Inhibition of thymidylate synthase by hydroxyurea in rapidly proliferating P815 mastocytoma cells

The incorporation of ¹⁴C]deoxycytidine, ³H]deoxyuridine, and ³H]thymidine, respectively into pyrimidine bases of DNA has been measured in rapidly proliferating P815 mouse mastocytoma cells in the presence of hydroxyurea. The incorporation of ¹⁴C]deoxycytidine-derived radioactivity into DNA cytosines is increased when compared to the incorporation into DNA thymines. The ³H]deoxyuridine-derived radioactivity is incorporated solely into DNA thymines and this incorporation is inhibited by hydroxyurea in a dose-dependent manner. This suggests an inhibitory effect of hydroxyurea on the thymidylate synthase which was proved in experiments in which the conversion of deoxyuridine monophosphate into deoxythymidine monophosphate catalysed by a crude enzyme preparation from P815 cells was inhibited in the presence of hydroxyurea. Enzymatic DNA methylation as measured by the conversion of incorporated ¹⁴C]deoxycytidine into 5-methylcytosines was not affected by hydroxyurea.