␥-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 (29). Each experimental point was performed in triplicate.

AT Fibroblast Strains Are Hypersensitive to the Toxic Effects of t-Butyl
Hydroperoxide-To examine the relative toxicity of reactive oxygen species exposure between normal and pATMdeficient fibroblasts, we treated different fibroblast strains with t-butyl hydroperoxide. We employed t-butyl hydroperoxide as a source of oxidative stress as it is poorly hydrolyzed by catalase (30). We reasoned that this is important since catalase activity has been reported to be low in ATM-deficient cells (31,32) 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 (7), exposure to increasing amounts of IR inhibited colony formation in ATM-deficient fibroblast strains more effectively than in NHFs (Fig. 1A). To compare the effects of t-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. 1B, the ATM-deficient fibroblasts were more sensitive to the colony-forming inhibiting effects of t-butyl hydroperoxide than the normal fibroblasts. LC 50 (lethal concentration for 50% of population) for both normal fibroblast strains was in the 40 -50 M t-butyl hydroperoxide range, whereas the LC 50 for the ATMdeficient dermal fibroblast strains was in the 6 -8 M range (Fig. 1B). 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 of t-butyl hydroperoxide (1-10 M), followed by less sensitivity at higher concentrations (Ͼ10 M). 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 of t-butyl hydroperoxide in both cell types (Fig. 1B, compare 1-10 M to 10 -300 M 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. 33). We pretreated fibroblasts with mannitol for 1 h prior to t-butyl hydroperoxide treatment in order to ascertain if the colonyinhibiting effects of t-butyl hydroperoxide could be reduced by co-treatment with an antioxidant. Mannitol, which is effective at scavenging hydroxyl radicals (34), was used as an antioxidant. As shown in Fig. 1C, pretreatment with mannitol partially inhibited the killing effect of t-butyl hydroperoxide on both normal and ATM-deficient fibroblast strains. For most concentrations examined, the differences between treatment with and without mannitol are significant. However, pretreatment with mannitol of the ATM-deficient cells showed less reduction of killing following treatment with the highest concentration of t-butyl hydroperoxide (100 M) (Fig. 1C). This is likely to be due to the very low number of ATM-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.
Fibroblasts Lacking pATM Function Fail to Exhibit G 1 Checkpoint Delay in Response to t-Butyl Hydroperoxide Exposure-G 1 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 M range resulted in a concentrationdependent suppression of S phase entry, as measured by flow cytometry (Table I and Fig. 2). When the ATM-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 (11,35), normal fibroblasts exhibit G 1 checkpoint arrest in response to IR, whereas ATM-deficient fibroblasts did not (Table I and Fig. 2).
p53 protein stabilization and activation, which is necessary for the elicitation of a full G 1 checkpoint response to cellular damage from IR exposure for more than a transient period (36 -38), is severely attenuated in ATM-deficient cells (35)(36)(37). Since ATM-deficient cells exhibit poor p53 induction in response to IR and fail to show a G 1 checkpoint function in response to both IR and t-butyl hydroperoxide treatment, we hypothesized that p53 induction in response to t-butyl hydroperoxide treatment would similarly be poor in AT fibro-  blasts compared with NHFs. When p53 immunoprecipitations were performed, followed by Western blotting with a second anti-p53 antibody, we found that p53 was induced in NHF1 cells in response to both t-butyl hydroperoxide and IR (Fig. 3). p53 induction in response to t-butyl hydroperoxide was slower than induction in response to IR, with significant induction following IR exposure by 1 h and induction by t-butyl hydroperoxide peaking later, ϳ1-2 h (Fig. 3). At the time points examined here, neither IR nor t-butyl hydroperoxide treatment significantly induced p53 protein levels in the ATM-deficient fibroblast strain GM02052 (Fig. 3).
