Implications of p53 in growth arrest and apoptosis on combined treatment of human Mammary epithelial cells with topotecan and UCN-01.

We previously reported (Cancer Chemother Pharmacol 45: 252-258, 2000) that UCN-01 (7-hydroxystaurosporine), a protein kinase inhibitor, which is under clinical trials as an anti-cancer agent in the USA and Japan, enhanced camptothecin-induced cytotoxicity in breast cancer cells that lack p53 function. This enhancement was mediated by the abrogation of G2 arrest of tumor cells. Subsequent studies from our laboratory also revealed that the combined use of both UCN-01 and camptothecin induced DNA double strand breaks in p53 mutant tumor cells but not in normal or p53 negative epithelial cells. In this study, we report the implication of p53 on growth arrest and apoptosis following the combined treatment of human mammary epithelial cells with topotecan, a specific topoisomerase I inhibitor, and UCN-01. Experiments were performed on the following cells: normal human mammary epithelial cells (HMEC) with wild type p53, HME cells transfected with HPV16 E6 protein which inactivates p53 (HMEC/E6), and MDA231 mammary tumor cells with p53 mutation. UCN-01 selectively enhanced the cytotoxicity of topotecan in both MDA231 and HMEC/E6 cells. In contrast, UCN-01 showed little pharmacological effect, if any, on HME cells. Median-effect analysis indicated that a synergistic cytotoxic interaction existed between UCN-01 and topotecan in both MDA231 and HMEC/E6 cells, whereas, in the normal HME cells, the growth inhibition was only additive. Detailed cell-cycle analyses revealed that UCN-01 abrogated S-phase accumulation induced by topotecan treatment in p53 defective MDA231 tumor cells and HMEC/E6 cells. No changes in the cell cycle profiles of the normal HME cells were observed. In combination, UCN-01 and topotecan induced maximum apoptotic response on both HMEC/E6 and MDA231 cells at 6 and 48 hrs, respectively. These data indicate that UCN-01 selectively enhances topotecan cytotoxicity in p53 defective cells through the induction of apoptotic signaling pathway(s), although the time course for the induction of cell death is not the same. UCN-01 may, therefore, provide a new modality for topotecan-based therapy, particularly in p53 defective cancer patients.