Regulation of dihydrofolate reductase gene transcription in methotrexate-resistant mouse fibroblasts

Abstract

We have used a methotrexate-resistant mouse 3T6 cell line (M50L3) that overproduces dihydrofolate reductase (DHFR) and its mRNA by a factor of about 300 to study the regulation of DHFR hnRNA synthesis. We have previously shown that when resting (G0) M50L3 cells are serum stimulated to reenter the cell cycle, the amount and rate of synthesis of DHFR and the content of DHFR mRNA all begin to increase as the cells enter the S phase of the cell cycle. The increase in DHFR mRNA content is due to an increase in the rate of mRNA production. In the presnt study, we have used the technique of DNA-excess filter hybridization to determine the rate of synthesis of DHFR hnRNA relative to total hnRNA at various times following serum stimulation. We found that the relative rate of DHFR hnRNA synthesis began to increase at about the same time (6 hours), and increased to about the same extent (three to fourfold by 15 hours following stimulation) as we observed previously for DHFR mRNA production. This suggests that the increase in DHFR mRNA production (and consequently DHFR gene expression) is controlled primarily, if not exclusively, at the level of transcription. We also studied the effect of addition of high concentrations of dibutyryl cAMP and theophylline on DHFR gene transcription. We found that addition of these drugs at the time of stimulation completely blocked the increase in DHFR hnRNA synthesis as well as entry into S phase. Addition of the drugs at either 13 or 20 hours following stimulation led to a rapid decrease in DHFR hnRNA synthesis. The drugs were found to have little effect on the ability of the cells to complete S phase when they were added at 13 hours following stimulation. Our results suggest that high intracellular concentrations of cAMP may affect DHFR gene expression not only by preventing the progression of cells through the G1 phase of the cell cycle but also by affecting the rate of DHFR gene transcription in a more direct manner.