Human recombinant interferon alpha-za plus 3′-azido-3′-deoxythymidine: Synergistic growth inhibition with evidence of impaired dna repair in human colon adenocarcinoma cells

Abstract

We reported that 3′-azidothymidine-3′-deoxythymidine (AZT) plus 5-fluorouracil or methotrexate produces additive cytotoxicity in HCT-8 cells: a reflection of increased AZT metabolism when de novo thymidylate (dTMP) synthesis was inhibited. We now report that AZT plus human recombinant Interferon alpha-2a (rIFN-α2a) produces synergistic growth inhibition in these cells. Evaluation of the effect of rIFN-α2a on dTMP metabolism revealed that exposure to rIFN-α2a (± AZT) did not affect dTMP synthase activity significantly but increased thymidine (dThd) kinase activity significantly. Consequently, AZT nucleotide production and incorporation into DNA were increased by coexposure to rIFN-α2a. This alone, however, cannot explain the observed synergism. Therefore, the effect of these agents on DNA excision/repair processes was assessed. Isotope clearance studies demonstrated that rIFN-aZa did not alter the rate of [ 3H]AZT excision from DNA. In contrast, filter-elution studies revealed that rIFN-α2a (± AZT) produced more DNA damage and delayed repair compared with the effects produced by AZT alone. Since DNA polymerases alpha and beta are directly involved in gap-filling repair synthesis, experiments next assessed the effect of rIFN-α2a and/or 3′-azido-3′-deoxythymidine-5′-triphosphate (AZTTP) on their activities. Polymerase alpha was inhibited slightly by AZTTP but not by rIFN-aZa. Polymerase beta activity, however, was inhibited dramatically by rIFN-α2a + AZTTP. Finally, western analysis revealed that a Z4-hr exposure to 5000 lU/mL rIFN-aZa (± 20 μ,M AZT) significantly reduced wild-type p53 expression compared with AZT-exposed cells. We conclude that rIFN-α2a enhances AZT-induced tumor cell growth inhibition by (i) increasing AZT metabolism, and (ii) inhibiting DNA repair and p53-mediated cell cycle control processes.