Doxorubicin Activates ATM-dependent Phosphorylation of Multiple Downstream Targets in Part through the Generation of Reactive Oxygen Species*

The requirement for the serine/threonine protein kinase ATM in coordinating the cellular response to DNA damage induced by ionizing radiation has been studied extensively. Many of the anti-tumor chemotherapeutics in clinical use today cause DNA double strand breaks; however, few have been evaluated for their ability to modulate ATM-mediated pathways. We have investigated the requirement for ATM in the cellular response to doxorubicin, a topoisomerase II-stabilizing drug. Using several ATM-proficient and ATM-deficient cell lines, we have observed ATM-dependent nuclear accumulation of p53 and ATM-dependent phosphorylation of p53 on seven serine residues. This was accompanied by an increased binding of p53 to its cognate binding site, suggesting transcriptional competency of p53 to activate its downstream effectors. Treatment of cells with doxorubicin led to the phosphorylation of histone H2AX on serine 139 with dependence on ATM for the initial response. Doxorubicin treatment also stimulated ATM autophosphorylation on serine 1981 and the ATM-dependent phosphorylation of numerous effectors in the ATM-signaling pathway, including Nbs1 (Ser343), SMC1 (Ser957), Chk1 (Ser317 and Ser345), and Chk2 (Ser33/35 and Thr68). Although generally classified as a topoisomerase II-stabilizing drug that induces DNA double strand breaks, doxorubicin can intercalate DNA and generate reactive oxygen species. Pretreatment of cells with the superoxide scavenger ascorbic acid had no effect on the doxorubicin-induced phosphorylation and accumulation of p53. In contrast, preincubation of cells with the hydroxyl radical scavenger, N-acetylcysteine, significantly attenuated the doxorubicin-mediated phosphorylation and accumulation of p53, p53-DNA binding, and the phosphorylation of H2AX, Nbs1, SMC1, Chk1, and Chk2, suggesting that hydroxyl radicals contribute to the doxorubicin-induced activation of ATM-dependent pathways.

The requirement for the serine/threonine protein kinase ATM in coordinating the cellular response to DNA damage induced by ionizing radiation has been studied extensively. Many of the anti-tumor chemotherapeutics in clinical use today cause DNA double strand breaks; however, few have been evaluated for their ability to modulate ATM-mediated pathways. We have investigated the requirement for ATM in the cellular response to doxorubicin, a topoisomerase II-stabilizing drug. Using several ATM-proficient and ATM-deficient cell lines, we have observed ATM-dependent nuclear accumulation of p53 and ATM-dependent phosphorylation of p53 on seven serine residues. This was accompanied by an increased binding of p53 to its cognate binding site, suggesting transcriptional competency of p53 to activate its downstream effectors. Treatment of cells with doxorubicin led to the phosphorylation of histone H2AX on serine 139 with dependence on ATM for the initial response. Doxorubicin treatment also stimulated ATM autophosphorylation on serine 1981 and the ATM-dependent phosphorylation of numerous effectors in the ATM-signaling pathway, including Nbs1 (Ser 343 ), SMC1 (Ser 957 ), Chk1 (Ser 317 and Ser 345 ), and Chk2 (Ser 33/35 and Thr 68 ). Although generally classified as a topoisomerase II-stabilizing drug that induces DNA double strand breaks, doxorubicin can intercalate DNA and generate reactive oxygen species. Pretreatment of cells with the superoxide scavenger ascorbic acid had no effect on the doxorubicin-induced phosphorylation and accumulation of p53. In contrast, preincubation of cells with the hydroxyl radical scavenger, N-acetylcysteine, significantly attenuated the doxorubicin-mediated phosphorylation and accumulation of p53, p53-DNA binding, and the phosphorylation of H2AX, Nbs1, SMC1, Chk1, and Chk2, suggesting that hydroxyl radicals contribute to the doxorubicin-induced activation of ATM-dependent pathways.
DNA double strand breaks (DSBs) 1 are among the most cytotoxic DNA lesions. They arise through both endogenous (e.g. oxidative respiration) and exogenous (e.g. ionizing radiation (IR)) sources. In response to DSBs, cells must react immediately to repair the lesion, arrest the cell cycle to facilitate repair, or, in cases when damage is too extensive, initiate apoptosis.
Ataxia-telangiectasia mutated (ATM) is a member of the phosphoinositide 3-kinase-like family of serine/threonine protein kinases (reviewed in Refs. [1][2][3]. ATM plays a central role in the cellular response to IR-induced DNA damage, essentially acting as a critical switch controlling whether and when a cell arrests following DNA damage. In response to DNA DSBs induced by IR, ATM, which exists in an unstimulated cell as an inactive homodimer or higher order multimer, autophosphorylates to generate the active, monomeric kinase (4). Activation of ATM results in the phosphorylation of a diverse array of downstream targets that participate in numerous cellular events, including DNA damage recognition and processing, regulation of three cell cycle checkpoints (G 1 , intra-S, and G 2 /M), and apoptosis (1)(2)(3). Among the most well studied targets are the tumor suppressor protein p53 and the checkpoint kinase Chk2.
