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/10T1/2 mouse embryo fibroblasts (10T1/2) cells. 5'-AdThd enhanced the uptake of IdUrd in SV-HU cells (greater than 400%), similar to what we have observed before for 647V cells. However, in 10T1/2 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 microM) compared to the sensitivity of
thymidine kinase to inhibition by
dTTP (IC50 2-4 microM). 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 10T1/2 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.(ABSTRACT TRUNCATED AT 400 WORDS)