Hypophosphorylated Rb, the product of the tumor suppressor gene associated with hereditary retinoblastoma, is thought to act as a suppressor of cell growth and proliferation during G1 phase. We investigated whether Rb expression was dependent on the expression level of the immediate early cell growth gene, c-fos, which is transiently expressed as cells re-enter G1 phase from quiescence. To explore the functional relationship between c-fos and Rb, a eukaryotic expression plasmid was constructed containing the c-fos gene under control of the SV40 promoter complex. This plasmid was co-transfected with plasmids encoding pRSV cat and G418 resistance, into HeLa S3 cells, and clonal populations of transfected (RSfos) cells selected. High levels of c-fos expression in transfected cells were confirmed by both western and northern blot. Rb protein content per cell was determined by flow cytometry using an Rb-specific primary antibody and a FITC-conjugated secondary antibody. Higher expression of Rb (2-6 fold/cell) in approximately 20% of RSfos transfected cells was observed in comparison to parental HeLa cells. Rb content per cell increased approximately 2-fold during the cell cycle in both parental HeLa cells and RSfos cell clones which overexpressed Rb. Rb accumulation occurred in a manner consistent with normal mass accumulation during the cell cycle. Overexpression of Rb in RSfos cells was also confirmed by western blot analysis. Because one possible function of RB may be to act as a brake on cell growth, it is possible that overexpression of RB acts as an inhibitory counter activity to overexpression of the growth promoting activity of c-fos. This possible balancing activity of Rb was further suggested when Rb protein expression levels were measured in different clonal lines of RSfos transfected cells overexpressing increasing levels of c-fos. Overexpression of Rb was proportional to the level of overexpression of c-fos in each clonal cell line. Such evidence suggests that Rb was proportional to the level of overexpression of c-fos in each clonal cell line. Such evidence suggests that Rb expression may be regulated in a manner which balances the transcription stimulatory effects of c-fos overexpression and its effects on the transcription of other genes during the cell cycle.

Antibodies↗

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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.

Base Sequence↗

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Interaction of the Rb tumor suppressor protein with the c-fos promoter in c-fos transfected cells overexpressing c-fos and Rb.

The Rb tumor suppressor protein is overexpressed in HeLa cell lines permanently transfected with a constitutively expressed c-fos gene. CP17-14 cell overexpression of Rb may be due to a balancing response to overexpression of the stimulatory effects of c-fos overexpression on transcription. The cis-acting retinoblastoma control element (RCE, -97 to -86 bp) in the human c-fos promoter is thought to allow regulation of c-fos by Rb. Gel-shift assays were performed with a 168 bp fragment encoding the c-fos RCE. Competition assays with increasing mass of unlabeled probe or dose-dependence assays using increasing mass of nuclear proteins, demonstrated sequence-specific complex formation. Indistinguishable complexes were formed between the c-fos RCE fragment in transfected cells, but at higher levels (> 50%), compared to proteins from parental cells. Supershift analysis utilizing epitope-specific Rb-monoclonal antibodies indicated the presence of Rb protein bound to the RCE-containing DNA fragment. In contrast, polyclonal anti-Rb antibodies enhanced the amounts of nuclear protein-DNA complexes detected but did not result in a supershift. These results suggested the presence of Rb and/or Rb-like peptides involved in complex formation and the presence of multiple variants of RCE-binding complexes in response to c-fos over-expression.

DNA-Binding Proteins↗