To date, most studies have investigated the effects of IR on the activation of ATM and ATM-dependent signaling pathways. IR is a potent DNA-damaging agent, inducing both DNA single strand breaks and DSBs, in large part through the actions of reactive oxygen species (ROS) generated by the ionization of water molecules in the cell and through lipid peroxidation. In addition to IR, many of the anti-tumor chemotherapeutics commonly used in the treatment of cancer induce, either directly or indirectly, DSBs, yet, at present, few DNAdamaging chemotherapeutics have been evaluated for their ability to activate ATM and ATM-dependent signaling pathways. It is well established, however, that numerous anticancer drugs induce the nuclear accumulation of p53 (5,6). The ability of these chemotherapeutics to induce p53 accumulation has been correlated directly with the DNA damaging capacity of the drug (5).
Several key pieces of evidence support a role for ATM in drug-induced DNA damage. Recently, arsenite, a potent human carcinogen that induces DSBs, was reported to induce p53 accumulation in an ATM-dependent manner (7). This increase in p53 was linearly correlated with strand break induction. Hexavalent chromium (Cr(VI)), a broad spectrum DNA-damaging agent, activates ATM kinase activity and induces the * This work was supported by National Cancer Institute of Canada Grant 011053 with funds from the Canadian Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  phosphorylation of p53 on serine 15 (8). Genistein, a tyrosine kinase inhibitor and topoisomerase II poison, activates ATM protein kinase activity and induces phosphorylation of ATM on serine 1981 and the ATM-dependent phosphorylation of histone H2AX on serine 139 and p53 on multiple serine residues (9, 10). Although not classically considered a DNA-damaging chemotherapeutic, the monofunctional DNA-alkylating agent N-methyl-NЈ-nitro-N-nitrosoguanidine stimulates ATM kinase activity and the ATM-dependent phosphorylation of p53 on serine 15, possibly triggered by the strand breaks created during the DNA repair process (11). Given the critical role for ATM in the cellular response to DSBs and the prominent, although not exclusive, role for ATM in the phosphorylation of p53 in response to DNA damage, we sought to examine the effects of the anti-tumor anthracycline, doxorubicin, on ATM and its downstream effectors.

EXPERIMENTAL PROCEDURES
Reagents-Doxorubicin, wortmannin, ascorbic acid, and N-acetylcysteine (NAC) were purchased from Sigma. Stock solutions of doxorubicin and wortmannin were prepared in dimethyl sulfoxide, protected from light, and stored at Ϫ20°C. Stock solutions of ascorbic acid and NAC were prepared fresh in 0.9% NaCl, with the pH of the NAC stock solution adjusted to pH 7.5.
Cells-ATM-proficient (BT and C3ABR) and ATM-deficient (L3 and AT1ABR) human lymphoblastoid cell lines were as previously described (9,10). Cells were maintained as suspension cultures in either RPMI 1640 (BT and L3) or Dulbecco's modified Eagle's medium/F-12 (C3ABR and AT1ABR) media (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 50 units/ml penicillin G, and 50 g/ml streptomycin sulfate. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO 2 . Chemotherapeutics, inhibitors, antioxidants, or equivalent volumes of carrier were added directly to the cell medium at the start of each experiment, unless otherwise stated. Where indicated, cells were irradiated in the presence of serum-containing medium using a Gammacell 1000 cesium-137 source (MDS Nordion, Ottawa, Canada).
Antibodies-The p53-specific monoclonal antibody DO-1, agaroseconjugated DO-1, and agarose-conjugated Pab1801 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Phosphospecific antisera to serines 6,9,15,20,37,46, and 392 of human p53, serines 317 and 345 of Chk1, and serines 33 and 35 and threonine 68 of Chk2 were purchased from Cell Signaling Technology (New England Biolabs, Beverly, MA), as was an antibody reactive for the total pool of Chk1. Phosphospecific antisera to serine 343 of Nbs1 and serine 957 of SMC1 were purchased from Novus Biologicals (Littleton, CO), as were antisera reactive to the total pools of Chk2, Nbs1, and SMC1. A polyclonal antibody to actin (A2066) was acquired from Sigma. A phosphospecific antiserum to serine 1981 of human ATM was purchased from Rockland Immunochemicals (Gilbertsville, PA). A phosphospecific mouse monoclonal antibody to serine 139 of human histone H2AX was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). A rabbit polyclonal antibody specific for human ATM (4BA) was a generous gift from Dr. Martin Lavin (Queensland Institute for Medical Research). DPK1, a polyclonal antibody specific for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) was raised against a recombinant protein fragment (amino acids 2018 -2136) and has been previously described (12).
