Dihydrofolate Reductase Gene Expression Research Articles

Control of cellular gene expression during adenovirus infection: induction and shut-off of dihydrofolate reductase gene expression by adenovirus type 2.

Infection of human cells by adenovirus results in multiple alterations of host gene expression. To examine the effects of viral infection on the expression of a single gene, a line of human cells was developed which is resistant to growth in methotrexate and which contains amplified RNA and protein specific for dihydrofolate reductase (DHFR). Cytogenetic evidence indicated the presence of amplified DNA. Adenovirus infection of these cells caused an induction and subsequent decline in the synthesis of DHFR protein. The maximum DHFR induction occurred 16 to 19 h after infection and reached a level 2.5-fold greater than that observed in uninfected cells. Induction of DHFR protein synthesis was accompanied by concomitant increases in the level of steady-state DHFR-specific cytoplasmic RNA. The relative rate of DHFR mRNA production (i.e., the appearance of DHFR-specific mRNA sequences in the cytoplasm) also increased 2.5-fold during induction. Later in infection, the relative rate of DHFR protein synthesis declined, reaching a level below that observed in uninfected cells. This decline was accompanied by a similar decline in the steady-state levels of DHFR RNA and in the relative rate of synthesis of DHFR mRNA. These data suggest that adenovirus infection controls DHFR gene expression by increasing and subsequently decreasing the relative rate at which DHFR-specific mRNA sequences appear in the cytoplasm and enter the pool of mRNA available for translation.

Inhibition of dihydrofolate reductase gene expression following serum withdrawal or db-cAMP addition in methotrexate-resistant mouse fibroblasts

We have used a methotrexate (MTX)-resistant mouse 3T6 cell line (M50L3), that overproduces dihydrofolate reductase (DHFR) and its mRNA by a factor of 300, to study the mechanism for turning off DHFR gene expression following withdrawal of serum factors or elevation of the intracellular level of cAMP. When resting (G0) M50L3 cells are serum-stimulated to reenter the cell cycle, the level of DHFR activity begins to increase at about the same time the cells begin synthesizing DNA. The increase in enzyme activity is preceded by increases in the synthesis rate of the enzyme, and the content and production rate of DHFR mRNA. These increases, as well as entry into S phase, are blocked when the cells are serum-stimulated in the presence of dibutyryl cyclic AMP (db-cAMP) and theophylline. In this study, we found that when these drugs were added, or the serum stimulus was withdrawn during S phase (20 h following stimulation), the subsequent increase in DHFR level was blocked. Immunoprecipitation of DHFR from pulse-labelled cells showed that both treatments led to a rapid decrease in synthesis rate of the enzyme. The effect on total protein synthesis was much less than on DHFR synthesis. In DNA-excess filter hybridization experiments, we found that the content of cytoplasmic DHFR mRNA decreased in parallel with the synthesis rate of the enzyme. This was due in part to a decrease in the production rate of DHFR mRNA relative to total mRNA. In addition, drug addition or serum withdrawal led to a significant destabilization of DHFR (as well as total) mRNA. About 85% of poly(A)(+) DHFR mRNA was associated with polysomes in resting, growing, or cAMP-treated cells, suggesting that DHFR gene expression was not controlled at the translational level under these conditions.

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

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.

Control of dihydrofolate reductase messenger ribonucleic acid production.

We used methotrexate-resistant mouse cells in which dihydrofolate reductase levels are approximately 500 times normal to study the effect of growth stimulation on dihydrofolate reductase gene expression. As a result of growth stimulation, the relative rate of dihydrofolate reductase protein synthesis increased threefold, reaching a maximum between 25 and 30 h after stimulation. The relative rate of dihydrofolate reductase messenger ribonucleic acid production (i.e., the appearance of dihydrofolate reductase messenger ribonucleic acid in the cytoplasm) increased threefold after growth stimulation and was accompanied by a corresponding increase in the relative steady-state level of dihydrofolate reductase ribonucleic acid in the nucleus. However, the increase in the nuclear level of dihydrofolate reductase ribonucleic acid was not accompanied by a significant increase in the relative rate of transcription of the dihydrofolate reductase genes. These data indicated that the relative rate of appearance of dihydrofolate reductase messenger ribonucleic acid in the cytoplasm depends on the relative stability of the dihydrofolate reductase ribonucleic acid sequences in the nucleus and is not dependent on the relative rate of transcription of the dihydrofolate reductase genes.

