The effects of
5-fluoro-2'-deoxyuridine (FdUrd) and
5,8-dideazaisofolic acid on the coordination of
thymidylate synthase activity and
DNA synthesis were examined in human CCRF-CEM leukemic cells following a continuous exposure to these agents. In logarithmically growing control
tumor cells, the rate of in situ
thymidylate synthase activity equaled the rate of
DNA synthesis. However, in
tumor cells incubated with growth-inhibitory concentrations of either FdUrd or
5,8-dideazaisofolic acid for 48 h, the rate of
thymidylate synthase activity was between 15- and 17-fold greater than the rate of
DNA synthesis. The loss in
tumor cell viability of FdUrd-treated cells was temporally related to this prolonged dissociation of thymidylate biosynthesis from
DNA biosynthesis. The dissociation of thymidylate from
DNA biosynthesis in cells incubated with FdUrd was not closely related to thymidylate depletion. The intracellular concentrations and activities of
thymidylate synthase were comparable in
tumor cells incubated for 24 or 48 h with either a growth-inhibitory or non-growth-inhibitory concentration of FdUrd, indicating no direct relationship among these parameters. Indirect thymidylate depletion induced by the combination of
2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine,
hypoxanthine, and
glycine inhibited in situ
thymidylate synthase activity and
DNA synthesis to an equal extent. In addition, the intracellular concentrations of all four deoxyribonucleoside 5'-triphosphates in
tumor cells incubated with FdUrd for 48 h were between 1.3- and 3.1-fold greater than their respective concentrations in control cells, reflecting their decreased utilization in
DNA synthesis in FdUrd-treated cells. These data indicated that inhibition of CCRF-CEM cell growth and
DNA synthesis following a continuous exposure to
cytostatic concentrations of either FdUrd or
5,8-dideazaisofolic acid resulted primarily from interference with thymidylate incorporation into
DNA, and not simple blockade of
thymidylate synthase.