Functional inactivation of the pRB pathway is a very frequent event in human cancer, resulting in deregulated activity of the E2F transcription factors. To understand the functional role of the E2Fs in cell proliferation, we have developed cell lines expressing E2F-1, E2F-2, and E2F-3 fused to the estrogen receptor ligand binding domain (ER). In this study, we demonstrated that activation of all three E2Fs could relieve the mitogen requirement for entry into S phase in Rat1 fibroblasts and that E2F activity leads to a shortening of the G 0 -G 1 phase of the cell cycle by 6 to 7 h. In contrast to the current assumption that E2F-1 is the only E2F capable of inducing apoptosis, we showed that deregulated E2F-2 and E2F-3 activities also result in apoptosis. Using the ERE2F-expressing cell lines, we demonstrated that several genes containing E2F DNA binding sites are efficiently induced by the E2Fs in the absence of protein synthesis. Furthermore, CDC25A is defined as a novel E2F target whose expression can be directly regulated by E2F-1. Data showing that CDC25A is an essential target for E2F-1, since its activity is required for efficient induction of S phase by E2F-1, are provided. Finally, our results show that expression of two E2F target genes, namely CDC25A and cyclin E, is sufficient to induce entry into S phase in quiescent fibroblasts. Taken together, our results provide an important step in defining how E2F activity leads to deregulated proliferation.

The E2F family of transcription factors regulate genes, whose products are essential for progression through the mammalian cell cycle. The transcriptional activity of the E2Fs is inhibited through the specific binding of the retinoblastoma protein, pRB, and the pRB homologs p107 and p130 to their transactivation domains. Seven members of the E2F transcription factor family have been isolated so far, and we were interested in investigating the possible contribution of the various E2Fs to cell cycle control. By presenting the results of the generation of cell lines with tetracycline-controlled expression of E2F-1 and E2F-4 and microinjection of expression plasmids for all members of the E2F family, we demonstrate here that the pRB-associated E2Fs (E2F-1, E2F-2, and E2F-3) all induce S phase in quiescent rat fibroblasts when expressed alone. In contrast, the p107/p130-associated E2Fs require the coexpression of the heterodimeric partner DP-1 to promote S-phase entry and accelerate G 1 progression. Furthermore, the pRB-associated E2Fs were all able to overcome a G 1 arrest mediated by the p16 INK4 tumor suppressor protein, and E2F-1 was shown to override a G 1 block mediated by a neutralizing antibody to cyclin D1. The p16 INK4-induced G 1 arrest was not affected by expression of E2F-4, E2F-5, or DP-1 alone, but simultaneous expression of E2F-4 and DP-1 could overcome this block. Our results demonstrate that the generation of E2F activity is rate limiting for G 1 progression, is sufficient to induce S-phase entry, and overcomes a p16-mediated G 1 block, and since E2F-1, E2F-2, and E2F-3 are associated with pRB, they are the most likely downstream effectors in the p16-cyclin D-pRB pathway. Furthermore, our data suggest that the two subsets of E2Fs are regulated by distinct mechanisms and/or that they have distinct functions in cell cycle control. Since E2F-4 and E2F-5 cannot promote S-phase entry by themselves, our results may provide an explanation for the apparent lack of aberrations in p107 or p130 in human cancer.Entry into and progression through the mammalian cell cycle are highly regulated processes, which at the molecular level involve a number of positively and negatively acting proteins. Many data suggest that among the negative regulators, the prototypic tumor suppressor, pRB, is crucial for proper cell cycle control (for a review, see reference 60). The gene for pRB, RB-1, was the first mammalian tumor suppressor gene to be cloned, and as such it has attracted much attention. Mutations in the RB-1 gene have been found not only in retinoblastomas but also in a variety of human tumors such as osteosarcomas, small-cell lung carcinomas, prostate carcinomas, and cervical cancers (60). The identification of RB-1 mutations in a diversity of human tumors was the first indication that pRB may have a more global regulatory role in cell proliferation. This indication has since been substantiated by a large number of experiments, and in particular it has been demonstrated that overexpression or microinjection of wild...

