p 53 Suppression of Arsenite-Induced Mitotic Catastrophe Is Mediated by p 21 CIP 1 / WAF 1

Arsenic trioxide, an acute promyelocytic leukemia chemotherapeutic, may be an efficacious treatment for other cancers. Understanding the mechanism as well as genetic and molecular characteristics associated with sensitivity to arsenite-induced cell death is key to providing effective chemotherapeutic usage of arsenite. Arsenite sensitivity correlates with deficient p53 pathways in multiple cell lines. The role of p53 in preventing arsenite-induced mitotic arrest-associated apoptosis (MAAA), a form of mitotic catastrophe, was examined in TR9-7 cells, a model cell line with p53 exogenously regulated in a tetracycline-off expression system. Arsenite activated G1 and G2 cell cycle checkpoints independently of p53, but mitotic catastrophe occurred preferentially in p53 ) cells. Cyclin B/CDC2(CDK1) stabilization and caspase-3 activation persisted in arsenite-treated p53 ) cells consistent with MAAA/ mitotic catastrophe. N-Benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a pan-caspase inhibitor, completely abolished arsenite-induced MAAA/mitotic catastrophe and greatly increased the mitotic index. WEE1 and p21 inhibit cyclin B/CDC2 by CDC2 tyrosine-15 phosphorylation and direct binding, respectively. CDC2-Y15-P was transiently elevated in arsenite-treated p53 ) cells but persisted in p53 ) cells. Arsenite induced p53-S15-P and p21 only in p53 ) cells. P21-siRNA-treated p53 ) cells were similar to p53 ) cells in mitotic index and cell cycle protein levels. p53-inducible proteins GADD45 and 14-3-3 are capable of inhibiting cyclin B/CDC2 but did not play a p53-dependent role in mitotic escape in TR9-7 cells. The data indicate that p53 mediates cyclin B/CDC2 inactivation and mitotic release directly via p21 induction. Arsenic is number 1 on the Agency for Toxic Substances and Diseases Registry list of hazardous chemicals and a common drinking water contaminant. Arsenic is a human carcinogen causing cancer in multiple tissues (National Research Council, 2001). Paradoxically, arsenic also shows promise as a chemotherapeutic agent. Arsenic has a long history of use in traditional Chinese medicine for leukemia and as a palliative treatment for chronic myelogenous leukemia in 19th to mid-20th century western medicine (Forkner and Scott, 1931). Arsenic trioxide (As2O3) is currently approved for use in treating all-trans retinoic acid refractory acute promyelogenous leukemia (Cohen et al., 2001) and is administered intravenously as a solution prepared in sodium hydroxide (i.e., as sodium arsenite). In vitro, arsenite induces apoptosis in other leukemia cell lines such as acute myeloid leukemia (Perkins et al., 2000) and multiple myeloma (Liu et al., 2003). Arsenite also induces apoptosis in solid tumor cell lines derived from neuroblastoma (Ora et al., 2000) and hepatocellular carcinoma (Zhang et al., 2003) and also gastric (Zhang et al., 1999), colon (Nakagawa et al., 2002), pancreatic (Li et al., 2003), and prostate and ovarian (Uslu et al., 2000) cancer cells in vitro. In vivo, arsenite inhibits growth of androgen-independent prostate cancer in an orthotopic metastasis model (Maeda et al., 2001). Thus, there is interest in using arsenic to treat other tumors, and several clinical trials have been initiated to test the effectiveness of arsenic in treating solid tumors as well as lymphoid malignancies