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 pe rmanently transfected with a constitutively expressed c-fos gene. CP17 -14 cell overexpression of Rb may be due to a balancing response to ov erexpression of the stimulatory effects of c-fos overexpression on tra nscription. 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 e ncoding the c-fos RCE. Competition assays with increasing mass of unla beled probe or dose-dependence assays using increasing mass of nuclear proteins, demonstrated sequence-specific complex formation. Indisting uishable complexes were formed between the c-fos RCE fragment in trans fected cells, but at higher levels (>50%), compared to proteins from p arental cells. Supershift analysis utilizing epitope-specific Rb-monoc lonal antibodies indicated the presence of Rb protein bound to the RCE -containing DNA fragment In contrast, polyclonal anti-Rb antibodies en hanced the amounts of nuclear protein-DNA complexes detected but did n ot 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 varients of RCE-binding complexes in response to c-fos ov er-expression.