p33 CDK2 /cyclin E-associated kinase activity has been shown to be required for progression through late G 1 and into S phase (39). To test the effect of t-butyl hydroperoxide on p33 CDK2 / cyclin E-associated kinase activity, we treated the NHF1 fibroblast strain with either 300 M t-butyl hydroperoxide or 3.0 Gy IR, followed by incubation for 1-10 h, and we measured p33 CDK2 /cyclin E histone H1 in vitro kinase activity. As shown in Fig. 4, both treatments resulted in p33 CDK2 /cyclin E histone H1 in vitro kinase activity suppression. Maximal inhibition occurred at 6 h, followed by partial recovery at 10 h. The inhibition observed following treatment of NHF1 cells with 300 M t-butyl hydroperoxide was less than that with exposure to 3.0 Gy IR and occurred with delayed kinetics compared with IR treatment (compare Fig. 4, A and B). When NHF1 cells were treated with t-butyl hydroperoxide over a 10 M to 1 mM range and incubated for 6 h, p33 CDK2 /cyclin E histone H1 in vitro kinase activity was found to fall in a concentration-dependent manner, with kinase activity reduced ϳ70% at concentrations of 100 M t-butyl hydroperoxide and above (data not shown). When the ATM-deficient dermal fibroblast strain GM02052 was treated with 300 M t-butyl hydroperoxide or 3.0 Gy, followed by incubation for 1-10 h, the ATM-deficient fibroblasts failed to exhibit significant inhibition of kinase activity by either agent (Fig. 4, A and B).

t-Butyl Hydroperoxide Exposure Induces a G 2 Checkpoint Response in Normal Fibroblasts That Is Defective in Fibro-
blasts Lacking pATM Function-Exposure of logarithmically growing cells from the normal fibroblast strain NHF1 to 1.5 Gy IR resulted in a rapid delay of entry into mitosis 2 h posttreatment, with a reduction in the mitotic index to only 3% (Ϯ10%) that of the mock-treated cells. ATM-deficient fibroblast cells (GM02052) showed no significant reduction in the mitotic index relative to mock-treated controls (94 Ϯ 2%), in agreement with previous findings (40). Similarly, exposure of NHF1 cells to 10 -300 M t-butyl hydroperoxide generated a strong G 2 checkpoint response (Fig. 5). Under the same conditions over the same concentration range of t-butyl hydroperoxide treatment, no significant inhibition of entry into mitosis was observed with the ATM-deficient fibroblast strain cells (GM02052) 2 h post-treatment (Fig. 5).
To explore further the G 2 checkpoint response to t-butyl hydroperoxide treatment, we performed p34 CDC2 /cyclin B histone H1 in vitro kinase activity assays on protein extracts from two normal and two ATM-deficient fibroblast strains. As shown in Fig. 6A, p34 CDC2 /cyclin B histone H1 in vitro kinase activity was suppressed in a concentration-dependent manner 2 h posttreatment with 1-300 M t-butyl hydroperoxide in both normal human fibroblast strains, NHF1 and GM03349. Similarly, exposure to 1.5 Gy IR inhibited p34 CDC2 /cyclin B histone H1 in vitro kinase activity from normal human fibroblasts, as we have previously reported (40, 41) (Fig. 6B). Neither treatment with t-butyl hydroperoxide at any concentration nor exposure to 1.5 Gy IR caused the two ATM-deficient fibroblast strains, GM02052 and GM03395, to display a significant suppression of p34 CDC2 /cyclin B histone H1 in vitro kinase activity (Fig. 6, A  and B). A representative p34 CDC2 /cyclin B histone H1 in vitro kinase assay for NHF1 and GM02052 fibroblasts is shown in Fig. 6C.
To investigate the possibility that the ATM-deficient fibroblast strains were capable of exhibiting a G 2 checkpoint function but with delayed kinetics in response to exposures of t-butyl hydroperoxide, a time course analysis of p34 CDC2 /cyclin B histone H1 in vitro kinase activity following treatment was performed. The results indicate that treatment with 300 M FIG. 3. Western blot of p53 shows induction in response to t-butyl hydroperoxide or ␥-IR treatment in normal and ATMdeficient fibroblasts. Cells were treated as indicated. p53 was immunoprecipitated with an anti-p53 antibody, and SDS-PAGE was performed. The resolved proteins were transferred to nitrocellulose paper and probed with a second anti-p53 antibody.

