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 (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. DNA double strand breaks (DSBs) 1The abbreviations used are: DSB, DNA double strand break; A-T, ataxia-telangiectasia; ATM, ataxia-telangiectasia mutated; DNA-PKcs, DNA-dependent protein kinase, catalytic subunit; Gy, gray(s); IR, ionizing radiation; NAC, N-acetylcysteine; PBS, phosphate-buffered saline; PDTC, pyrrolidinedithiocarbamate; ROS, reactive oxygen species. 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. 1Goodarzi A.A. Block W.D. Lees-Miller S.P. Prog. Cell Cycle Res. 2003; 5: 393-411PubMed Google Scholar, 2Shiloh Y. Nat. Rev. Cancer. 2003; 3: 155-168Crossref PubMed Scopus (2157) Google Scholar, 3Kurz E.U. Lees-Miller S.P. DNA Repair (Amst.). 2004; 3: 889-900Crossref PubMed Scopus (397) Google Scholar). 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 (4Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2691) Google Scholar). 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 (G1, intra-S, and G2/M), and apoptosis (1Goodarzi A.A. Block W.D. Lees-Miller S.P. Prog. Cell Cycle Res. 2003; 5: 393-411PubMed Google Scholar, 2Shiloh Y. Nat. Rev. Cancer. 2003; 3: 155-168Crossref PubMed Scopus (2157) Google Scholar, 3Kurz E.U. Lees-Miller S.P. DNA Repair (Amst.). 2004; 3: 889-900Crossref PubMed Scopus (397) Google Scholar). 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 DNA-damaging 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 (5Fritsche M. Haessler C. Brandner G. Oncogene. 1993; 8: 307-318PubMed Google Scholar, 6Ashcroft M. Taya Y. Vousden K.H. Mol. Cell. Biol. 2000; 20: 3224-3233Crossref PubMed Scopus (321) Google Scholar). The ability of these chemotherapeutics to induce p53 accumulation has been correlated directly with the DNA damaging capacity of the drug (5Fritsche M. Haessler C. Brandner G. Oncogene. 1993; 8: 307-318PubMed Google Scholar). 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 (7Yih L.H. Lee T.C. Cancer Res. 2000; 60: 6346-6352PubMed Google Scholar). 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 phosphorylation of p53 on serine 15 (8Ha L. Ceryak S. Patierno S.R. J. Biol. Chem. 2003; 278: 17885-17894Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). 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 (9Ye R. Bodero A. Zhou B.B. Khanna K.K. Lavin M.F. Lees-Miller S.P. J. Biol. Chem. 2001; 276: 4828-4833Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 10Ye R. Goodarzi A.A. Kurz E.U. Saito S. Higashimoto Y. Lavin M.F. Appella E. Anderson C.W. Lees-Miller S.P. DNA Repair (Amst.). 2004; 3: 235-244Crossref PubMed Scopus (56) Google Scholar). Although not classically considered a DNA-damaging chemotherapeutic, the monofunctional DNA-alkylating agent N-methyl-N′-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 (11Adamson A.W. Kim W.J. Shangary S. Baskaran R. Brown K.D. J. Biol. Chem. 2002; 277: 38222-38229Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). 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. Reagents—Doxorubicin, wortmannin, ascorbic acid, and N-acetylcysteine (NAC) of doxorubicin and in and at of ascorbic acid and in with the of the to and and ATM-deficient and human cell as (9Ye R. Bodero A. Zhou B.B. Khanna K.K. Lavin M.F. Lees-Miller S.P. J. Biol. Chem. 2001; 276: 4828-4833Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 10Ye R. Goodarzi A.A. Kurz E.U. Saito S. Higashimoto Y. Lavin M.F. Appella E. Anderson C.W. Lees-Miller S.P. DNA Repair (Amst.). 2004; 3: 235-244Crossref PubMed Scopus (56) Google Scholar). as in either and or and with and at in a or of directly to the cell at the of cells in the of a and to 15, and of human p53, and of Chk1, and and and of Chk2 Cell as was an reactive for the of to serine of Nbs1 and serine of SMC1 as reactive to the of Chk2, Nbs1, and to was to serine 1981 of human ATM was to serine 139 of human histone H2AX was for human ATM was a Lavin for a for the catalytic of the DNA-dependent protein kinase was a protein and has been Lees-Miller S.P. S. S.P. G. Khanna K.K. Lavin M.F. J. PubMed Scopus Google Scholar). and nuclear protein as (9Ye R. Bodero A. Zhou B.B. Khanna K.K. Lavin M.F. Lees-Miller S.P. J. Biol. Chem. 2001; 276: 4828-4833Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). the protein as the of protein by and with to p53 or a to p53 at serine and of p53 phosphorylation at of p53 and as S. Goodarzi A.A. Higashimoto Y. Y. Lees-Miller S.P. Appella E. Anderson C.W. