Regulation of dihydrofolate reductase gene expression in mouse fibroblasts during the transition from the resting to growing state

✍ Scribed by Lee F. Johnson; Carolyn L. Fuhrman; Leanne M. Wiedemann

• Publisher: John Wiley and Sons
• Year: 1978
• Tongue: English
• Weight: 784 KB
• Volume: 97
• Category: Article
• ISSN: 0021-9541
• DOI: 10.1002/jcp.1040970314

✦ Synopsis

Abstract

The rate of accumulation of dihydrofolate reductase (DHFR) was studied in resting, growing and serum stimulated mouse 3T6 fibroblasts by first exposing the cells briefly to 10^−6^ M methotrexate (MTX) to inactivate specifically and irreversibly the pre‐existing enzyme, then determining the rate of recovery of reductase activity after removal of MTX. DHFR activity was quantitated by measuring the ability of a cell extract to reduce ^3^H‐folic acid or to bind ^3^H‐MTX. In all cases, recovery of enzyme activity was inhibited by cyclo‐heximide, indicating that the recovery was due to de novo synthesis of reductase.

We found that the rate of accumulation of DHFR was high in exponentially growing cells, as expected, but about 40‐fold lower in resting (G~0~) 3T6 cells. When resting 3T6 cells were induced to re‐enter the cell cycle following serum stimulation, we found that the rate of accumulation of DHFR increased sharply about ten hours after serum stimulation. DNA replication also began at this time. When resting cells were serum stimulated in the presence of inhibitors of DNA synthesis (hydroxyurea or cytosine arabinoside), the increase in DHFR synthesis was the same as in control stimulated cells. This indicates a lack of tight coupling between DNA synthesis and reductase gene expression. The increase in DHFR accumulation was inhibited by Actinomycin D (5 μg/ml) if the drug was added 7.5 hours after stimulation, but was not inhibited if the drug was added 15 hours after stimulation. This is consistent with the idea that DHFR gene expression is regulated at the level of transcription, and that reductase mRNA is transcribed only between 7.5 and 15 hours following stimulation.