Effect of interferon on 5-fluorouracil-induced perturbations in pools of deoxynucleotide triphosphates and DNA strand breaks.

Abstract

Interferon (IFN) augments the anabolism of 5-fluorouracil (5FU) to its active metabolite, fluorodeoxyuridylate (FdUMP), which inhibits thymidylate synthase (TS). We sought to determine whether this resulted in greater perturbations of nucleotide pools and if so, whether this was associated with an increase in cell lethality, specifically focussing on the lethal cellular lesion, DNA double strand breaks (dsb). To determine whether combination therapy with 5FU + IFN resulted in greater depletion of thymidine nucleotide pools than 5FU alone, a highly sensitive DNA polymerase assay was used. In two human colon cancer cell lines, treatment with 5FU + IFN resulted in a rapid decrease in levels of dTTP by 95%. The addition of IFN to 5FU resulted in greater depletion of dTTP levels over treatment with 5FU alone by up to fourfold, and markedly augmented the dATP/dTTP ratio. The addition of IFN to 5FU had no effect on 5FU-induced perturbations in dCTP, dGTP or dATP pools at 8 and 12 h. Measurement of DNA dsb demonstrated that treatment of HT-29 cells with 10 microM 5FU for 24 h did not increase DNA dsb versus control. The combination of 5FU + 500 U/ml IFN, however, resulted in an increased number of dsb versus both 5FU and untreated control cells (P < 0.01), equivalent to 0.74 +/- 0.12 Gy. The addition of IFN to 5FU resulted in a selective further depletion of pools of dTTP and an increase in the number of DNA dsb versus 5FU treatment alone.