Induction of ATM activation, histone H2AX phosphorylation and apoptosis by etoposide - Relation to cell cycle phase

被引:87
作者
Tanaka, Toshiki
Halicka, H. Dorota
Traganos, Frank
Seiter, Karen
Darzynkiewicz, Zbigniew
机构
[1] New York Med Coll, Brander Canc Res Inst, Dept Biol, Valhalla, NY 10595 USA
[2] Yamaguchi Univ, Sch Med, Dept Surg 1, Ube, Yamaguchi, Japan
[3] New York Med Coll, Dept Med, Valhalla, NY 10595 USA
关键词
VP-16; podophyllotoxin; anthraquinone; novantrone; mitoxantrone; cell cycle; apoptosis; DNA topoisomerase; oxidative stress; DNA replication; DNA damage; ROS;
D O I
10.4161/cc.6.3.3835
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
Etoposide (VP-16) belongs to the family of DNA topoisomerase II (topo2) inhibitors, drugs widely used in cancer chemotherapy. Their presumed mode of action is stabilization of "cleavable complexes" between topo2 and DNA; collisions of DNA replication forks with these complexes convert them into DNA double-strand breaks (DSBs), potentially lethal lesions that may trigger apoptosis. Immunocytochemical detection of activation of ATM (ATM-S1981P) and histone H2AX phosphorylation (gamma H2AX) provides a sensitive probe of the induction of DSBs in individual cells. Using multiparameter cytometry we measured the expression of ATM-S1981P and gamma H2AX as well as initiation of apoptosis (caspase-3 activation) in relation to the cell cycle phase in etoposide-treated human lymphoblastoid TK6 cells. The induction of ATM-S1981P and gamma H2AX was seen in all phases of the cell cycle. The G(1)-phase cells, however, preferentially underwent apoptosis. The extent of etoposide-induced H2AX phosphorylation was partially reduced by N-acetyl-L-cysteine (NAC), a scavenger of reactive oxygen species (ROS). The maximal reduction of H2AX phosphorylation by NAC, seen in G(1)-phase cells, was nearly 50%. NAC also protected a fraction of G(1) cells from etoposide-induced apoptosis, but had no such effect on S or G(2)M cells. However, no significant rise in the intracellular level of ROS upon treatment with etoposide was detected. The effects of etoposide were compared with the previously investigated effects of another topo2 inhibitor, mitoxantrone. The latter was seen to induce a maximal level of ATM-S1981P and gamma H2AX (partially abrogated by NAC) in G(1)-phase cells, but unlike etoposide, triggered apoptosis exclusively of S-phase cells. The data suggest that in addition to the generally accepted mechanism involving collisions of replication forks with the "cleavable complexes", other mechanisms which appear to be different for etoposide vs. mitoxantrone, may contribute to formation of DSBs and to triggering of apoptosis.
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页码:371 / 376
页数:6
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