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). for the of p53 phosphorylation at these as R. Goodarzi A.A. Kurz E.U. Saito S. Higashimoto Y. Lavin M.F. Appella E. Anderson C.W. Lees-Miller S.P. DNA Repair (Amst.). 2004; 3: 235-244Crossref PubMed Scopus (56) Google Scholar). the of ATM phosphorylation at serine 1981 and the phosphorylation of downstream effectors of ATM, cell cells in phosphate-buffered and by in and of a protein as a ATM, or SMC1, of protein on and to in at for of protein on and to as the addition of to the nuclear as (9Ye R. Bodero A. Zhou B.B. Khanna K.K. Lavin M.F. Lees-Miller S.P. J. Biol. Chem. 2001; 276: 4828-4833Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). for H2AX the phosphorylation of histone H2AX on serine ATM-proficient and ATM-deficient cells as for and in PBS, and cells in a of in for at by in for in for to with in at for and in with in for at by with in for in PBS, of a an of cells was for for serine H2AX as a of the of cells the To in to the was to the treatment and and cells in the to in the the of cells was and the and of multiple an and The ATM-proficient and ATM-deficient cells or the effect of with was for an was In the of phosphorylation events, phosphorylation to protein by the of the by the of the the of Doxorubicin ATM-dependent and of p53 on studies have that phosphorylation of p53 in response to IR is by the ATM protein kinase S. L. S. Taya Y. Anderson C.W. L. C. Y. Y. Y. PubMed Scopus Google Scholar, Taya Y. Appella E. Kastan M.B. PubMed Scopus Google and that ATM is for p53 and for the of p53 at Doxorubicin has been to the nuclear accumulation and phosphorylation of p53 (5Fritsche M. Haessler C. Brandner G. Oncogene. 1993; 8: 307-318PubMed Google Scholar, S. Goodarzi A.A. Higashimoto Y. Y. Lees-Miller S.P. Appella E. Anderson C.W. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google of these studies investigated the requirement for ATM in these we that doxorubicin-induced and phosphorylation of p53 on serine 15 at following treatment in the of the ATM protein kinase and Treatment of ATM-proficient cells with doxorubicin induced phosphorylation of p53 on serine 15 at to a with that observed with to IR Gy, The phosphorylation was in the ATM-deficient cells at the however, in a manner to IR Taya Y. Appella E. Kastan M.B. PubMed Scopus Google doxorubicin induced a accumulation of p53 and phosphorylation at serine 15 in ATM-deficient cells at and not results in with a of ATM-proficient and ATM-deficient cell not To the role of ATM in the phosphorylation and accumulation of p53 in response to doxorubicin, cells with wortmannin, a that to the binding of phosphoinositide 3-kinase-like kinases Cancer Res. Google Scholar, L. Mol. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). ATM-proficient cells for with of to the addition of doxorubicin, and for a in of cells with both the accumulation and serine 15 phosphorylation of p53 to results observed with a ATM-proficient human cell not Doxorubicin ATM-dependent of p53 on 15, and response to IR, p53 on at serine residues at 15, and and has been that ATM is for the phosphorylation of the at 15, and with phosphorylation in ATM-deficient cells at S. Goodarzi A.A. Higashimoto Y. Y. Lees-Miller S.P. Appella E. Anderson C.W. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). To whether p53 is also at multiple serine residues in response to doxorubicin and to the of p53 was ATM-proficient and ATM-deficient cells and with to p53. Doxorubicin treatment induced the phosphorylation of p53 at and in phosphorylation was ATM-dependent In contrast, phosphorylation was observed at a in ATM-deficient results in and cells not Doxorubicin p53-DNA in whether the doxorubicin also stimulated p53 to its cognate DNA binding site, nuclear ATM-proficient and ATM-deficient cells by an Treatment of ATM-proficient cells with doxorubicin was to increase the ability of p53 to its cognate In contrast, binding of p53 to DNA was significantly in ATM-deficient Doxorubicin of ATM on of ATM kinase activity following has been to autophosphorylation of ATM on serine 1981 (4Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2691) Google Scholar). To examine whether doxorubicin also induce autophosphorylation of ATM on serine ATM-proficient and ATM-deficient cells with doxorubicin for to and with a to serine 1981 of to doxorubicin induced phosphorylation of ATM at serine to a with that induced by to IR Gy, Doxorubicin ATM-dependent of in the ATM p53 is an of ATM, activation of ATM results in the phosphorylation of a diverse array of downstream targets that participate in multiple cellular of ATM-dependent phosphorylation of an of the of the cellular response. To a on the requirement for ATM in the cellular response to doxorubicin ATM-proficient and ATM-deficient cells with doxorubicin and for or to and with to downstream effectors of to doxorubicin induced the ATM-dependent phosphorylation of the to in phosphorylation and phosphorylation 15 of p53 of Chk2 and serine of Nbs1 in an ATM-dependent manner of the of doxorubicin In contrast, ATM-dependent phosphorylation of Chk1 on and Chk2 on and and SMC1 on serine doxorubicin treatment ATM-dependent phosphorylation of Chk1 at and in response to either doxorubicin or IR was also accompanied by increased with the for the cellular of Chk1 in a manner to p53, protein and accumulation or whether the protein on a with higher for the to its Chk2 a of seven ATM phosphorylation S. G. A. Y. S. A. 2000; PubMed Scopus Google Scholar, Cancer Res. 2000; 60: Google Scholar, R. A. Nat. Cell Biol. 2000; PubMed Scopus Google Scholar). of Chk2 is the of are to a and the are at in for the phosphorylation of Chk2 by ATM Block R. Khanna K.K. Goodarzi A.A. J. Taya Y. Lavin M.F. Lees-Miller S.P. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of Chk2 is accompanied by a in the of Chk2. In response to treatment with doxorubicin, phosphorylation of Chk2 at is and the of an species of Chk2 suggesting that is of the residues in Chk2 to following to This is in to the phosphorylation of Chk2 at serine serine that is and in the of Chk2 with In an manner to that of Chk1, the of of Chk2 is accompanied by with the for the cellular of Chk2 Given the increased of the of the is to protein a in the with for the In a manner to Chk2, Nbs1 is in an ATM-dependent manner at multiple serine including serine Kim J. Kastan M.B. Nature. 2000; PubMed Scopus Google Scholar, M. A. S. Lavin M.F. Khanna Nat. 2000; PubMed Scopus Google Scholar, S. E. Y. Y. Lee Nature. 2000; PubMed Scopus Google Scholar, G. Nature. 2000; PubMed Scopus Google Scholar). This is accompanied by a in the of In response to treatment with doxorubicin, phosphorylation of Nbs1 at serine is of treatment and the of a of Nbs1 suggesting that serine is of the residues in Nbs1 to following treatment with Doxorubicin of H2AX in an ATM-dependent and and of is the phosphorylation of histone H2AX on serine 139 J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C. A. A. Cell Biol. 2003; PubMed Scopus Google Scholar). H2AX to as can as by In response to IR, phosphorylation has been to in a manner by ATM and DNA-PKcs, with ATM a role in the treatment and M. M. Cancer Res. 2004; PubMed Scopus Google Scholar). To whether doxorubicin induces phosphorylation of histone H2AX and the for ATM in and cells and by with a for phosphorylation of histone H2AX at serine phosphorylation of histone H2AX at serine 139 was observed of doxorubicin with to in the ATM-proficient cells In contrast, H2AX phosphorylation was in the ATM-deficient at and a to ATM-proficient cells at that doxorubicin induces in ATM-deficient to M. M. Cancer Res. 2004; PubMed Scopus Google ATM an role at protein kinases ATM contribute in a or manner to the doxorubicin-induced phosphorylation of histone and of p53 and p53-DNA generally classified as a topoisomerase II-stabilizing drug that induces DSBs, doxorubicin can intercalate DNA and generate through the of its with and C.J. and The of Scholar). To a role for in