FIG. 4. Time course showing the effect of t-butyl hydroperoxide and IR treatment on p33 CDK2 /cyclin E kinase activity in a normal and ATM-deficient fibroblast strain.
Cells in log phase were exposed for 15 min to 300 M t-butyl hydroperoxide (A) or exposed to 3.0 Gy ␥-IR (B). The cells were then cultured for the indicated times, lysed, and immunoprecipitated with anti-cyclin E antibody to obtain p33 CDK2 /cyclin E protein complexes following the addition of protein G-agarose beads. p33 CDK2 /cyclin E kinase activity was measured by in vitro kinase assay using exogenous histone H1 protein as the substrate in the presence of [␥-32 P]ATP. SDS-PAGE was performed to resolve proteins; the gel was dried, and kinase activity was quantified by phosphorimaging.
t-butyl hydroperoxide and exposure to 1.5 Gy IR were both effective at suppressing p34 CDC2 /cyclin B histone H1 kinase activity in NHF1 cells at 1 h post-treatment, with maximal suppression of kinase activity occurring at 2 h. Activity recovered to roughly untreated levels by 6 h (Figs. 7, A and B). These kinetics closely resemble those reported previously for the suppression of mitotic entry by 1.5 Gy IR (40). Neither ATMdeficient fibroblast strain (GM02052 and GM03395) showed significant suppression of p34 CDC2 /cyclin B histone H1 in vitro kinase activity in response to either treatment with 300 M t-butyl hydroperoxide or exposure to 1.5 Gy IR at any time points examined (Fig. 7, A and B). Mannitol pretreatment reduced the p34 CDC2 /cyclin B histone H1 in vitro kinase activity inhibition initiated by 100 M t-butyl hydroperoxide treatment (data not shown). Thus an antioxidant may partially reverse the suppressive effects of t-butyl hydroperoxide on the G 2 checkpoint function as indicated by the inhibition of p34 CDC2 /cyclin B kinase activity.
t-Butyl Hydroperoxide Treatment Activates pATM-associated Kinase Activity-IR treatment of melanoma and lymphoblast cell lines was found to increase pATM-associated kinase activity toward the PHAS-1 and p53 proteins in in vitro kinase activity assays (16,17). IR-inducible kinase activity was found in pATM normal cell lines but not in cells lines derived from AT patients. In these studies, pATM kinase activity was reported to be induced by roughly 2-fold following treatment with IR (16,17). Based on the data above, we hypothesized that oxidative damage generated by treatment with t-butyl hydroperoxide should induce pATM-associated kinase activity.
A rabbit polyclonal antiserum to a peptide corresponding to pATM residues 826 -840 was raised and affinity-purified to the cognate peptide. Based on Western blot analysis of whole cell and/or nuclear protein extracts from NHF1 and HeLa cells, it was determined that the anti-ATM 7 affinity-purified antibody (␣ATM 7 a.p.) recognizes a protein of ϳ350 kDa, the predicted size of pATM. This band was not detected in dermal fibroblasts derived from individuals with AT (Fig. 8).
NHF1 cells and the ATM-deficient dermal fibroblast strain AG03058 treated with either 6.0 Gy IR or 15 min with 300 M t-butyl hydroperoxide were examined for pATM kinase activity 90 min following treatment. As shown in Fig. 9A, low levels of pATM-associated in vitro kinase activity were found to be associated with immunoprecipitated protein complexes from ex-tracts from untreated NHFs using the ␣ATM 7 a.p. antibody. This activity was roughly 50% higher than the background level of kinase activity associated with protein G-agarose bead mock immunoprecipitations (using no antibody) with extracts from IR-treated NHFs. When protein extracts from NHF1 cells treated with either IR or t-butyl hydroperoxide were assayed for pATM-associated in vitro kinase activity toward PHAS-1 protein, the level of activity in the immunocomplexes was significantly increased. t-Butyl hydroperoxide treatment induced pATM-associated kinase activity an average of 2.2-fold over the level found associated with pATM immunocomplexes from untreated NHF1 cells in 3 independent experiments. IR treatment increased pATM-associated kinase activity an average of 2.1-fold (average of 6 independent experiments) (Fig. 9A). A representative pATM kinase assay is shown in Fig. 9B. To examine the pATM-dependent specificity of these in vitro kinase assays, ␣ATM 7 a.p. antibody was preincubated with either the peptide that it was raised against (ATM-N826) or an irrelevant peptide of the same length (data not shown). As shown in Fig. 9A, the ATM-N826 peptide largely blocked IR- Cells were treated as indicated, fixed by the addition of cold methanol, and stained with DAPI. The percentage of mitotic cells was counted by fluorescence microscopy. The results are expressed as the relative mitotic index, which is the mitotic index of the treated population expressed as a percentage of the mitotic index of the mock-treated population.
