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 μM 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/30min. Increasing concentrations of both thymidine and deoxyuridine inhibited the phosphorylation of [ 3H]d4T with ic 50 values of 5.7 and 35 μM respectively. d4T was found to be a weak substrate for purified H9 cytosolic thymidine kinase ( K m = 138 μM) and a weak competitive inhibitor of thymidine and deoxyuridine phosphorylation by this enzyme ( K i = 1.37 and 0.33 mM respectively).