doxorubicin-mediated effects on p53, ATM-proficient cells with to treatment with Pretreatment of cells with the superoxide scavenger A. J. Cancer PubMed Scopus Google ascorbic acid had no effect on the doxorubicin-induced and phosphorylation of p53 on serine 15 In contrast, preincubation of cells with the hydroxyl radical scavenger A. J. Cancer PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google significantly attenuated the doxorubicin-mediated and phosphorylation of p53 on serine 15 suggesting that hydroxyl radicals a role in doxorubicin-induced activation of ATM-dependent pathways. with the phosphorylation and accumulation of p53 in cells with NAC, p53 in these cells a ability to its cognate results observed with in Pretreatment with the ATM-dependent of in the ATM a on the role of hydroxyl radicals in the cellular response to doxorubicin ATM-proficient cells for with to the addition of doxorubicin The was for a or and and with to downstream effectors of Pretreatment of cells with significantly attenuated or the doxorubicin-induced phosphorylation of and ATM, Nbs1 and SMC1, with attenuated the observed for Chk1 and Chk2, led to a of the doxorubicin-induced results observed with in the ATM-proficient cell results observed in cells with hydroxyl radical In addition to its can also as an and in cells with ATM, Nbs1, Chk1 and Chk2 phosphorylation was observed not with doxorubicin not induce phosphorylation of ATM or was SMC1 phosphorylation with of and doxorubicin not Pretreatment with the of the role of hydroxyl radicals in the doxorubicin-mediated phosphorylation of histone H2AX, ATM-proficient cells for with to the addition of doxorubicin and for an or and by with a for at serine Pretreatment of cells with attenuated the phosphorylation of histone H2AX at effect was no observed at a when protein to histone H2AX M. M. Cancer Res. 2004; PubMed Scopus Google Scholar). This the of doxorubicin and that DNA damage is to the generated by doxorubicin, the the DSBs generated through the doxorubicin-mediated of topoisomerase It these that to the protein that H2AX with The serine/threonine protein kinase, ATM, plays a critical role in the cellular response to DNA damage. to IR DSBs, to the activation of ATM in the many of the anticancer drugs in clinical use today also have the capacity to induce however, is the role of ATM in response to the damage induced by these We the that doxorubicin, a topoisomerase II poison, induces ATM autophosphorylation and the ATM-dependent phosphorylation of multiple downstream effectors the DNA damage response We evidence that ROS, hydroxyl participate in the doxorubicin-mediated activation of has that p53 is at serine residues in an ATM-dependent manner in response to IR S. Goodarzi A.A. Higashimoto Y. Y. Lees-Miller S.P. Appella E. Anderson C.W. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). In contrast, doxorubicin induced the phosphorylation of p53 at 15, and in phosphorylation was In response to a p53 is at serine residues 15, and in an ATM-dependent the induced phosphorylation at these in a manner R. Goodarzi A.A. Kurz E.U. Saito S. Higashimoto Y. Lavin M.F. Appella E. Anderson C.W. Lees-Miller S.P. DNA Repair (Amst.). 2004; 3: 235-244Crossref PubMed Scopus (56) Google Scholar). It is that phosphorylation is a and of the activity of the at a cell can DNA binding and among L. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In the of p53, initial studies that phosphorylation of is on the phosphorylation of serine 15 PubMed Scopus Google Scholar, Saito S. Higashimoto Y. S. Anderson C.W. Appella E. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). In of p53 at and the phosphorylation of p53 at serine 15, and the phosphorylation of stimulates these S. Goodarzi A.A. Higashimoto Y. Y. Lees-Miller S.P. Appella E. Anderson C.W. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). have evidence for in the phosphorylation of residues in p53 S. Higashimoto Y. C. Y. Appella E. Anderson C.W. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). phosphorylation of serine 15 to for the phosphorylation of serine serine and and are for phosphorylation the phosphorylation of residues in the of p53 S. Higashimoto Y. C. Y. Appella E. Anderson C.W. