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.
Pai
S R
SR
Department of Pathobiology, Auburn University, Alabama 36849-5519, USA.
Bird
R C
RC
D004268:DNA-Binding Proteins / Q000378:metabolism; D003852:Deoxyribonucleoproteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics*; D016160:Retinoblastoma Protein / Q000378:metabolism*; D014162:Transfection
D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins; D009687:Nuclear Proteins; D016760:Proto-Oncogene Proteins c-fos; D016160:Retinoblastoma Protein