Immunoblots and Immunoprecipitation-Crude nuclear protein extracts (500 mM NaCl extraction) were prepared from logarithmically growing cells, as previously described (9). Protein concentrations were determined using the Bradford-based Bio-Rad protein assay (Bio-Rad) using bovine serum albumin as the standard. For immunoblots, 30 g of protein were resolved by SDS-PAGE and probed with antibodies to p53 (DO-1), actin, or a phosphospecific antiserum to p53 phosphorylated at serine 15. Detection and analysis of p53 phosphorylation at serines 6,9,20,37,46, and/or 392 were performed after immunoprecipitation of p53 using agarose-conjugated DO-1 and Pab1801 antibodies as described previously (13). Immunoprecipitation/immunoblot experiments for the detection of p53 phosphorylation at these sites were carried out as described (10).
Electrophoretic Mobility Shift Assay-Electrophoretic mobility shift assays using crude nuclear extracts (500 mM NaCl extraction) were performed as described previously (9).
Immunofluorescence Microscopy for Histone H2AX Phosphorylation-To evaluate the phosphorylation of histone H2AX on serine 139, logarithmically growing ATM-proficient BT and ATM-deficient L3 cells were treated as described above for immunoblots. Cells were harvested and washed twice in PBS, and 5 ϫ 10 4 cells in a volume of 200 l were centrifuged onto coverslips (800 rpm, 5 min). Cells were fixed in 3.7% (w/v) formaldehyde for 10 min at room temperature, followed by permeabilization in PBS containing 0.5% (v/v) Triton X-100 for 10 min. Samples were blocked in PBS containing 1% (w/v) bovine serum albumin for 30 min prior to incubation with primary antibody (1:400 in blocking buffer) at room temperature for 2 h and extensive washing in PBS containing 0.05% (v/v) Tween 20. Cells were then incubated with Alexa 488-conjugated goat anti-mouse (Molecular Probes, Inc., Eugene, OR) secondary antibody (1:500 in blocking buffer) for 30 min at room temperature, followed by further PBS/Tween 20 washes. Nuclei were counterstained with 4Ј,6-diamidino-2-phenylindole (1 g/ml in PBS) (Sigma) for 10 min. After extensive washing in PBS, coverslips were mounted using Vectashield (Vector Laboratories, Burlingame, CA). For detection of immunofluorescence, slides were viewed using a Leica DMRXA2 microscope. For each experimental point, an average of 250 cells was scored for reactivity for serine 139-phosphorylated H2AX as a percentage of the total number of cells within the field. To avoid bias in selecting fields to score, the observer was blinded to the experimental treatment and selected fields and counted cells in the 4Ј,6-diamidino-2-phenylindole channel prior to observing them in the fluorescein isothiocyanate channel. For each field, the percentage value of ␥H2AXpositive cells was calculated, and the mean and S.D. of values from multiple fields within an experimental point were determined and represented graphically using Prism (version 3.0) software (GraphPad Software, San Diego, CA). The differences between ATM-proficient and ATM-deficient cells or the effect of pretreatment with NAC was analyzed for significance using an unpaired, one-tailed Student's t test. p values less than 0.005 were deemed statistically significant.
Image Analysis-Image analysis was performed using ImageQuant software (Amersham Biosciences). In the evaluation of specific phosphorylation events, phosphorylation levels were normalized to total protein levels by dividing the intensity of the phosphospecific signal by the intensity of the signal measured from blots using antibodies recognizing the total pool of protein.

Doxorubicin Induces ATM-dependent Stabilization and Phosphorylation of p53 on Serine 15-Previous studies have
shown that phosphorylation of p53 in response to IR is mediated by the ATM protein kinase (14,15) and that ATM is important for p53 stabilization and for stimulating the transactivation functions of p53 at early times after IR. Doxorubicin has previously been shown to stimulate the nuclear accumulation and phosphorylation of p53 (5, 13), but neither of these studies investigated the requirement for ATM in these events. Here, we demonstrate that doxorubicin-induced stabilization and phosphorylation of p53 on serine 15 at early time points following treatment occur only in the presence of the ATM protein kinase (Fig. 1, A and B). Treatment of ATM-proficient BT cells with doxorubicin (1 M) induced phosphorylation of p53 on serine 15 within 60 min, further increasing at 120 min to a level comparable with that observed with exposure to IR (10 Gy, 2 h). The phosphorylation was absent in the ATMdeficient L3 cells at the times examined; however, in a manner similar to IR (16), doxorubicin induced a modest accumulation of p53 and phosphorylation at serine 15 in ATM-deficient cells at later time points (4 h and longer) (data not shown). Similar results were obtained in experiments with a second pair of ATM-proficient (C3ABR) and ATM-deficient (AT1ABR) cell lines (data not shown).
