Increase in c-fos and c-myc mRNA levels in untransformed and SV40-transformed 3Y1 fibroblasts after addition of serum: its relationship to the control of initiation of S phase.

When rat 3Y1 fibroblasts were exposed to serum after 7.5 h of S, G2, and M phases in the absence of serum, the c-fos and c-myc mRNA levels markedly increased. This marked increase was also observed when density-arrested cells were stimulated with fresh serum to initiate proliferation. Increase in the c-fos and c-myc mRNA levels was not observed in cells that had traversed 7.5 h in these phases in the presence of serum. Cells passing through S, G2, and M phases in the absence of serum delayed entry into the next S phase approximately 8 h compared to control cells incubated in the presence of serum. Also, when density-arrested cells were stimulated with serum for 5 h, then deprived of serum for 8 h, and then incubated in serum again, the c-fos and c-myc mRNA levels increased. In this last case, the total excess time of serum exposure required to enter S phase was only 2 h, indicating that cells had not returned to the initial density-arrested state during the serum deprivation period. The increase in c-fos and c-myc mRNA levels following addition of serum after incubation in the absence of serum was also observed in SV40-transformed 3Y1 cells. The entry of SV40-transformed cells into S phase was not markedly affected by the absence of serum. These results can be explained by assuming that there is a process leading to the initiation of S phase that is operating or accumulating continuously in all cell cycle phases. In 3Y1 cells the expression of the c-fos and c-myc genes is required at any cell cycle phase, and the increase in c-fos and c-myc mRNA levels in response to changes in serum concentration simply reflects the possible overexpression due to the delay of a hypothesized negative feedback regulation. In SV40-transformed 3Y1 cells, the process leading to the initiation of S phase operates normally in response to growth factors, and the SV40 large T antigen supplements or enhances the process in the absence of the growth factors.