Control of dihydrofolate reductase messenger ribonucleic acid production.

We used methotrexate-resistant mouse cells in which dihydrofolate reductase levels are approximately 500 times normal to study the effect of growth stimulation on dihydrofolate reductase gene expression. As a result of growth stimulation, the relative rate of dihydrofolate reductase protein synthesis increased threefold, reaching a maximum between 25 and 30 h after stimulation. The relative rate of dihydrofolate reductase messenger ribonucleic acid production (i.e., the appearance of dihydrofolate reductase messenger ribonucleic acid in the cytoplasm) increased threefold after growth stimulation and was accompanied by a corresponding increase in the relative steady-state level of dihydrofolate reductase ribonucleic acid in the nucleus. However, the increase in the nuclear level of dihydrofolate reductase ribonucleic acid was not accompanied by a significant increase in the relative rate of transcription of the dihydrofolate reductase genes. These data indicated that the relative rate of appearance of dihydrofolate reductase messenger ribonucleic acid in the cytoplasm depends on the relative stability of the dihydrofolate reductase ribonucleic acid sequences in the nucleus and is not dependent on the relative rate of transcription of the dihydrofolate reductase genes.

Polyoma virus and cyclic AMP-mediated control of dihydrofolate reductase mRNA abundance in methotrexate-resistant mouse fibroblasts.

As a model cell culture system for studying polyoma-mediated control of host gene expression, we isolated methotrexate-resistant 3T6 cells in which one of the virus-induced enzymes, dihydrofolate reductase, is a major cellular protein. In highly methotrexate-resistant cell lines dihydrofolate reductase synthesis accounts for over 10% that of soluble portein, corresponding to an increase of approximately 100-fold over the level in parental cells. This increase in dihydrofolate reductase synthesis is due to a corresponding increase in the abundance of dihydrofolate reductase mRNA and gene sequences. We have used these cells to show that infection with polyoma virus results in a 4- to 5-fold increase in the relative rate of dihydrofolate reductase synthesis and a corresponding increase in dihydrofolate reductase mRNA abundance. The increase in dihydrofolate reductase synthesis begins 15 to 20 h after infection and continues to increase until cell lysis. These observations represent the first direct evidence that viral infection of eukaryotic cells results in the increased synthesis of a specific cellular enzyme and an increase in the abundance of a specific cellular mRNA. In order to gain additional insight into the control of dihydrofolate reductase synthesis we examined other parameters affecting dihydrofolate reductase synthesis. We found that the addition of fresh serum to stationary phase cells results in a 2-fold stimulation of dihydrofolate reductase synthesis, beginning 10 to 12 h after serum addition. Serum stimulation of dihydrofolate reductase synthesis is completely inhibited by the presence of dibutyryl cyclic AMP as well as by theophylline or prostaglandin E1, compounds which cause an increase in intracellular cyclic AMP levels. In fact, the presence of dibutyryl cyclic AMP and theophylline results in a 2- to 3-fold decrease in the rate of dihydrofolate reductase synthesis and the abundance of dihydrofolate reductase mRNA. However, in contrast to the effect on serum stimulation, dibutyryl cyclic AMP and theophylline do not inhibit polyoma virus induction of dihydrofolate reductase synthesis or dihydrofolate reductase mRNA levels. These observations suggest that dihydrofolate reductase gene expression is controlled by at least two regulatory pathways: one involving serum that is blocked by high levels of cyclic AMP and another involving polyoma induction that is not inhibited by cyclic AMP.

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

AbstractThe 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 3H‐folic acid or to bind 3H‐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 (G0) 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.