To confirm further the role of ATM in the phosphorylation and accumulation of p53 in response to doxorubicin, cells were pretreated with wortmannin, a fungal metabolite that binds irreversibly to the ATP binding site of phosphoinositide 3-kinase-like kinases (17,18). ATM-proficient BT cells were pretreated for 30 min with increasing concentrations of wortmannin prior to the addition of doxorubicin, and incubations were continued for a further 120 min. As shown in Fig. 1C, pretreatment of cells with 10 M wortmannin reduced both the accumulation and serine 15 phosphorylation of p53 to basal levels. Similar results were observed with a second ATM-proficient human lymphoblastoid cell line (C3ABR; data not shown).
Doxorubicin Induces ATM-dependent Phosphorylation of p53 on Serines 6,9,15,20,37,46, and 392-In response to IR, p53 becomes phosphorylated on at least eight serine residues (located at amino acids 6, 9, 15, 20, 33, 46, 315, and 392), and it has been demonstrated that ATM is required for the early phosphorylation of the amino-terminal serines at positions 9, 15, 20, and 46, with only weak phosphorylation evident in ATM-deficient cells at later time points (13). To evaluate whether p53 is also phosphorylated at multiple serine residues in response to doxorubicin and to evaluate the ATM-dependence of any such post-translational modifications, p53 was immunoprecipitated from doxorubicin-treated ATM-proficient (BT) and ATM-deficient (L3) cells and immunoblotted with phosphospecific antisera to p53. Doxorubicin treatment induced the phosphorylation of p53 at serines 6, 9, 20, 37, 46, and 392, and, in all cases, phosphorylation was ATM-dependent ( Fig. 2). In contrast, only very weak, if any, phosphorylation was observed at a later time point (4 h) in ATM-deficient cells. Very similar results were obtained in C3ABR (ATM-proficient) and AT1ABR (ATM-deficient) cells (data not shown).
Doxorubicin Stimulates p53-DNA Binding in ATM-competent Cells-To determine whether the doxorubicin also stimulated p53 to bind its cognate DNA binding site, nuclear extracts isolated from doxorubicin-treated ATM-proficient (BT) and ATM-deficient (L3) cells were analyzed by an electrophoretic mobility shift assay. Treatment of ATM-proficient cells with doxorubicin was found to increase dramatically the ability of p53 to bind its cognate DNA-binding site (Fig. 3). In contrast, binding of p53 to DNA was significantly reduced in ATM-deficient cells.
Doxorubicin Induces Phosphorylation of ATM on Serine 1981-Stimulation of ATM kinase activity following irradiation has recently been demonstrated to occur after autophosphorylation of ATM on serine 1981 (4). To examine whether doxorubicin could also induce autophosphorylation of ATM on serine 1981, ATM-proficient (BT) and ATM-deficient (L3) cells were treated with doxorubicin (1 M) for 120 min prior to extract preparation and immunoblotting with a phosphospecific antiserum to serine 1981 of ATM. Exposure to doxorubicin induced marked phosphorylation of ATM at serine 1981, to a level comparable with that induced by exposure to IR (10 Gy, 2 h) (Fig. 4A).
Doxorubicin Induces ATM-dependent Phosphorylation of Multiple Downstream Effectors in the ATM Signaling Pathway-Although p53 is an important target of ATM, activation of ATM results in the phosphorylation of a diverse array of downstream targets that participate in multiple cellular processes. Analysis of ATM-dependent phosphorylation of one substrate cannot provide an accurate picture of the complexity of the cellular response. To gain a broader perspective on the requirement for ATM in the early cellular response to doxorubicin treatment, ATM-proficient (BT) and ATM-deficient (L3) cells were treated with doxorubicin (1 M) and incubated for 60 or 120 min prior to extract preparation and immunoblotting with phosphospecific antisera to known downstream effectors of ATM. Exposure to doxorubicin induced the ATM-dependent phosphorylation of all substrates tested. Interestingly, the sub- and harvested at the indicated times. Nuclear extracts were prepared and analyzed by sequential immunoblotting using a phosphospecific antiserum to serine 15 of p53 (p53 pS15), a panspecific antibody (DO-1) to p53 (p53), and a polyclonal antiserum to actin. An extract from BT cells irradiated with 10-Gy IR and allowed to recover for 2 h served as a positive control (IR). B, the immunoblots shown in A were scanned and quantitated, and serine 15 phosphorylation was normalized to total levels of p53 (as judged by immunoreactivity with the DO-1 antibody and described under "Experimental Procedures"). strates appear to stratify into two groups, those manifesting in early phosphorylation events (Յ60 min) and late phosphorylation events (Ͼ60 min). Serine 15 of p53 (Fig. 1A), threonine 68 of Chk2 (Fig. 4B) and serine 343 of Nbs1 (Fig. 4B) were all phosphorylated in an ATM-dependent manner within 60 min of the initiation of doxorubicin