Regulation of thymidylate synthetase activity in cultured mammalian cells

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Abstract
Changes in thymidylate synthetase specific activity in Don Chinese hamster cells grown in vitro have been examined during the culture cycle and after exposure of lag- and log-phase cultures to drugs which inhibit DNA, RNA, and protein synthesis. During the culture cycle enzyme activity was low during lag phase, rose 6- to 8-fold before log phase, fluctuated between 5.5 and 9 nmol dTMP/h/107 cells during log phase, and declined to base level during stationary phase. Puromycin prevented all increases in enzyme specific activity and caused a decrease in enzyme activity when applied to log-phase cultures. Actinomycin D prevented the initial rise in enzyme activity if applied during early lag phase but caused a pronounced increase in enzyme activity above control levels when applied during log phase. High thymidine concentration (1 mM) stopped cell division in log-phase cultures but did not alter the log-phase plateau level of thymidylate synthetase activity. Fluorodeoxyuridine stopped cell division and depressed enzyme activity to varying degrees depending upon its concentration, but at concentrations less than 10-6M enzyme activity eventually returned to normal log-phase levels and cell division resumed if puromycin was not present. Methotrexate stopped cell division and caused a 3- to 4-fold increase in enzyme activity above control levels if puromycin was not present. This increase occurred in the presence of actinomycin D but was retarded by addition of thymidine when actinomycin D was not present. These experiments suggest that the regulation of thymidylate synthetase activity in log-phase cells is complex and may involve thymidine triphosphate.