Human recombinant interferon alpha-2a plus 3'-azido-3'-deoxythymidine. Synergistic growth inhibition with evidence of impaired DNA repair in human colon adenocarcinoma cells.
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-alpha 2a) produces synergistic growth inhibition in these cells. Evaluation of the effect of rIFN-alpha 2a on dTMP metabolism revealed that exposure to rIFN-alpha 2a (+/-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-alpha 2a. 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-alpha 2a did not alter the rate of [3H]AZT excision from DNA. In contrast, filter-elution studies revealed that rIFN-alpha 2a (+/-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-alpha 2a and/or 3'- azido-3'-deoxythymidine-5'-triphosphate (AZTTP) on their activities. Polymerase alpha was inhibited slightly by AZTTP but not by rIFN-alpha 2a. Polymerase beta activity, however, was inhibited dramatically by rIFN-alpha 2a + AZTTP. Finally, western analysis revealed that a 24-hr exposure to 5000 IU/mL rIFN-alpha 2a (+/-20 microM AZT) significantly reduced wild-type p53 expression compared with AZT-exposed cells. We conclude that rIFN-alpha 2a enhances AZT-induced tumor cell growth inhibition by (i) increasing AZT metabolism, and (ii) inhibiting DNA repair and p53-mediated cell cycle control processes.
Darnowski
J W
JW
Department of Medicine, Brown University, Rhode Island Hospital, Providence 02903, USA.
Davol
P A
PA
Goulette
F A
FA
D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D053938:DNA Fragmentation; D004260:DNA Repair / Q000187:drug effects; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000008:administration & dosage*; D019384:Nucleic Acid Synthesis Inhibitors; D011994:Recombinant Proteins; D013937:Thymidine Kinase / Q000187:drug effects; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000032:analysis; D015215:Zidovudine / Q000008:administration & dosage*
D000077190:Interferon alpha-2; D016898:Interferon-alpha; D019384:Nucleic Acid Synthesis Inhibitors; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D015215:Zidovudine; D013937:Thymidine Kinase