treatment. In contrast, ATM-de- FIG. 2. Doxorubicin induces ATM-dependent phosphorylation of p53 on serines 6, 9, 20, 37, 46, and 392. A, ATM-proficient (BT) and ATM-deficient (L3) cells were treated with doxorubicin (1 M) and harvested at the indicated times. To determine the phosphorylation state of p53 at the indicated serines, p53 was immunoprecipitated from whole cell extracts and analyzed by immunoblotting, as described under "Experimental Procedures," using phosphospecific antisera, followed by incubation with the monoclonal antibody DO-1 for total p53. B, the immunoblots shown in A were scanned and quantitated, and p53 phosphorylation at individual sites was normalized to total p53 (as judged by immunoreactivity with the DO-1 antibody). Hatched bars, ATM-proficient BT cells; solid bars, ATM-deficient L3 cells. In the case of phosphorylation of p53 at serine 37, an elevated, uneven background on the immunoblot precluded an accurate quantitative assessment of p53 phosphorylation at this residue in the ATM-deficient L3 cells. Hence, data for this are not included in the graph. pendent phosphorylation of Chk1 on serines 317 and 345, Chk2 on serines 33 and 35, and SMC1 on serine 957 were only detectable 120 min after initiating doxorubicin treatment (Fig.  4B). Interestingly, ATM-dependent phosphorylation of Chk1 at serines 317 and 345 in response to either doxorubicin or IR was also accompanied by increased immunoreactivity with the antibody for the total cellular pool of Chk1 (Chk1; Fig. 4B). Whether this, in a manner similar to p53, represents protein stabilization and accumulation or whether the phosphorylated protein takes on a secondary structure with higher affinity for the antiserum remains to be determined.
Within its amino-terminal domain, Chk2 contains a cluster of seven potential ATM phosphorylation sites (19 -21). Whereas threonine 68 of Chk2 is the primary site of ATMdirected phosphorylation, other sites within this cluster are phosphorylated to a lesser extent, and the amino-terminal 57 amino acids are required, at least in vitro, for the efficient phosphorylation of Chk2 by ATM (22). Phosphorylation of Chk2 is accompanied by a reduction in the electrophoretic mobility of Chk2. In response to treatment with doxorubicin, phosphorylation of Chk2 at threonine 68 is readily detectable within 60 min and precedes the appearance of an electrophoretically retarded, hyperphosphorylated species of Chk2 (Fig. 4B), suggesting that threonine 68 is one of the first residues in Chk2 to be phosphorylated following exposure to doxorubicin. This is in contrast to the phosphorylation of Chk2 at serine 33 and/or serine 35 that is detectable only after 120 min and only in the hyperphosphorylated form of Chk2 with reduced electrophoretic mobility (Fig. 4B). In an inverse manner to that of Chk1, the appearance of this hyperphosphorylated form of Chk2 is accompanied by reduced immunoreactivity with the antibody for the total cellular pool of Chk2 (Chk2; Fig.  4B). Given the increased abundance of the phosphorylated forms of the protein, this is unlikely to represent protein destabilization but rather may reflect a change in the secondary structure with reduced affinity for the antiserum.

FIG. 3. Doxorubicin stimulates p53-DNA binding in ATM-proficient (BT) cells.
Oligonucleotides containing a consensus p53 binding site were annealed and end-labeled with [␥-32 P]ATP. Nuclear extracts (9 g of protein) from untreated or doxorubicin-treated (1 M, 2 h) ATM-proficient (BT) or ATM-deficient (L3) cells were assayed for binding activity to the 32 P-labeled binding site in the presence of 1 g of poly(dI-dC)⅐poly(dI-dC) and 4 l of the p53 monoclonal antibody Pab421 (to stabilize the binding of p53 to its cognate binding site) (56). The DNA-protein complex (bound) was separated from free probe (free) by electrophoresis through a nondenaturing, 4.5% polyacrylamide gel.

FIG. 4. Doxorubicin induces the autophosphorylation of ATM on serine 1981 and ATM-dependent phosphorylation of multiple downstream effectors in the ATM signaling pathway.
A, ATMproficient (BT) and ATM-deficient (L3) cells were treated with doxorubicin (1 M, 2 h) or exposed to 10-Gy IR and allowed to recover for 2 h prior to harvest. Whole cell extracts were prepared and analyzed by sequential immunoblotting using a phosphospecific antiserum to serine 1981 of ATM, a panspecific antiserum to ATM (4BA), and a polyclonal antiserum to DNA-PKcs (to verify the loading of comparable protein levels in all lanes). B, ATM-proficient (BT) and ATM-deficient (L3) cells were treated with doxorubicin (1 M) and harvested at the indicated times. Whole cell extracts were prepared, and the phosphorylation status of downstream effectors within the ATM signaling network was analyzed by immunoblotting with available phosphospecific antisera. An extract from BT cells irradiated with 10-Gy IR and allowed to recover for 2 h served as a positive control (IR).
