Studies on Thymidine Kinase, Thymidylate Kinase, and Deoxycytidylate Deaminase of Chang Liver Cells

Abstract

The activity levels of thymidylate kinase and deoxycytidylate deaminase in Chang human liver cells in tissue culture were measured during a normal 5-day growth cycle. The activity of both enzymes showed considerable variations during different phases of growth, being high in rapidly proliferating and low in nonproliferating cultures. Data are presented which indicate that deoxycytidylate deaminase as well as thymidylate kinase is subject to feedback inhibition by deoxythymidine nucleotides. The activity levels of thymidylate kinase, deoxycytidylate deaminase, and deoxythymidine kinase were measured in cultures treated with various selective inhibitors of deoxyribonucleic acid synthesis. The inhibitors were used in concentrations which were sufficient to arrest cell multiplication. Treatment of the liver cells with amethopterin, mitomycin C, or high concentrations of deoxythymidine did not affect the activities of thymidylate kinase and deoxycytidylate deaminase. Hydroxyurea had no effect on the levels of these enzymes during the first 24 hours after addition, whereas after 48 hours the enzyme activities were considerably lower in the treated than in the untreated cultures. A marked increase in deoxythymidine kinase activity was observed in amethopterin-treated cultures, an effect which could be prevented by actinomycin D. The activity levels of kinases responsible for the phosphorylation of deoxyadenosine, deoxyguanosine, and deoxycytidine were not stimulated. Inhibition of growth by hydroxyurea was likewise associated with a strong increase in deoxythymidine kinase activity, an effect which was inversely related to the growth rate of the cell cultures. The hydroxyurea-induced rise in the kinase level could be prevented by puromycin and actinomycin D, but not by deoxynucleosides exogenously supplied. Deoxythymidine kinase activity was also found to increase when cell growth was inhibited by high concentrations of deoxythymidine, whereas treatment with mitomycin C did not affect the enzyme level.