The effect of polyoma virus, serum factors, and dibutyryl cyclic AMP on dihydrofolate reductase synthesis, and the entry of quiescent cells into S phase

Abstract

Three procedures were used to induced dihydrofolate reductase synthesis in quiescent cultures of methotrexate resistant mouse fibroblasts: 1) lytic infection with polyoma virus, 2) growth stimulation by replating cells at lower density in fresh cell culture medium, and 3) the addition of fresh medium to confluent cells. Following polyoma infection, an increase in the percentage of S-phase cells began at approximately 20 hours; dihydrofolate reductase synthesis also increased following a lag of 20 hours or more, and continued to increase throughout the late phase of lytic infection, reaching values nearly fivefold greater than that originally present in the quiescent cells. When quiescent cells received fresh medium (with or without replating), the percentage of cells in S phage began to increase by 10 hours and was accompanied by an increase in dihydrofolate reductase synthesis which reached a maximum by approximately 25 hours. These observations show that the initial entry of cells into S phase following mitogenic stimulation is associated with an induction of dihydrofolate reductase synthesis. Dibutyryl cyclic AMP blocked the stimulation of dihydrofolate reductase synthesis and the increase in the percentage of S-phase cells that resulted from the addition of fresh medium to confluent cells. When dibutyryl cyclic AMP was added at various times following the addition of fresh medium, the block in the induction of dihydrofolate reductase synthesis was correlated with a corresponding block in the increase in S-phase cells. These results suggest that dibutyryl cyclic AMP blocks cells at a point in G1 prior to either the induction of dihydrofolate reductase synthesis of the beginning of S phase. The relationship between the control of dihydrofolate reductase synthesis and entry into S phase suggests some form of coordinate control over these two parameters.

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

“…4 mock-infected cells. The observed decrease in mock-infected cells was consistent with that observed in other cell lines progressing from a growing into a resting state (12,15,41).…”

“…This is a difficult task because of the low levels of synthesis of the products from any individual gene and the previous unavailability of gene-specific probes (12,15,19,23,41 which time the MTX concentration was increased. The cell lines used in this study, S3-100, S3-300, and S3-500, were grown in 100, 300 and 500 ,uM MTX, respectively.…”

“…Furthermore, the protein and RNA species produced in the resistant line are identical to those reported for unamplified human cells (29). By analogy with results with other MTX-resistant cell lines (12,15,19,23,41), we assume that the changes in DHFR gene expression observed during infection of the S3-500 cells accurately reflect the changes which occur in normal cells. Thus, these cells are well suited for use as a model system to study the effects of adenovirus on DHFR gene expression.…”

“…We Lytic infection of MTX-resistant quiescent murine cells with polyoma virus also results in an induction of DHFR gene expression (12,19). After an early lag, the level of induction progressively increases during the late states of infection and reaches a value several times that present in uninfected quiescent cells.…”

Growth-dependent expression of dihydrofolate reductase mRNA from modular cDNA genes.

“…Treatment of stationary cells with cyclic AMP (cAMP) further reduces the level of DHFR mRNA (20,24). Stimulation of cells by the addition of serum or virus infection results in increased synthesis of DHFR mRNA (19). Synthesis of DHFR mRNA appears to be specific to the early part of the S phase in the cell cycle (28), which perhaps explains the higher levels of this mRNA in growing cells.…”