In a manner similar to Chk2, Nbs1 is phosphorylated in an ATM-dependent manner at multiple serine residues, including serine 343 (23)(24)(25)(26). This is accompanied by a modest reduction in the electrophoretic mobility of Nbs1. In response to treatment with doxorubicin, phosphorylation of Nbs1 at serine 343 is detectable within 60 min of treatment and precedes the appearance of a reduced mobility form of Nbs1 (Fig. 4B), suggesting that serine 343 is one of the first residues in Nbs1 to be phosphorylated following treatment with doxorubicin.
Doxorubicin Induces Phosphorylation of Histone H2AX in Both an ATM-dependent and ATM-independent Manner-A very early and sensitive marker of DSB induction is the phosphorylation of histone H2AX on serine 139 (27,28). Phosphorylated H2AX (also referred to as ␥H2AX) can be visualized as foci by immunofluorescence using phosphospecific antibodies. In response to IR, this phosphorylation event has been shown to be mediated in a redundant manner by ATM and DNA-PKcs, with ATM playing a more predominant role in the very early times after treatment and under certain growth conditions (29). To determine whether doxorubicin induces phosphorylation of histone H2AX and the preference for ATM in this process, cytospins were prepared from logarithmically growing, doxorubicin-treated BT (ATM-proficient) and L3 (ATM-deficient) cells and examined by immunofluorescence with a phosphospecific antibody for phosphorylation of histone H2AX at serine 139. Robust phosphorylation of histone H2AX at serine 139 was observed within 30 min of doxorubicin treatment, with modest further increases up to 120 min in the ATM-proficient BT cells (Fig. 5). In contrast, H2AX phosphorylation was delayed in the ATM-deficient L3 cells, rising above background at 60 min and reaching a level similar to ATM-proficient cells at 120 min (Fig. 5). These results, demonstrating that doxorubicin induces phosphorylation, albeit delayed, in ATM-deficient cells, suggest that, similar to other published reports (29), ATM may play an important role at early times after treatment, but protein kinases other than ATM contribute in a complementary or redundant manner to the doxorubicin-induced phosphorylation of histone H2AX.
N-Acetylcysteine Abrogates Doxorubicin-mediated Stabilization and Phosphorylation of p53 and Attenuates p53-DNA Binding-Although generally classified as a topoisomerase II-stabilizing drug that induces DSBs, doxorubicin can intercalate DNA and generate ROS through the reaction of its quinone moiety with cytochrome P450 reductase and NAD(P)H (30). To evaluate a possible role for ROS in doxorubicin-mediated effects on p53, ATM-proficient BT cells were pretreated with antioxidants prior to treatment with doxorubicin. Pretreatment of cells with the superoxide scavenger (31) ascorbic acid had no effect on the doxorubicin-induced stabilization and phosphorylation of p53 on serine 15 (Fig. 6A). In contrast, preincubation of cells with the hydroxyl radical scavenger (31, 32) NAC significantly attenuated the doxorubicin-mediated stabilization and phosphorylation of p53 on serine 15 (Fig. 6B),  3-6) and further incubation for 2 h. Nuclear extracts were prepared and analyzed by immunoblotting as for Fig. 1. C, electrophoretic mobility shift assays were carried out with the extracts from lanes 1, 3, and 6 in B as described for Fig. 3. suggesting that hydroxyl radicals may play a role in doxorubicin-induced activation of ATM-dependent pathways. Consistent with the reduced phosphorylation and accumulation of p53 in doxorubicin-treated cells pretreated with NAC, p53 in extracts prepared from these cells showed a dramatically reduced ability to bind its cognate DNA-binding site (Fig. 6C). Similar results were observed with antioxidant pretreatment in C3ABR (ATM-proficient) cells.
Pretreatment with N-Acetylcysteine Attenuates the Doxorubicin-mediated, ATM-dependent Phosphorylation of Multiple Downstream Effectors in the ATM Signaling Pathway-To gain a broader perspective on the role of hydroxyl radicals in the early cellular response to doxorubicin treatment, ATM-proficient BT cells were pretreated for 30 min with NAC prior to the addition of doxorubicin (1 M). The incubation was continued for a further 60 or 120 min, and extracts were then prepared and immunoblotted with phosphospecific antisera to known downstream effectors of ATM. Pretreatment of cells with NAC significantly attenuated or delayed the doxorubicin-induced phosphorylation of all substrates tested (Fig. 7, A and B). For ATM, Nbs1 and SMC1, pretreatment with NAC only partially attenuated the observed phosphorylation, whereas for Chk1 and Chk2, NAC pretreatment led to a nearly complete abrogation of the doxorubicin-induced phosphorylation. Similar results were observed with NAC pretreatment in the ATM-proficient C3ABR cell line.
