Locus of the Interaction among 5-FIuorouraciI, Leucovorin, and Interferon-a2a in Colon Carcinoma Cells1

Prior studies from these laboratories demonstrated 3.2-fold potentia- tion of 5-fluorouracil (FUra) cytotoxicity by recombinant human inter-feron-a2a (rIFN-a2a) in GC.(/cl colon adenocarcinoma cells that was sig nificantly enhanced to 14-fold when FUra was combined with rIFN-a2a + a mixture of the diasteroisomers of the biologically active (65) and inactive (6Ä)leucovorin or 5-formyl-H4PteGlu (LV), events that were reversible by thymidine (dThd). In GCj/clTS-c3/c3 cells, deficient in thymidylate synthase, rIFN-a2a cytotoxicity was not influenced by the concentration of dThd, indicating no direct effect at the level of dThd-less stress. Direct assays of thymidylate synthase indicated no significant difference between FUra-induced accumulation of total thymidylate synthase or free or un bound thymidylate synthase in cells receiving FUra + modulators. In addition, the cytotoxic activity of CB3717, a specific quinazoline-based inhibitor of thymidylate synthase, was not potentiated by rIFN-a2a. These studies suggested that thymidylate synthase was not the primary target site for rIFN-a2a activity. Since data indicated that a 5-fluoropyrimidine was required in the interaction among FUra, LV, and r!FN-«2a, attention was focused at the level of UNA. Both DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs) induced by FUra were signifi cantly elevated by rIFN-«2a and LV administered as single modulators and were influenced by the concentrations of both FUra and rIFN-«2a. However, when FUra was combined with LV, rIFN-a2a further potenti ated the frequency of DNA SSBs, and data correlated with the relative cytotoxic activity of FUra-LV-rIFN-«2a combinations. No effect on CB37I7-induced DNA SSBs or DSBs by rIFN-a2a was demonstrated. Drug exposure for 48 h was required to detect measurable differences in DNA SSB frequency among FUra-LV-rIFN-«2a treatment groups and correlated with decreased clonogenic survival under these conditions. Continuous exposure to FUra (72 h) allowed shorter exposures to LV and/or rIFN-a2a (48 h) to maintain maximal cytotoxicity. Shorter expo sure 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-a2a lies at the level of DNA. rIFN-«2a may exert its effects via enhancement of FUra base excision or incorporation into DNA, events that subsequently become influenced by thymidylate synthase inhibition and dThd-less stress and are further potentiated by LV. Further studies will define the exact target of rIFN-«2a action. the cytotoxic activity was potentiated lu to 14-fold (13). Data suggested at least additive effects of LV and rIFN-a2a, that tumors unresponsive to FUra-LV may respond to the three-agent combination, and that lower concentrations of rIFN-a2a may be effective with a FUra-LV combination in comparison to FUra administered alone. Effects were reversed by dThd, although the activity of CB3717, a quinazoline-based specific inhibitor of thymi dylate synthase, was not potentiated by rIFN-a2a. Results indicated that the primary interaction site may be dThd or thymidylate synthase independent with a requirement for a 5-fluoropyrimidine but that modulation by rIFN-a2a required some level of thymidylate synthase inhibition. The results of experiments described in the current manu script demonstrate the correlation between FUra-LV-rIFN-a2a cyto toxicity and DNA SSBs and DSBs, eliminate thymidylate synthase as the primary target site, indicate that the primary locus of the interac tion lies at the level of DNA, and that the interaction is enhanced by dThd-less stress mediated via the inhibition of thymidylate synthase. The SSB frequency cor related with the formation of DNA DSB, following drug treatment for all treatment groups, which are likely to be the lesions associated with the cytotoxic action of FUra-LV-rIFN-a2a regimens. Of was that CB3717 induced DNA strand breaks that were not potentiated by rIFN-a2a, correlating with data derived from clonogenic assays, in which r!FN-«2a failed to potentiate CB3717 cytotoxicity (13). This result again indicated the necessity for interaction of the halogenated nucleotide at the level of DNA.