Effect of 3'-deoxythymidin-2'-ene (d4T) on nucleoside metabolism in H9 cells.
The effect of 3'-deoxythymidin-2'-ene (d4T) on the metabolism of exogenously supplied radiolabeled nucleosides was investigated. Following a 24-hr exposure to 250 microM d4T, we observed no significant effect on the incorporation of [3H]thymidine or [3H]deoxycytidine into DNA. In contrast, the amounts of [3H]uridine, [3H]deoxyuridine, and [3H]cytidine were significantly lower than those incorporated by control cultures. d4T had no significant effect on the incorporation of [3H]uridine or [3H]cytidine into RNA, or the incorporation of 3H-labeled amino acids into protein. In d4T-treated cells the relative proportions of [3H]dTMP, [3H]dTDP, and [3H]dTTP formed did not change but their absolute concentrations were increased. d4T significantly reduced the level of [3H]dUMP, and a parallel decrease in [3H]dTMP derived from [3H]dUMP was also evident. d4T increased the amounts of labeled deoxycytidine metabolites formed, with increased dCMP levels the most prominent. In a cell-free extract, [3H]d4T was phosphorylated at a rate of 1.6 pmol/30 min. Increasing concentrations of both thymidine and deoxyuridine inhibited the phosphorylation of [3H]d4T with IC50 values of 5.7 and 35 microM respectively. d4T was found to be a weak substrate for purified H9 cytosolic thymidine kinase (Km = 138 microM) and a weak competitive inhibitor of thymidine and deoxyuridine phosphorylation by this enzyme (Ki = 1.37 and 0.33 mM respectively).
Marongiu
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ME
Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510.
August
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Prusoff
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WH
D000596:Amino Acids / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured / Q000187:drug effects; D003562:Cytidine / Q000378:metabolism; D003841:Deoxycytidine / Q000378:metabolism; D015224:Dideoxynucleosides / Q000494:pharmacology*; D006801:Humans; D009705:Nucleosides / Q000378:metabolism*; D018119:Stavudine; D013936:Thymidine / Q000378:metabolism; D013937:Thymidine Kinase / Q000378:metabolism; D014529:Uridine / Q000378:metabolism
D000596:Amino Acids; D015224:Dideoxynucleosides; D009705:Nucleosides; D003841:Deoxycytidine; D003562:Cytidine; D018119:Stavudine; D013937:Thymidine Kinase; D013936:Thymidine; D014529:Uridine