TI - Doxorubicin activates ATM -dependent phosphorylation of multiple downstream targets in part through the generation of reactive oxygen species . AB - 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 .