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 (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.
Cancer Biology Research Group and Department of Biochemistry and Molecular Biology, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada. kurz@ucalgary.ca
Ebba U
EU
Kurz
Pauline
P
Douglas
Susan P
SP
Lees-Miller
D000111:Acetylcysteine / Q000737:chemistry; D000730:Androstadienes / Q000494:pharmacology; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D001205:Ascorbic Acid / Q000378:metabolism; D064007:Ataxia Telangiectasia Mutated Proteins; D001665:Binding Sites; D018797:Cell Cycle Proteins; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006657:Histones / Q000737:chemistry; D006801:Humans; D017665:Hydroxyl Radical; D007091:Image Processing, Computer-Assisted; D015151:Immunoblotting; D047468:Immunoprecipitation; D008856:Microscopy, Fluorescence; D009841:Oligonucleotides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000502:physiology*; D011839:Radiation, Ionizing; D017382:Reactive Oxygen Species; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025521:Tumor Suppressor Proteins; D000077191:Wortmannin
D000730:Androstadienes; D000903:Antibiotics, Antineoplastic; D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C468783:H2AX protein, human; D006657:Histones; D009841:Oligonucleotides; D017382:Reactive Oxygen Species; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D013912:Threonine; D017665:Hydroxyl Radical; D012694:Serine; D004317:Doxorubicin; D004247:DNA; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D001205:Ascorbic Acid; D000111:Acetylcysteine; D000077191:Wortmannin