Positive and negative regulation of cell proliferation by E2F-1: influence of protein level and human papillomavirus oncoproteins.
E2F-1 is a member of a family of transcription factors implicated in the activation of genes required for the progression through the S phase of the cell cycle. We have examined the biological activities of E2F-1 with short-term colony-forming assays and long-term immortalization assays. High levels of E2F-1, produced by transfection of the E2F-1 cDNA under the control of a strong promoter, reduced colony formation in normal human foreskin keratinocytes (NHFKs). This inhibition could not be overcome by wild-type human papillomavirus type 16 (HPV16) E6 and E7, two proteins which cooperate to immortalize NHFKs, or by a transdominant p53 mutant. High levels of E2F-1 also inhibited growth of primary and established fibroblasts. The growth-inhibitory activity required the DNA binding function of E2F-1 but not its transactivation or pRB binding activities. A positive role for lower levels of E2F-1 in NHFK immortalization was established by examining the ability of E2F-1 to complement HPV16 E7 mutants that were unable to cooperate with HPV16 E6 to immortalize NHFKs. Although E2F-1 was unable by itself to cooperate with E6, it did, in conjunction with E6, complement a p24GLY mutant of E7 that is defective for immortalization and binding of pRB and pRB-related proteins. By contrast, E2F-1 was unable to complement two other E7 mutants, p2PRO and p31/32ARG/PRO, which are also defective in the immortalization assay, although their proteins display wild-type binding of pRB in vitro. Since the binding of E7 to pRB results in disruption of pRB-E2F interaction and release of transcriptionally active E2F, the data support the hypothesis that binding of pRB by E7 and the consequence increase in E2F, the data support the hypothesis that binding of pRB by E7 and the consequence increase in E3F activity are important but not sufficient for E7-induced keratinocyte immortalization.
Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, Maryland 20892.
R M
RM
Melillo
K
K
Helin
D R
DR
Lowy
J T
JT
Schiller
D016475:3T3 Cells; D000818:Animals; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002455:Cell Division; D002471:Cell Transformation, Neoplastic; D002472:Cell Transformation, Viral; D004268:DNA-Binding Proteins / Q000378:metabolism; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D005816:Genetic Complementation Test; D006801:Humans; D066298:In Vitro Techniques; D015603:Keratinocytes / Q000166:cytology; D008297:Male; D051379:Mice; D009856:Oncogene Proteins, Viral / Q000502:physiology*; D027383:Papillomaviridae; D050725:Papillomavirus E7 Proteins; D012097:Repressor Proteins; D016160:Retinoblastoma Protein / Q000378:metabolism; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000502:physiology*; D016335:Zinc Fingers
C533431:Arid4a protein, mouse; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; C493754:E2f1 protein, mouse; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012097:Repressor Proteins; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; C059731:oncogene protein E7, Human papillomavirus type 16