Basis for the Differential Modulation of the Uptake of 5-Iododeoxyuridine by 5′-Aminothymidine among Various Cell Types

We have previously reported that 5′-aminothymidine (5′-AdThd), an antagonist of the feedback inhibition exerted by dTTP that regulates thymidine kinase, enhances the uptake and cytotoxicity of 5-iododeoxyuridine in various human bladder cancer cell lines but not in normal human urothelial cells (HU) propagated in vitro . In this work we have analyzed the factors that could potentially account for the differential effect of 5′-AdThd among various cell types: 647V (a human bladder cancer cell line); HU; SV-HU (a SV40-transformed human urothelial cell line), and C3H/10T½ mouse embryo fibroblasts (10T½) cells. 5′-AdThd enhanced the uptake of IdUrd in SV-HU cells (>400%), similar to what we have observed before for 647V cells. However, in 10T½ and HU cells, 5′-AdThd only minimally increased the uptake of 5-iododeoxyuridine (about 160%). Thymidine kinases purified from the different sources were similarly sensitive to inhibition by dTTP or 5′-AdThd and to deinhibition of the dTTP-induced regulation of enzyme activity by 5′-AdThd. Furthermore, [3H]-5′-AdThd permeated and accumulated intracellularly in all cell types. In none of these cultures was nucleoside phosphorylase activity detected, as indicated by the inability of the cells to produce thymine or iodouracil after exposure to the appropriate nucleosides. Also, 5′-AdThd did not affect the breakdown of dTMP by crude preparations of cytosolic 5′-nucleotidase from the different cells. We found that intracellular dTTP pools in the various cell types were substantially high (15–26 µm) compared to the sensitivity of thymidine kinase to inhibition by dTTP (IC50 2–4 µm). This suggests that thymidine kinase is in a strongly inhibited state in situ . To test the sensitivity of thymidine kinase ( in situ ) to regulation by dTTP we investigated: ( a ) the effect of depleting intracellular dTTP pools with methotrexate on the uptake of thymidine (dThd); and ( b ) the effect of pH on the uptake of dThd and its perturbation by 5′-AdThd, since the inhibition of thymidine kinase activity by dTTP is known to be pH dependent. We found that a 47% reduction of dTTP pools by methotrexate in 10T½ and HU cells did not result in an increase in thymidine kinase activity, as indicated by the lack of an effect on the uptake of dThd. However, we have previously shown that, under similar conditions, 647V cells show a substantial increase in dThd uptake. On the other hand, incubation of HU or 10T½ cells in the more acidic medium (pH 6.5), conditions which potentiate inhibition of thymidine kinase by dTTP, resulted in an increase in the degree of stimulation of dThd uptake by 5′-AdThd, compared to conditions at pH 7.5. Substantial increases were also observed in SV-HU and 647V cells. Possible explanations for these differences are discussed. These results indicate that regulation of thymidine kinase activity can differ among different cell types. This could provide a rationale for selectivity in the use of dThd analogues in cancer chemotherapy.