Effect of staurosporine and ucn-01 on gemcitabine cytotoxicity in relation to cell cycle effects and p53 status

Gemcitabine (dFdC) is an anti-cancer agent that is affected by cell cycle modulation. Staurosporine and 7-hydroxystaurosporine (UCN-01) are potent protein kinase C (PKC) inhibitors as well as inhibitors of cyclin dependent kinase 2 (cdk2) and were therefore investigated for potential synergism, cell cycle modulation, cell death induction, and the role of the p53 protein. Lovo colon cancer cell line variants with wild type and mutant p53 (Lovo 175x2) or inactive (Lovo Li) were used for this purpose. Combinations of dFdC with staurosporine were most synergistic when cells were exposed to dFdC prior to staurosporine, while for UCN-01 the simultaneous exposure was most synergistic. This synergism appeared to be related with abrogation of the cell cycle and cell cycle proteins. For dFdC (1.0 μM), a gradual time dependent increase of cells in S phase (up to 40%) was observed in all Lovo variants. Staurosporine (0.05 μM) initially induced accumulation of cells in the G2/M phase (after 24 hr), while 48 hr exposure showed accumulation in the S-phase. UCN-01 (0.5 μM) caused an arrest in G0/G1 after 24 hr exposure, while after 48 hr cells accumulated in the S phase. Simultaneous exposure to dFdC combinations showed an average cell cycle distribution of both drugs when used alone. In sequential addition of dFdC combinations, the first drug dominated the cell cycle distribution. Synergism of gemcitabine combinations was associated with induction of cell death by UCN-01, which was 2-fold higher in Lovo 175x2 (mutant p53) compared to other Lovo variants. Additive effects in induction of cell death were observed at the simultaneous addition of dFdC and UCN-01, but exposure to dFdC prior to UCN-01 caused a 2-fold higher induction of cell death than the sum of each compound alone in Lovo 175x2 cells, in contrast to the other lines. Accumulation of cells in the G2/M phase prevented repair of DNA damage, resulting in increased apoptosis. These data demonstrate that staurosporine and UCN-01 affect dFdC cytotoxicity via modulation of the cell cycle.