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 t he activation of genes required for the progression through the S phas e 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 c DNA under the control of a strong promoter, reduced colony formation i n normal human foreskin keratinocytes (NHFKs). This inhibition could n ot be overcome by wild-type human papillomavirus type 16 (HPV16) E6 an d E7, two proteins which cooperate to immortalize NHFKs, or by a trans dominant p53 mutant. High levels of E2F-1 also inhibited growth of pri mary and established fibroblasts. The growth-inhibitory activity requi red the DNA binding function of E2F-1 but not its transactivation or p RB binding activities. A positive role for lower levels of E2F-1 in NH FK immortalization was established by examining the ability of E2F-1 t o complement HPV16 E7 mutants that were unable to cooperate with HPV16 E6 to immortalize NHFKs. Although E2F-1 was unable by itself to coope rate with E6, it did, in conjunction with E6, complement a p24GLY muta nt 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 i n the immortalization assay, although their proteins display wild-type binding of pRB in vitro. Since the binding of E7 to pRB results in di sruption of pRB-E2F interaction and release of transcriptionally activ e E2F, the data support the hypothesis that binding of pRB by E7 and t he consequent increase in E2F activity are important but not sufficien t for E7-induced keratinocyte immortalization.