Acute apoptosis by cisplatin requires induction of reactive oxygen species but is not associated with damage to nuclear DNA

Abstract

Cisplatin is a broad-spectrum anticancer drug that is also widely used in experimental studies on DNA damage-induced apoptosis. Induction of apoptosis within 24-48 hr requires cisplatin concentrations that are at least one order of magnitude higher than the IC 50 . Here, we show that such high, apoptosis-inducing cisplatin concentrations induce cellular superoxide formation and that apoptosis is inhibited by superoxide scavengers. The same concentration limit and the requirement for superoxide are also true for induction of caspase activation in enucleated cells (cytoplasts), showing that cisplatin-induced apoptosis occurs independently of nuclear DNA damage. In contrast, lower cisplatin concentrations, which do not induce acute apoptosis, are sufficient for induction of DNA damage signaling. We propose that the antiproliferative effects of cisplatin at IC 50 doses involve premature senescence and secondary, nonstress-induced apoptosis. The higher doses currently used in in vitro studies lead to acute, stress-induced apoptosis that involves induction of superoxide but is largely DNA damage-independent. ' 2006 Wiley-Liss, Inc.Key words: cisplatin; DNA damage; apoptosis; senescence Cis-diamminedichloroplatinum (II) (cisplatin) is a commonly used anticancer agent, especially effective in the treatment of testicular carcinoma and also used to treat other malignancies such as ovarian, cervix, head-and-neck and small-cell lung cancer. 1 Cisplatin forms covalent bonds to the N7 positions of DNA purines to form intra-or interstrand crosslinks, and DNA is generally acknowledged as the primary target of cisplatin. 2,3 Cisplatin is a commonly used model agent for induction of DNA damage-dependent apoptosis in vitro. Acute apoptosis is induced within 24-36 hr, and major efforts have been performed to elucidate apoptotic signaling pathways induced by cisplatin within this time frame. These various studies have highlighted the roles of c-ABL, stressactivated protein kinases and p53 as downstream mediators of cisplatin-induced DNA damage and as mediators of apoptotic signaling (recently reviewed in 3 ). In addition to induction of apoptosis, cisplatin also induces premature senescence. 4 Premature senescence is currently thought to be related to replicative senescence, which has been demonstrated to be a DNA damage response. 5 During replicative senescence, signaling pathways involving ATM (Ataxia telangiectasia mutated) and p53 are activated by telomere uncapping. 5,6 The same signaling mechanisms are believed to be involved in premature senescence induced by DNA damaging drugs. 6,7 Apoptosis and senescence are obviously mutually exclusive cellular outcomes. The factors that decide whether cisplatin will trigger apoptosis or senescence are not clearly understood. In one scenario, senescence and apoptosis represent graded responses to increasing DNA damage. In more complex scenarios, cisplatin induces senescence and apoptosis by different (or partially different) mechanisms.As inducer of apoptosis, cisplatin is used at very d...

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