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). the phosphorylation of p53 is in an and The role of ATM in process is to a of chemotherapeutics and DNA-damaging a of downstream Although as a topoisomerase II poison, numerous cellular effects of doxorubicin are through its of Recently, has been that treatment of cells with doxorubicin to an increase in p53 protein by the transcriptional of superoxide and S.P. A. S. M. S. L. S. M. M. Cancer Res. 2004; PubMed Scopus Google Scholar). This was with an increased of ROS, and with was to significantly the of the use of we have that hydroxyl radicals a role in the doxorubicin-induced activation of ATM-dependent pathways. It is to that the of histone H2AX, ATM, Nbs1, and SMC1 phosphorylation by and the of doxorubicin, that of phosphorylation is to the generated by doxorubicin, the the DSBs generated by doxorubicin through its of topoisomerase the that the phosphorylation of Chk1 and Chk2 an oxidative response a DNA damage response. we the that the observed effects of on H2AX, Nbs1, and SMC1 phosphorylation are in to the of ATM In the ATM is for the to response to DNA damage, phosphoinositide 3-kinase-like as can the response at or, in the of cells ATM, for its damage must an when the role of ATM in response E.U. Lees-Miller S.P. DNA Repair (Amst.). 2004; 3: 889-900Crossref PubMed Scopus (397) Google Scholar, Y. L. Y. L. Y. J. 2003; PubMed Scopus Google Scholar). in of cell Several studies have the role of ATM in the cellular response to of these used and of the by the activation of R. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, 2001; Google Scholar, A. Mol. Cancer 2004; 3: Google Scholar). studies have to and have that doxorubicin induces the ATM-dependent phosphorylation of p53 at serine 15 A. L. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google and activates a protein kinase to of kinase activity and activation of the in an ATM-dependent manner S. L. M. M. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). has been well studied that the and of are in response to Cell. 2002; PubMed Scopus Google although the role for in the doxorubicin-induced activation of to in the for ATM to of ataxia-telangiectasia with the central role of ATM in cell cycle regulation in response to DNA damage, is by to IR, cancer and a of to (reviewed in Refs. 2Shiloh Y. Nat. Rev. Cancer. 2003; 3: 155-168Crossref PubMed Scopus (2157) Google and M.F. Y. Rev. PubMed Scopus Google Scholar). of studies have a role for in of ATM as well as the of (reviewed in Refs. G. Y. PubMed Scopus Google Scholar, A. G. Y. DNA Repair (Amst.). 2002; PubMed Scopus Google Scholar, 2003; 8: PubMed Scopus Google Scholar). It has been that ATM a of in or oxidative damage, the activation of for cells G. Y. PubMed Scopus Google Scholar, 2003; 8: PubMed Scopus Google Scholar). the of ATM in cells a of oxidative with are that cells and significantly of following Cancer Res. Google and of A. Y. G. A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google and of numerous of oxidative damage C. A. S. A. PubMed Scopus Google Scholar). We that hydroxyl radicals a role in the activation of ATM and ATM-dependent signaling which the ATM and doxorubicin has been in to induce an and in in and and the of or C.J. J. Google Scholar). It is to and the in cells Cancer Res. Google that ATM a either directly or indirectly, in the Although is studies that of the is for in ATM, and clinical and evidence to an increased cancer K.K. J. Cancer 2000; PubMed Scopus Google Scholar). In these have an to IR J. Cancer. Full Text Full Text PDF PubMed Scopus Google Scholar, S.P. J. M. Lavin M.F. Nat. 2002; PubMed Scopus Google Scholar). many of the anti-tumor chemotherapeutics used in the treatment of cancer have the capacity to induce DSBs or generate cancer for in ATM, to these drugs or IR to of effects or the increased of of drugs that not activate ATM to treatment for these with the of effects and the of We Lavin for for the and cell and and are to for the of the cell We of the Lees-Miller for critical of the

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