The E2F transcription factors are essential for regulating the correct timing of activation of several genes whose products are implicated in cell proliferation and DNA replication. The E2Fs are targets for negative regulation by the retinoblastoma protein family, which includes pRB, p107, and p130, and they are in a pathway that is frequently found altered in human cancers. There are five members of the E2F family, and they can be divided into two functional subgroups. Whereas, upon overexpression, E2F-1, -2, and -3 induce S phase in quiescent fibroblasts and override G 1 arrests mediated by the p16 INK4A tumor suppressor protein or neutralizing antibodies to cyclin D1, E2F-4 and -5 do not. Using E2F-1 and E2F-4 as representatives of the two subgroups, we showed here, by constructing a set of chimeric proteins, that the amino terminus of E2F-1 is sufficient to confer S-phase-inducing potential as well as the ability to efficiently transactivate an E2F-responsive promoter to E2F-4. We found that the E2F-1 amino terminus directs chimeric proteins to the nucleus. Surprisingly, a short nuclear localization signal derived from simian virus 40 large T antigen could perfectly substitute for the presence of the E2F-1 amino terminus in these assays. Thus, nuclearly localized E2F-4, when overexpressed, displayed biological activities similar to those of E2F-1. Furthermore, we showed that nuclear localization of endogenous E2F-4 is cell cycle regulated, with E2F-4 being nuclear in the G 0 and early G 1 phases and mainly cytoplasmic after the pRB family members have become phosphorylated. We propose a novel mechanism for the regulation of E2F-dependent transcription in which E2F-4 regulates transcription only from G 0 until mid-to late G1 phase whereas E2F-1 is active in late G 1 and S phases, until it is inactivated by cyclin A-dependent kinase in late S phase.E2F was originally defined as a cellular activity required for the transactivation of the adenovirus E2 promoter by the E1A oncoproteins (34). E1A binds directly to pRB, the product of the retinoblastoma susceptibility gene, and to two pRB relatives, p107 and p130 (40). These proteins, often referred to as pocket proteins, are regulators of the E2F family of transcription factors. Five E2F family members have so far been isolated by virtue of their ability to bind directly to pocket proteins and by homology cloning (38). The affinity of the E2Fs toward pocket proteins and DNA is greatly enhanced by their binding to one of two heterodimeric partners, DP-1 and DP-2/3 (38). The DNA tumor virus oncoproteins E1A, human papillomavirus E7, and simian virus 40 (SV40) large T antigen all regulate E2F-dependent transcription by binding and dissociating the pocket proteins from the E2F heterodimers (4).The E2Fs are believed to regulate the correct timing of transcription of several genes whose products are required for DNA replication (dyhydrofolate reductase, DNA polymerase ␣, and thymidine kinase) and progression through the cell cycle (cyclin A, cyclin E, CDC2, E2F-1, B-Myb...

In current models of the mammalian cell cycle, the pRB family proteins act as key regulators of the GI phase of the cell cycle, where fundamental decisions are taken as to whether a cell should proliferate, differentiate, or undergo apoptosis. Of the numerous cellular proteins that interact with the pRB family proteins, the best characterized are the E2F transcription factors, and it is widely believed that the ability of the pRB family proteins to restrict cell proliferation is dependent on the inhibition of E2F transcriptional activity.To understand the functional role of the E2Fs for cell proliferation in more detail, we have developed cell lines expressing E2F-I , E2F-2 and E2F-3 fused to the estrogen receptor ligand binding domain (ER). Activation of all three E2Fs can relieve the mitogen requirement for entiy into S phase, and activation of the E2Fs leads to a shortening of the GO-G1 phase of the cell cycle by 6-7 hours. In contrast to the current assumption that E2F-I is the only E2F that can induce apoptosis, we show that deregulated E2F-2 and E2F-3 activities also result in apoptosis. By using the ERE2F expressing cell lines, we demonstrate that several genes containing E2F DNA binding sites are efficiently induced by the E2Fs in the absence of protein synthesis. The direct targets for the E2Fs are involved in the regulation of initiation of DNA replication, and in cell cycle and growth control. Data will be presented showing the identification of two novel targets for the E2Fs, namely CDC25A (essential for activation of GI CDKs) and CDC6 (essential for the initiation of DNA replication). Furthermore, results will be presented showing that CDC6 is a substrate for the Cyclin NCDK2 kinase, and it is targeted for ubiquitin-mediated proteolysis. The implications of these results will be discussed at the meeting. 66 Regulation of p53 stability. The role of Mdm2 and nuclear export.