The cellular interaction of 5-fluorouracil and cisplatin in a human colon carcinoma cell line

The combination of 5-fluorouracil (5-FU) and cisplatin (CDDP) has been shown to have synergistic cytotoxicity in human tumours, but the biochemical mechanism for this interaction remains unclear. Therefore, the aim of this study was to investigate the interaction of 5-FU and CDDP in a human colon carcinoma cell line, NCI H548. A 24 h exposure to 5-FU resulted in a 5-FU IC50 value of 24.2 +/- 4.5 microM, Dm 22.6 microM; exposure to CDDP for 2 h resulted in a IC50 value of 20.8 +/- 8.0 microM, Dm 21.9 microM. When cells were exposed simultaneously to 5-FU for 24 h and CDDP for the initial 2 h, or when cells were treated with CDDP for 2 h followed by various concentrations of 5-FU for 24 h, no greater than additive cytotoxicity was observed. In contrast, when cells were treated with 5-FU for 24 h prior to CDDP for 2 h, a greater than additive interaction was noted (5-FU IC50 1.2 +/- 0.6 microM, Dm 1.3 microM, CI 0.45). Thymidine 10 microM partially reversed the growth inhibitory effects of the 5-FU/ CDDP combination. Using both immunological and biochemical assays, no notable differences in the total amount of TS enzyme or the fraction of bound TS enzyme could be detected in the absence or presence of CDDP. No notable differences could be detected in intracellular reduced folate pools, FdUMP or FUTP pools, or 5-FU incorporation into RNA or DNA with the addition of CDDP to 5-FU. DNA fragmentation studies revealed that the combination of 5-FU followed by CDDP demonstrated a greater degree of single-stranded DNA fragments in parental (P = 0.024) and newly synthesised DNA (P = 0.025) compared with the administration of CDDP prior to 5-FU or either drug alone. The increase in smaller DNA fragment size was reversed with the addition of thymidine (10 microM). These findings suggest that the interaction of 5-FU and CDDP is associated with a greater degree of fragmentation of both nascent and parental DNA.