Qualitatively similar results were observed in cells pretreated with pyrrolidinedithiocarbamate (PDTC), another hydroxyl radical scavenging antioxidant. In addition to its antioxidant properties, paradoxically, PDTC can also function as an oxidant, and in cells treated with PDTC alone, ATM, Nbs1, Chk1 (serine 345), and Chk2 (threonine 68) phosphorylation was observed (data not shown). Interestingly, subsequent incubation with doxorubicin did not induce further phosphorylation of ATM or Chk2; nor was SMC1 phosphorylation detectable with sequential incubations of PDTC and doxorubicin (data not shown).
Pretreatment with N-Acetylcysteine Only Partially Attenuates the Early, Doxorubicin-mediated Phosphorylation of Histone H2AX-To gain some insight into the role of hydroxyl radicals in the doxorubicin-mediated phosphorylation of histone H2AX, logarithmically growing, ATM-proficient BT cells were pretreated for 30 min with NAC prior to the addition of doxorubicin (1 M) and continued incubation for an additional 60 or 120 min. Cytospins were then prepared and examined by immunofluorescence with a phosphospecific antibody for ␥H2AX at serine 139. Pretreatment of cells with NAC partially, but significantly, attenuated the phosphorylation of histone H2AX at 60 min (Fig. 8), but this effect was no longer observed at 120 min, a time when redundant protein kinase(s) become engaged to phosphorylate histone H2AX (29). This result may reflect the dual nature of doxorubicin and suggests that some DNA damage is attributable to the ROS generated by doxorubicin, whereas the remainder may represent the DSBs generated through the doxorubicin-mediated stabilization of topoisomerase II-DNA cleavable complexes. It may be these cleavable complexes that signal to the complementary protein kinase(s) that phosphorylates H2AX with slightly delayed kinetics.

DISCUSSION
The serine/threonine protein kinase, ATM, plays a critical role in the cellular response to DNA damage. Exposure to IR generates DSBs, leading to the rapid activation of ATM in the cell. Interestingly, many of the anticancer drugs in active clinical use today also have the capacity to induce DSBs; however, little is known about the role of ATM in response to the damage induced by these drugs. We present here the finding that doxorubicin, a topoisomerase II poison, induces ATM autophosphorylation and the ATM-dependent phosphorylation of multiple downstream effectors within the DNA damage response pathway. We further present evidence that ROS, specifically hydroxyl radicals, participate in the doxorubicin-mediated activation of this complex pathway.
A previous study has shown that p53 is phosphorylated at four serine residues in an ATM-dependent manner in response to IR (13). In contrast, doxorubicin induced the phosphorylation of p53 at serines 6, 9, 15, 20, 37, 46, and 392, and, in all cases, phosphorylation was ATM-dependent. In response to genistein, a plant isoflavonoid, p53 is phosphorylated at six serine residues (serines 6, 9, 15, 20, 46, and 392) in an ATM-dependent manner, whereas the related bioflavonoid quercetin induced phosphorylation at these sites in a strictly ATM-independent manner (10). It is becoming clear that multisite phosphorylation is a dynamic and powerful method of delicately modulating the activity of proteins within the cell. Phosphorylation at different regions within a cell can control localization, stability, protein-protein interaction, DNA binding activity, and enzymatic activity, among others (33). In the case of p53, initial studies demonstrated that casein kinase-1-dependent phosphorylation of threonine 18 is dependent on the prior phosphorylation of serine 15 (34,35). In addition, acetylation of p53 at lysines 320 and 383 requires the prior phosphorylation of p53 at serine 15, and the phosphorylation of additional amino-terminal sites further stimulates these acetylation events (13). Recent reports have presented evidence for much more extensive interdependence in the phosphorylation of amino-terminal residues in p53 (36). Prior phosphorylation of serine 15 appears to be required for the efficient phosphorylation of serine 9, serine 20, and threonine 18, whereas serines 6 and 9 are dependent upon one another for phosphorylation without affecting the phosphorylation of other residues in the amino terminus of p53 (36). Clearly, the phosphorylation of p53 is regulated in an intricate and dynamic manner. The role of ATM in this process is equally complex, responding to a specific subset of chemotherapeutics and DNA-damaging agents, each triggering a unique pattern of downstream post-translational modifications.
