E2F-Rb complexes assemble and inhibit cdc25A transcription in cervical carcinoma cells following repression of human papillomavirus oncogene expression

Expression of the bovine papillomavirus E2 protein in cervical carcinoma ce lls represses expression of integrated human papillomavirus (BPV) E6/E7 onc ogenes, followed by repression of the cdc25A gene and other cellular genes required for cell cycle progression, resulting in dramatic growth arrest. T o explore the mechanism of repression of cell cycle genes in cervical carci noma cells following E6/E7 repression, we analyzed regulation of the cdc25A promoter, which contains two consensus E2F binding sites and a consensus E 2 binding site. The wild-type E2 protein inhibited expression of a lucifera se gene linked to the cdc25A promoter in HT-3 cervical carcinoma cells. Mut ation of the distal E2F binding site in the cdc25A promoter abolished E2-in duced repression, whereas mutation of the proximal E2F site or the E2 site had no effect. None of these mutations affected the activity of the promote r in the absence of E2 expression. Expression of the E2 protein also led to posttranscriptional increase in the level of E2F4, p105(Rb), and p130 and induced the formation of nuclear E2F4-p130 and E2F4-p105(Rb) complexes. Thi s resulted in marked rearrangement of the protein complexes that formed at the distal E2F site in the cdc25A promoter, including the replacement of fr ee E2F complexes with E2F4-p105(Rb) complexes. These experiments indicated that repression of E2F-responsive promoters following HPV E6/E7 repression was mediated by activation of the Rb tumor suppressor pathway and the assem bly of repressing E2F4-Rb DNA binding complexes. Importantly, these experim ents revealed that HPV-induced alterations in E2F transcription complexes t hat occur during cervical carcinogenesis are reversed by repression of HPV E6/E7 expression.