Cyclin A is a functional target of retinoblastoma tumor suppressor protein-mediated cell cycle arrest

Although RE inhibits the G(1)-S transition, the mechanism through which RE prevents cell cycle advancement remains unidentified. To delineate the mech anism(s) utilized by RE to exert its anti-proliferative activity, constitut ively active RE proteins (which cannot be inactivated by phosphorylation) o r p16ink4a (which prevents RE inactivation) were utilized. Both proteins in hibited the G(1)-S transition, whereas wildtype RE did not. We show that ac tive RE acts to attenuate cyclin A promoter activity, and that overexpressi on of cyclin E reverses RE-mediated repression of the cyclin A promoter. Al though cyclin A is an E2F-regulated gene, and it has been long hypothesized that RE mediates cell cycle advancement through binding to E2F and attenua ting its transactivation potential, cyclin E does not reverse dominant nega tive E2F-mediated repression of the cyclin A promoter. Although active RE r epressed both cyclin A and two other paradigm E2F-regulated promoters, only cyclin A transcription was restored upon co-expression of cyclin E. Additi onally, we show that RE but not dominant negative E2F regulates the cyclin A promoter through the CCRE element. These data identify cyclin A as a down stream target of RB-mediated arrest. Consistent with this idea, ectopic exp ression of cyclin A reversed RE-mediated G(1) arrest. The findings presente d suggest a pathway wherein cyclin A is a downstream target of RE, and cycl in E functions to antagonize this aspect of RE-mediated G(1)-S inhibition.