Although primarily regarded as a topoisomerase II poison, numerous cellular effects of doxorubicin are mediated through its generation of ROS. Recently, it has been demonstrated that prolonged treatment of cells with doxorubicin (0.86 M, 24 -120 h) leads to an increase in p53 protein levels, followed by the p53-mediated transcriptional up-regulation of manganese superoxide dismutase and glutathione peroxidase-1 (37). This was associated with an increased production of ROS, and cotreatment with NAC was shown to reduce significantly the number of apoptotic cells. Through the use of chemical antioxidants, we have shown that hydroxyl radicals play a role in the doxorubicin-induced activation of ATM-dependent pathways. It is tempting to speculate that the partial suppression of histone H2AX, ATM, Nbs1, and SMC1 phosphorylation by NAC pretreatment (Figs. 7 and 8) reflects the multifaceted nature of doxorubicin, that some level of phosphorylation is attributable to the ROS generated by doxorubicin, whereas the balance reflects the DSBs generated by doxorubicin through its stabilization of topoisomerase II-DNA cleavable complexes. Were this the case, it would suggest that the phosphorylation of Chk1 and Chk2 reflects an NAC-sensitive oxidative stress response more than a direct DNA damage response. However, we cannot exclude the possibility that the observed effects of NAC pretreatment on H2AX, Nbs1, and SMC1 phosphorylation are attributable, in part, to the NAC-induced suppression of ATM autophosphorylation.
In the cell, ATM is predominantly required for the early (minutes to hours) response to DNA damage, whereas other phosphoinositide 3-kinase-like kinases, such as ATR, can complement the response at later time points or, in the case of cells lacking ATM, compensate for its absence. Therefore, time after damage must be an important experimental consideration when studying the role of ATM in any given response (3,38). Whole cell extracts were prepared, and the effect of antioxidant pretreatment on the phosphorylation status of downstream effectors within the ATM signaling network was analyzed by immunoblotting with phosphospecific antisera. B, the immunoblots shown in A were scanned and quantitated, and phosphorylation at each site was normalized to total levels of each respective protein analyzed. To account for any basal effect of the pretreatment alone, data for each condition (doxorubicin alone or NAC pretreatment) were then expressed as the fold induction of phosphorylation over the doxorubicin-untreated control within each set (lanes 1 and 4). Hatched bars, treatment with doxorubicin alone (no pretreatment); cross-hatched bars, pretreatment with NAC (50 mM, 30 min). have examined early time points (up to 4 h) and have demonstrated that doxorubicin induces the ATM-dependent phosphorylation of p53 at serine 15 (42) and activates a mitogenactivated protein kinase/extracellular signal-regulated kinase pathway leading to stimulation of IB kinase activity and activation of the prosurvival transcription factor NF-B in an ATM-dependent manner (43). Interestingly, it has been well studied that the expression and function of NF-B are upregulated in response to ROS (44), although the role for ROS in the doxorubicin-induced activation of NF-B remains to be studied.
Inherited defects in the gene coding for ATM lead to development of ataxia-telangiectasia (A-T). Consistent with the central role of ATM in cell cycle regulation in response to DNA damage, this autosomal recessive disorder is characterized by profound sensitivity to IR, cancer predisposition, immunodeficiency, genomic instability, and a progressive loss of motor control due to cerebellar ataxia (reviewed in Refs. 2 and 45). A multitude of studies have supported a role for ROS in aspects of ATM function as well as the pathogenesis of A-T (reviewed in Refs. 46 -48). It has been suggested that ATM could be a sensor of perturbations in redox homeostasis or oxidative damage, triggering the activation of signal transduction pathways responsible for protecting cells from such insults (46,48). Thus, the absence of functional ATM would result in cells under a continuous state of oxidative stress. Consistent with this are observations that A-T cells and tissues exhibit significantly reduced rates of GSH resynthesis following depletion (49) and show reduced levels of nicotine adenine dinucleotide (50) and elevated levels of numerous biomarkers of oxidative damage (51). We demonstrate here that hydroxyl radicals play a role in the rapid activation of ATM and ATM-dependent signaling pathways, which further supports the hypothesized link between ATM function and ROS. Interestingly, doxorubicin has been demonstrated in mice to induce an immediate and acute reduction in GSH levels in erythrocytes, liver, and cardiac tissue, and the administration of thiol donors (cysteamine or NAC) prevents this fall (52). It is tempting to speculate, given this and the impaired recovery from GSH depletion in A-T cells (49), that ATM may play a role, either directly or indirectly, in modulating the GSH biosynthesis/recycling pathway.
Although A-T is rare, studies suggest that 1-2% of the general population is heterozygous for mutations in ATM, and clinical and epidemiological evidence points to an increased cancer risk, particularly breast cancer, within this carrier pop-ulation (53). In addition, these carriers have an intermediate sensitivity to IR (54,55). Interestingly, many of the anti-tumor chemotherapeutics used in the treatment of breast cancer have the capacity to induce DSBs or generate ROS. For breast cancer patients heterozygous for mutations in ATM, exposure to these drugs or IR could lead to more profound manifestations of side effects or the increased incidence of secondary, treatmentrelated malignancies. Identification of drugs that do not activate ATM could lead to modified treatment protocols for these patients with the aim of reducing side effects and improving the long term outcome of therapy.