No abstract

Background: Gemcitabine (dFdC) is an active antitumour agent with radiosensitising properties, shown both in preclinical and clinical studies. In the present study, the relation between deoxycytidine kinase (dCK) activity and the radiosensitising effect of gemcitabine was investigated in eight different human tumour cell lines.

The action of 5-Fluorouracil (5-FU) is mediated by inhibition of thymidylate synthase (TS), which is regulated by cell cycle proteins controlled by protein phosphorylation. We studied the effects of staurosporine and its analogue UCN-01, inhibitors of protein kinase C (PKC) on 5-FU cytotoxicity in Lovo colon cancer cells. Each drug contributes equally to the cell cycle effects of the 5-FU combinations. In sequential drug administration, the cell cycle distribution was determined by the first drug. Simultaneous 5-FU combinations induced additive effects in induction of apoptosis. When staurosporine was used as the second drug, induction of apoptosis was 2-fold higher than the sum of both drugs alone. Based on induction of apoptosis 5-FU addition prior to the PKC inhibitors seemed preferable.

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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 G 2 /M phase (after 24 hr), while 48 hr exposure showed accumulation in the S-phase. UCN-01 (0.5 μM) caused an arrest in G 0 /G 1 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.