Modulation of Fluorouracil Cytotoxicity by Interferon-α and -γ

Abstract

Because interferons (IFN)-α and -γ individually have increased fluorouracil (FUra) cytotoxicity in several in vitromodels, we studied the effects of FUra combined with IFN-α + γ in HT29 colon cancer cells. A 96-hr exposure to IFN-α (500 units/ml) plus IFN-γ (10 units/ml) and a 72-hr exposure to 0.25–1 μm FUra (hr 24–96) inhibited cell growth and colony formation in an additive or more-than-additive fashion. When cells were exposed to IFN-α + γ and FUra, free FdUMP levels became detectable, whereas [³H]FUra-RNA incorporation decreased. Exposure to IFN-α + γ, FUra, or the combination decreased dTTP pools to 58%, 43%, and 17% of control, respectively. A marked increase in the dATP to dTTP ratio was seen with FUra with or without IFN-α + γ. Thymidylate synthase catalytic activity was reduced to 28% and 24% of control with FUra with or without IFN-α + γ, suggesting that the enhanced dTTP depletion must be due to another mechanism. FUra-mediated thymidylate synthase inhibition was accompanied by a 124-fold increase in total deoxyuridylate immunoreactivity and a 31-fold increase in dUTP pools, but the addition of IFN-α + γ attenuated the accumulation. Treatment with IFN-α + γ and FUra individually interfered with nascent DNA chain elongation, whereas the three-drug combination produced the most striking effects. IFN-α + γ plus FUra produced the greatest amount of single-strand breaks in nascent DNA and dramatically decreased net DNA synthesis. IFN-α + γ with or without FUra produced double-strand breaks in parental DNA. These results suggest that dTTP depletion, dATP/dTTP imbalance, pronounced inhibition of DNA synthesis, and damage to nascent and parental DNA contribute to the enhanced cytotoxicity with the triple combination.