FIG. 6. Concentration-response curve showing the effect of t-butyl hydroperoxide and ␥-IR treatment on p34 CDC2 /cyclin B1 kinase activity in two ATM-normal and two ATM-deficient fibroblast strains. Cells were harvested for analysis 2 h following treatment with either increasing concentrations of t-butyl hydroperoxide (A) or 1.5 Gy ␥-radiation (B). Cyclin B1-associated kinase activity was analyzed by immunoprecipitation of protein with anti-cyclin B1 antibody, followed by in vitro kinase analysis using histone H1 as an exogenous substrate. C shows a representative p34 CDC2 /cyclin B1 kinase assay with pATM-normal NHFs and pATM-deficient GM02052 fibroblasts. Proteins were resolved by SDS-PAGE, and incorporation of ␥-32 P into histone H1 protein was quantified by PhosphorImager analysis.
induced pATM-associated in vitro kinase activity to PHAS-1. Finally, pretreatment with 1 mM mannitol significantly lowered t-butyl hydroperoxide-induced pATM-associated kinase activity toward the PHAS-1 protein (data not shown), indicating that an antioxidant could reverse the pATM-activating effects of t-butyl hydroperoxide in this assay.
Further confirmation that the in vitro kinase activity toward PHAS-1 was associated with pATM was found in that in vitro kinase activity assayed from protein extracts from fibroblasts lacking pATM showed no significant difference whether anti-pATM antibody was present in the assays or not. Furthermore, with extracts from IR-or t-butyl hydroperoxide-treated ATMdeficient fibroblasts, the addition of ATM-N826 peptide to the assay did not suppress the background level of in vitro kinase activity (Fig. 9B).
Heme Oxygenase-1 Induction Is Normal in AT Fibroblasts-Heme oxygenase-1 (HO-1) is induced in human skin fibroblasts by peroxides (42). To determine whether AT fibroblasts were deficient in this response to oxidative stress, we treated four fibroblast strains (1 normal and 3 from individuals with AT) with 0, 1, 3, 10, 30, and 100 M t-butyl hydroperoxide for 4 h and performed Western blot analysis for HO-1 protein. As shown in Fig. 10, t-butyl hydroperoxide treatment resulted in an induction of HO-1 protein at 10 M concentrations and above in all four fibroblast strains. There was no significant difference in the response of any of the fibroblasts to HO-1 induction. Western blot analyses demonstrated that t-butyl hydroperoxide maximally induced HO-1 protein at 4 -5 h in each cell type (data not shown). Pretreatment of NHF1 fibroblasts with mannitol for 1 h followed by treatment with 10 M t-butyl hydroperoxide for 4 h in the continued presence of mannitol resulted in a significant inhibition of HO-1 protein induction, demonstrating that an antioxidant could partially reverse the effects of t-butyl hydroperoxide (data not shown).
