title: Activation of ATM and histone H2AX phosphorylation induced by mitoxantrone but not by topotecan is prevented by the antioxidant N-acetyl-L-cysteine.
abstract: The DNA topoisomerase II (topo2) inhibitor mitoxantrone (MXT) and topo1 inhibitor topotecan (TP) are antitumor drugs widely used to treat different types of cancer. Their mechanism of action is thought to stabilize otherwise transient ("cleavable") complexes between topo2 or topo1 and DNA; the collisions of the DNA replication fork during replication, or RNA polymerase during transcription, with these complexes convert them into double-strand DNA breaks (DSBs), potentially lethal lesions that may trigger apoptosis. In the present study we observed that treatment of human lung carcinoma A549 or promyelocytic leukemic HL-60 cells with MXT led to ATM activation and phosphorylation of histone H2AX on Ser-139, the reporters of induction of DSBs, in all phases of the cell-cycle. Only S-phase cells, however, underwent apoptosis after treatment with MXT, which implied that DSBs in the cells replicating DNA were more effective in triggering apoptosis than DSBs in G(1) or G(2)M phase cells. Unlike MXT, the treatment with TP induced ATM activation and H2AX phosphorylation almost exclusively in S-phase cells and only S phase cells underwent apoptosis. The induction of both ATM activation and H2AX phosphorylation by MXT was prevented to a large extent by N-acetyl-L-cysteine (NAC), a scavenger of reactive oxygen species (ROS). The protective effect of NAC was observed for cells in all phases of the cell cycle. NAC offered no protection at all against TP. The induction of DSBs by MXT, thus, appears to be predominantly mediated through ROS, while DSBs generated during treatment with TP most likely are a consequence of collisions of replication forks with the "cleavable" complexes.
authors.affiliation: Brander Cancer Research Institute, New York Medical College, Valhalla, New York 10595, USA.
authors.forename: Xuan
authors.initials: X
authors.lastname: Huang
authors.forename: Akira
authors.initials: A
authors.lastname: Kurose
authors.forename: Toshiki
authors.initials: T
authors.lastname: Tanaka
authors.forename: Frank
authors.initials: F
authors.lastname: Traganos
authors.forename: Wei
authors.initials: W
authors.lastname: Dai
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Darzynkiewicz
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000627:therapeutic use; D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000378:metabolism*; D004249:DNA Damage / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D018922:HL-60 Cells / Q000187:drug effects / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000378:metabolism; D008942:Mitoxantrone / Q000627:therapeutic use*; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D019772:Topotecan / Q000627:therapeutic use*; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D000975:Antioxidants; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D025521:Tumor Suppressor Proteins; D019772:Topotecan; D008942:Mitoxantrone; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D000111:Acetylcysteine