IN-VITRO SYNERGISM BETWEEN 5-FLUOROURACIL AND NATURAL BETA-INTERFERONIN HUMAN COLON-CARCINOMA CELLS

The combination of 5-fluorouracil (FUra) plus IFN-beta was studied in vitro using a human colon carcinoma cell line, HCT-8. Continuous expos ure to high concentrations of IFN-beta is cytotoxic to these cells (ED (50), 600 +/- 50 IU/ml). A strong synergism (P < 0.002) was observed w hen a shortterm (l-h), high-concentration exposure to fluoropyrimidine (300 or 1000 mu M) was followed by IFN-beta given continuously. In fa ct, the mean ratio between the expected (product of the survival of ea ch agent alone) and the observed clonogenic cell survival rates of the combination was 3.4 (range, 2.4-4.9). Longer exposures to the fluorop yrimidine (24 h, 7 days) produced less than additive effects with IFN- beta, indicating strong schedule dependency for this synergism. The me chanism of interaction was studied at four levels. First, thymidylate synthase (TS) activity, inhibition, and recovery were measured by an i n situ assay in cells treated with FUra, IFN-beta, and their combinati on. When the prolonged infusion of IFN-beta followed a l-h exposure to FUra, the observed TS inhibition and recovery, at each time point, we re very similar to the expected values, indicating that the interactio ns between these drugs at the level of TS are not the determinant of t he synergism. Second, cell cycle analysis was done. During the continu ous exposure to IFN-beta, a significant accumulation of HCT-8 cells in S-phase was observed at 24, 48, and 72 h compared to untreated contro ls (P = 0.003); however, under the same experimental conditions produc ing synergy in the clonogenic assay, no significant cell cycle perturb ations were produced by the combination of FUra followed by IFN-beta c ompared to those caused by each agent alone. Third, using the alkaline elution test, we also demonstrated that the synergism is not due to e nhanced FUra-induced DNA single-strand break frequency in high molecul ar weight DNA. Finally, nucleic acid incorporation experiments, using tritiated FUra, showed that the cytokine, given continuously (300 IU/m l), enhanced the amount of FUra incorporated into nucleic acids 24 h a fter a l-h exposure to 300 and 1000 mu M of FUra. The median percentag e of enhancement values were 31.6 +/- 11.5% for the lower drug concent ration and 18.4 +/- 8.1% for the higher drug concentration tested. The se results suggest that the mechanism of this synergism may be related to the ability of IFN-beta to promote the incorporation of intracellu lar FUra metabolites into nucleic acids and/or to inhibit the cleavage of FUra nucleotides from RNA/DNA.