Thymine Glycol Formation Induced by t-Butyl Hydroperoxide Treatment Is the Same in pATM-normal and pATM-deficient Fibroblasts-NHF1 cells and ATM-deficient fibroblasts (AG03058) were treated with t-butyl hydroperoxide or IR and examined for thymine glycol formation. As shown in Table II, treatment of either fibroblast type with 100 or 300 M t-butyl hydroperoxide (A) or 6.0 Gy ␥-IR (B) resulted in essentially equal thymine glycol formation between the fibroblast types. Thus, the initial damage produced by t-butyl hydroperoxide and IR treatment in pATM normal and deficient fibroblasts is not significantly different, as measured by thymine glycol formation. DISCUSSION We have examined the role of pATM in cellular responses to oxidative damage generated by t-butyl hydroperoxide treatment of normal human fibroblasts and ATM-deficient dermal fibroblasts. We undertook this study for several reasons as follows. 1) ATM-deficient cells have been reported to be unusually sensitive to oxidants such as nitric oxide, superoxide, and hydrogen peroxide in colony-forming efficiency assays compared with normal cells (21,(23)(24)(25). 2) ATM-deficient cells re-synthesize glutathione unusually slowly after depletion with diethyl maleate (22). 3) Cellular damage by IR exposure has been suggested to involve damage due to reactive oxygen species (43). 4) The RAD9 gene product, which has similar functions to pATM, has been found to be necessary for checkpoint arrest in response to peroxide exposure (44). 5) BRCA1, which has been demonstrated to play a role in protecting cells from damage by hydrogen peroxide (45), has recently been found to FIG. 7. Time course showing the effect of t-butyl hydroperoxide and ␥-IR treatment on p34 CDC2 /cyclin B1 kinase activity in two ATM-normal and two ATM-deficient fibroblast strains. Cells were exposed to either 300 M t-butyl hydroperoxide (A) or 1.5 Gy ␥-radiation (B) and harvested at the indicated times. Cyclin B1-associated kinase activity was analyzed using histone H1 as an exogenous substrate.
FIG. 8. pATM Western analysis. HeLa cells, NHF1 cells, and two dermal fibroblast strains from individuals with AT were analyzed by immunoblot analysis for pATM. Whole cell extracts were prepared and subjected to SDS-PAGE followed by transblotting onto nitrocellulose. The blots were then probed for pATM using ␣ATM 7 a.p., a rabbit polyclonal antibody that was peptide affinity-purified. be phosphorylated by activated pATM (46). 6) ATM-deficient mice have been found to have elevated markers of oxidative damage, such as nitrotyrosine and hemeoxygenase activity, in organs known to be affected by the AT phenotype. Interestingly, cerebellar HO levels were found to be 600% greater in ATM-deficient mice than in ATM-normal mice (26). This last observation is particularly interesting as cerebellar pathologies are a hallmark of AT (47)(48)(49). Finally, pATM has been suggested to function, in part, as a sensor of oxidative stress (20).
We report that compared with pATM normal fibroblasts, ATM-deficient fibroblasts exhibit the following characteristics after oxidative damage: 1) greater sensitivity to the toxic effects of t-butyl hydroperoxide treatment in colony-forming efficiency assays; 2) failure to delay late G 1 phase progression, as measured by flow cytometry; 3) failure to exhibit G 1 checkpoint function, as measured by inhibition of p33 CDK2 /cyclin E-associated in vitro kinase activity; 4) failure to induce p53; 5) failure to exhibit inhibition of p34 CDC2 /cyclin B histone H1 in vitro kinase activity; and 6) failure to delay entry into mitosis. Also t-butyl hydroperoxide stimulates pATM-associated kinase activity. Thus, in response to oxidative damage, ATM-deficient cells appear to lack many critical cell cycle checkpoint re-sponses, in a similar manner to that observed with IR-induced damage (for review see Ref. 33).
In contrast to these findings, HO-1 protein induction in response to t-butyl hydroperoxide appeared normal in ATM-deficient fibroblasts, indicating that some cellular responses to oxidative damage are regulated normally in ATM-deficient cells. Similarly, both ATM-deficient and -normal cells exhibited a biphasic response in colony-forming efficiency assays, with an initial sensitivity to low concentrations of t-butyl hydroperoxide (1-10 M), followed by lesser sensitivity at higher concentrations (Ͼ10 M). Whereas ATM-deficient fibroblasts were clearly more sensitive to the colony-forming inhibitory effects of t-butyl hydroperoxide, the appearance of the biphasic response in both normal and AT cells suggests that higher concentrations of t-butyl hydroperoxide (Ͼ10 M) induce a resistance to the effects of t-butyl hydroperoxide that is both pATM-independent and similar to that seen in normal cells. The biphasic colony-inhibitory response we found here is similar to one reported with mouse embryonic stem cells exposed to hydrogen peroxide (45), suggesting that this biphasic response may be a common adaptive event following peroxide exposure.
