Prior studies from these laboratories demonstrated 3.2-fold potentiation of 5-fluorouracil (FUra) cytotoxicity by recombinant human interferon-α2a (rIFN-α2a) in GC3/cl colon adenocarcinoma cells that was significantly enhanced to 14-fold when FUra was combined with rIFN-α2a + a mixture of the diasteroisomers of the biologically active (6S) and inactive (6R) leucovorin or 5-formyl-H4PteGlu (LV), events that were reversible by thymidine (dThd). In GC3/clTS-c3/c3 cells, deficient in thymidylate synthase, rIFN-α2a 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 unbound 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-α2a. These studies suggested that thymidylate synthase was not the primary target site for rIFN-α2a activity. Since data indicated that a 5-fluoropyrimidine was required in the interaction among FUra, LV, and rIFN-α2a, attention was focused at the level of DNA. Both DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs) induced by FUra were significantly elevated by rIFN-α2a and LV administered as single modulators and were influenced by the concentrations of both FUra and rIFN-α2a. However, when FUra was combined with LV, rIFN-α2a further potentiated the frequency of DNA SSBs, and data correlated with the relative cytotoxic activity of FUra-LV-rIFN-α2a combinations. No effect on CB3717-induced DNA SSBs or DSBs by rIFN-α2a was demonstrated. Drug exposure for 48 h was required to detect measurable differences in DNA SSB frequency among FUra-LV-rIFN-α2a 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-α2a (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-α2a lies at the level of DNA. rIFN-α2a 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-α2a action.

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Supported by National Cancer Institute awards CA23099, CA32613, CA21765 and by the American Lebanese Syrian Associated Charities.

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