Cell cycle regulation of thymidylate synthetase gene expression in cultured mouse fibroblasts.

This study describes the regulation of thymidylate synthetase activity in mouse 3T6 fibroblasts during serum-induced transitions between the resting (G0) and growing state. The specific activity of thymidylate synthetase was about 17 times lower in resting 3T6 cells than in exponentially growing cells. When resting cells were serum-stimulated to re-enter the cell cycle, thymidylate synthetase activity remained at the level found in resting cells until 12 h following stimulation (G0-G1 phase), then increased sharply as the cells entered S phase. Enzyme activity increased about 20-fold over that of resting cells by 30 h following stimulation and continued to increase linearly for at least another 30 h. Studies with inhibitors of protein and RNA synthesis indicated that the increase in enzyme activity was due to de novo synthesis of the enzyme, and that the increase was controlled at the level of transcription. When cells were stimulated in the presence of inhibitors of DNA synthesis, thymidylate synthetase activity increased in the same manner as in control-stimulated cells. This indicated that the induction of gene expression was not directly coupled to DNA replication. When the serum stimulus was withdrawn during S phase (20 h following stimulation) the cells completed the cell cycle and divided, but then reverted back to the resting state. Thymidylate synthetase activity stopped increasing at about the same time the cells stopped synthesizing DNA, and decreased thereafter with a half-life of 20 to 25 h.