LOCUS OF THE INTERACTION AMONG 5-FLUOROURACIL, LEUCOVORIN, AND INTERFERON-ALPHA-2A IN COLON-CARCINOMA CELLS

Prior studies from these laboratories demonstrated 3.2-fold potentiati on of 5-fluorouracil (FUra) cytotoxicity by recombinant human interfer on-alpha2a (rIFN-alpha2a) in GC3/cl colon adenocarcinoma cells that wa s significantly enhanced to 14-fold when FUra was combined with rIFN-a lpha2a + a mixture of the diasteroisomers of the biologically active ( 6S) and inactive (6R) leucovorin or 5-formyl-H4PteGlu (LV), events tha t were reversible by thymidine (dThd). In GC3/clTS-c3/c3 cells, defici ent in thymidylate synthase, rIFN-alpha2a cytotoxicity was not influen ced by the concentration of dThd, indicating no direct effect at the l evel of dThd-less stress. Direct assays of thymidylate synthase indica ted no significant difference between FUra-induced accumulation of tot al thymidylate synthase or free or unbound thymidylate synthase in cel ls receiving FUra + modulators. In addition, the cytotoxic activity of CB3717, a specific quinazoline-based inhibitor of thymidylate synthas e, was not potentiated by rIFN-alpha2a. These studies suggested that t hymidylate synthase was not the primary target site for rIFN-alpha2a a ctivity. Since data indicated that a 5-fluoropyrimidine was required i n the interaction among FUra, LV, and rIFN-alpha2a, attention was focu sed at the level of DNA. Both DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs) induced by FUra were significantly elevate d by rIFN-alpha2a and LV administered as single modulators and were in fluenced by the concentrations of both FUra and rIFN-alpha2a. However, when FUra was combined with LV, rIFN-alpha2a further potentiated the frequency of DNA SSBs, and data correlated with the relative cytotoxic activity of FUra-LV-rIFN-alpha2a combinations. No effect on CB3717-in duced DNA SSBs or DSBs by rIFN-alpha2a was demonstrated. Drug exposure for 48 h was required to detect measurable differences in DNA SSB fre quency among FUra-LV-rIFN-alpha2a treatment groups and correlated with decreased clonogenic survival under these conditions. Continuous expo sure to FUra (72 h) allowed shorter exposures to LV and/or rIFN-alpha2 a (48 h) to maintain maximal cytotoxicity. Shorter exposure times for FUra during continuous exposure to the modulators were less cytotoxic. Data suggest that the primary locus of the interaction among FUra, LV , and rIFN-alpha2a lies at the level of DNA. rIFN-alpha2a may exert it s effects via enhancement of FUra base excision or incorporation into DNA, events that subsequently become influenced by thymidylate synthas e inhibition and dThd-less stress and are further potentiated by LV. F urther studies will define the exact target of rIFN-alpha2a action.