One possible interpretation of our findings could be that ATM-deficient cells may respond normally to oxidative damage generated by t-butyl hydroperoxide treatment but not exhibit normal checkpoint responses or p53 induction due to their receiving less damage than normal fibroblasts after exposure to the same concentration of t-butyl hydroperoxide. To address FIG. 9. pATM in vitro kinase assay. A, NHFs (ATM-normal) and the ATM-deficient dermal fibroblasts AG03058 were treated with either 300 M t-butyl hydroperoxide or 6.0 Gy ␥-IR, as indicated, incubated for 90 min, then harvested, and lysed, and protein extracts were subjected to immunoprecipitation with ␣ATM 7 a.p. antibody. An in vitro kinase analysis was performed using PHAS-1 protein as an exogenous substrate in the presence of [␥-32 P]ATP. Error bars in this figure represent standard deviation in three successive pATM in vitro kinase assays. The incorporation of ␥-32 P into PHAS-1 protein was quantified by SDS-PAGE followed by PhosphorImager analysis. B, a representative pATM in vitro kinase assay used to generate figure A.  10. Western blot analysis of heme oxygenase induction in response to t-butyl hydroperoxide treatment. One normal and three ATM-deficient fibroblast strains were treated as indicated; whole cell lysates were prepared, and protein extracts were subjected to SDS-PAGE. The resolved proteins were then transblotted onto nitrocellulose and probed using an anti-heme oxygenase antibody. a Cells were harvested immediately after treatment and quick-frozen until analysis. Thymine glycol quantification was performed as reported previously (29). b ND, not detectable. c Ϯ, standard deviation. this issue we quantified DNA damage induced by IR and tbutyl hydroperoxide exposure by quantifying thymine glycol formation in normal and ATM-deficient fibroblasts. We found that thymine glycol formation was not significantly different between the fibroblast types directly following exposure to either agent. Based on these data, we conclude that the initial damage received by normal and ATM-deficient fibroblasts following IR and t-butyl hydroperoxide exposure is approximately the same and that, in fact, it is the absence of pATM function that accounts for the differences seen between the cells types. A major hallmark of cells from individuals with AT is the ablation of the G 1 , S, and G 2 checkpoint functions in response to IR, as well as other IR response-related events, such as p53 and p21 induction (11,12,36,37,50). Here we have shown that after exposure to t-butyl hydroperoxide, ATM-deficient fibroblasts lack G 1 and G 2 checkpoint responses, lack p53 induction, and show enhanced toxicity, reminiscent of the cellular responses seen with ATM-deficient fibroblasts exposed to IR. Thus our data support the hypothesis that pATM plays a role in resistance to oxidative stress (20).
Cell cycle checkpoints are thought to function, in part, to allow cells time to repair damage, particularly damage to DNA, before re-entering the cell cycle and completing subsequent cellular events such as DNA replication or mitosis. Ablation of cellular checkpoint functions, such as occurs in ATM-deficient cells or in cells treated with methylxanthines, can result in greater lethality following exposure to toxic agents (for review see Ref. 33). One question we have not addressed is whether or not pATM responds to oxidative damage to DNA or responds directly to a change in intracellular redox state independent of DNA damage. Although our data do not address this question specifically, we do demonstrate that ATM-deficient fibroblasts fail to exhibit normal G 1 and G 2 checkpoint responses following exposure to oxidative stress. Thus the inability of ATM-deficient cells to exhibit G 1 and G 2 checkpoint responses following oxidative damage is likely to be one mechanism that predisposes these cells to enhanced toxicity in response to exposure to oxidative stress. Our results suggest that there is an inability to respond to oxidative stress in cells from individuals with AT, a consequence of which may be relevant to the mechanisms of Purkinje cell death and other pathological changes observed in AT patients.