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Molecular and Cellular Biology, October 2000, p. 7059-7067, Vol. 20, No. 19
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

E2F-Rb Complexes Assemble and Inhibit cdc25A Transcription in Cervical Carcinoma Cells following Repression of Human Papillomavirus Oncogene Expression

Lingling Wu,¹ Edward C. Goodwin,¹ Lisa Kay Naeger,^1, Elena Vigo,² Konstantin Galaktionov,³ Kristian Helin,² and Daniel DiMaio^1,*

Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510¹; Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy²; and Department of Molecular and Human Genetics, BCM-Ben Taub Research Center, Baylor College of Medicine, Houston, Texas 77030³

Received 4 April 2000/Returned for modification 15 May 2000/Accepted 5 July 2000

Expression of the bovine papillomavirus E2 protein in cervical carcinoma cells represses expression of integrated human papillomavirus (HPV) E6/E7 oncogenes, followed by repression of the cdc25A gene and other cellular genes required for cell cycle progression, resulting in dramatic growth arrest. To explore the mechanism of repression of cell cycle genes in cervical carcinoma cells following E6/E7 repression, we analyzed regulation of the cdc25A promoter, which contains two consensus E2F binding sites and a consensus E2 binding site. The wild-type E2 protein inhibited expression of a luciferase gene linked to the cdc25A promoter in HT-3 cervical carcinoma cells. Mutation of the distal E2F binding site in the cdc25A promoter abolished E2-induced repression, whereas mutation of the proximal E2F site or the E2 site had no effect. None of these mutations affected the activity of the promoter 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. This resulted in marked rearrangement of the protein complexes that formed at the distal E2F site in the cdc25A promoter, including the replacement of free 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 assembly of repressing E2F4-Rb DNA binding complexes. Importantly, these experiments revealed that HPV-induced alterations in E2F transcription complexes that occur during cervical carcinogenesis are reversed by repression of HPV E6/E7 expression.

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^* Corresponding author. Mailing address: Department of Genetics, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510. Phone: (203) 785-2684. Fax: (203) 785-7023. E-mail: daniel.dimaio{at}yale.edu.

Present address: Gilead Sciences, Foster City, CA 94404.

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Molecular and Cellular Biology, October 2000, p. 7059-7067, Vol. 20, No. 19
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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