c-fos expression is required during all phases of the cell cycle during exponential cell proliferation

Transient transcription of the c-fos gene is induced by serum stimulation of quiescent cells during the earliest part of Gl phase, reaching maximum levels of mRNA within 30 min. To determine whether expression of c-fos is required, or has any regulatory role in continuous exponential cell proliferation following re-entry into the cell cycle, a chimeric plasmid was constructed containing the human c-fos gene such that transcription was under control of the SV40 promoter complex. The plasmid was co-transfected into HeLa S3 cells (RSfos cells) along with plasmids encoding pRSVcat and G418 resistance followed by clonal propagation. The regulatory role of c-fos in an exponentially growing transfected RSfos cell clone (CP17-14) and parental HeLa cells was assessed through studies of c-fos overexpression or suppression of c-fos translation by treatment with a c-fos antisense 16-mer oligonucleotide. Transfected cells grew normally despite excess expression of c-fos. In contrast, antisense oligonucleotide treatment efficiently suppressed proliferation in normal exponentially growing cells by more than 70% for approximately 60 hr. Beyond this time oligonucleotides were ineffective likely due to degradation/depletion. In contrast, transfected cells grew normally, indicating overexpression of c-fos was sufficient to neutralize the effects of c-fos antisense oligonucleotides. Flow cytometric analysis of cell cycle phase distribution and determination of proliferation rate in oligonucleotide treated HeLa cells revealed a virtually complete inhibition of cell proliferation without a block in any specific cell cycle phase. In addition, no effect of oligonucleotide treatment on cell cycle phase distribution was observed in CP17-14 cells. Overexpression of c-fos rendered these cells resistant as the antisense c-fos oligonucleotides were unable to impose proliferative inhibition. These results demonstrate that c-fos expression is required during all phases of the continuous cell cycle in exponentially growing cells suggesting an important maintenance role for c-fos in addition to its role during re-entry into the cell cycle from quiescence.