Cell cycle analysis of Krox-20, c-fos, and JE expression in proliferating NIH3T3 fibroblasts.

We have shown that early growth response genes which were identified on the basis of their expression during the G0 to G1 transition can also be induced in proliferating fibroblasts. The expression of Krox-20 and c-fos mRNAs increased dramatically upon stimulation of cell populations of increasing density and correlated with the percentage of cells in the G0-G1 stages of the cell cycle. However, fractionation of serum-stimulated cultures into cell cycle stage-specific subpopulations using fluorescence-activated cell sorting revealed that the levels of Krox-20 and c-fos mRNAs were equal in all stages of the cell cycle. This result was corroborated by serum and cycloheximide stimulation of stage-specific fractions separated by centrifugal elutriation. Expression of the immediate-early gene JE was also induced in all stages of the cell cycle in the elutriated fractions. Thus, although the amount of induced Krox-20, c-fos, and JE mRNA correlated with the number of quiescent cells in a proliferating population, expression of all three genes occurred to the same extent in all stages of the cell cycle at a given culture density. Possible explanations for the density-associated increase in induced expression are discussed. We have demonstrated that there are both transcriptional and posttranscriptional components to the stimulated expression of the Krox-20 and c-fos genes in proliferating fibroblasts. Since the increased expression of these genes has the same steady-state and transcription rate kinetics as serum response factor-mediated induction, we are currently investigating the role of serum response factor in serum-induced expression in proliferating cells.