1. ¹Department of Pharmacology and Toxicology, Dartmouth Medical School, Lebanon, NH 03756, USA
2. ²Norris Cotton Cancer Center, One Medical Center Drive, Rubin Building Level 6, Lebanon, NH 03756, USA

Correspondence: A Eastman, E-mail: [email protected]

³These two authors contributed equally to this work

Received 09 January 2004; Revised 10 December 2004; Accepted 10 December 2004; Published online 14 March 2005.

Abstract

The topoisomerase I inhibitor SN38 arrests cell cycle progression primarily in S or G₂ phases of the cell cycle in a p53-independent manner. The Chk1 inhibitor, 7-hydroxystaurosporine (UCN-01), overcomes both S and G₂ arrest preferentially in cells mutated for p53, driving cells through a lethal mitosis and thereby enhancing cytotoxicity. The mechanism by which p53 maintains S and G₂ arrest was investigated here. The p53 wild-type MCF10A cells were arrested in S phase by incubation with SN38 for 24 h. Subsequent incubation with UCN-01 failed to abrogate arrest. To examine the impact of p53, MCF10A cells were developed, which express the tetramerization domain of p53 to inhibit endogenous p53 function. These cells were attenuated in SN38-mediated induction of p21^WAF1, and UCN-01 induced S, but not G₂ progression. In contrast, MCF10A cells expressing short hairpin RNA to ablate p53 expression underwent both S and G₂ phase progression with UCN-01. The difference in G₂ progression was attributed to p53-mediated gene repression; the MCF10A cells expressing the tetramerization domain retained p53 protein and repressed both cyclin B and Chk1, while cells ablated for p53 did not repress these proteins. Hence, inhibition of p53 activator function permits S phase abrogation, while additional inhibition of p53 repressor function is required for abrogation of G₂ arrest. These studies provide a mechanistic explanation for how this therapeutic strategy can selectively target tumor cells.