E2F transcription activity is composed of a family of heterodimers encoded by distinct genes. Through the overproduction of each of the five known E2F proteins in mammalian cells, we demonstrate that a large number of genes encoding proteins important for cell cycle regulation and DNA replication can be activated by the E2F proteins and that there are distinct specificities in the activation of these genes by individual E2F family members. Coexpression of each E2F protein with the DP1 heterodimeric partner does not significantly alter this specificity. We also find that only E2F1 overexpression induces cells to undergo apoptosis, despite the fact that at least two other E2F family members, E2F2 and E2F3, are equally capable of inducing S phase. The ability of E2F1 to induce apoptosis appears to result from the specific induction of an apoptosis-promoting activity rather than the lack of induction of a survival activity, because co-expression of E2F2 and E2F3 does not rescue cells from E2F1-mediated apoptosis. We conclude that E2F family members play distinct roles in cell cycle control and that E2F1 may function as a specific signal for the initiation of an apoptosis pathway that must normally be blocked for a productive proliferation event.Various studies have led to the delineation of a pathway controlling the progression of cells from quiescence, through G 1 , and into S phase that involves the activation of G 1 cyclin-dependent kinases (cdk), inactivation of Rb and related proteins, and accumulation of E2F transcription factor activity (for reviews see refs. 1-7). It is also now clear that the disruption of components of this control pathway, either the activation of positive acting components such as the G 1 cyclins or the inactivation of negative components such as p53, Rb, and the cyclin-dependent kinase inhibitors (CKI), can lead to the loss of cell growth control underlying the development of various forms of human cancer (7,8).Like many other signal transduction activities, E2F consists of a family of related proteins that include five distinct E2F members and at least two heterodimer partners, DP1 and DP2. The complexity of the E2F activity, as generated by the formation of a variety of heterodimeric protein complexes, suggests a complexity of function whereby the individual family members might play distinct roles in cellular growth control. For instance, the individual E2F family members might integrate distinct signaling pathways within the cell to facilitate the orderly progression through the growth cycle. That is, individual E2F genes might respond to different components of a growth signaling process, either distinct extracellular growth factors or simply distinct signal transduction pathways that integrate a complex growth response. Additionally, but not exclusive of the first instance, the individual E2F proteins could activate distinct target genes, the total of which constitutes the range of activities necessary for cells to progress into and through S phase. Finally, the complexity...

Previous work has demonstrated the important role of E2F transcription activity in the induction of S phase during the transition from quiescence to proliferation. In addition to the E2F-dependent activation of a number of genes encoding DNA replication activities such as DNA Pol ␣, we now show that the majority of genes encoding initiation proteins, including Cdc6 and the Mcm proteins, are activated following the stimulation of cell growth and are regulated by E2F. The transcription of a subset of these genes, which includes Cdc6, cyclin E, and cdk2, is also regulated during the cell cycle. Moreover, whereas overall E2F DNA-binding activity accumulates during the initial G 1 following a growth stimulus, only E2F3-binding activity reaccumulates at subsequent G 1 /S transitions, coincident with the expression of the cell-cycle-regulated subset of E2F-target genes. Finally, we show that immunodepletion of E2F3 activity inhibits the induction of S phase in proliferating cells. We propose that E2F3 activity plays an important role during the cell cycle of proliferating cells, controlling the expression of genes whose products are rate limiting for initiation of DNA replication, thereby imparting a more dramatic control of entry into S phase than would otherwise be achieved by post-transcriptional control alone.

Considerable evidence points to a role for G1 cyclin-dependent kinase (CDK) in allowing the accumulation of E2F transcription factor activity and induction of the S phase of the cell cycle. Numerous experiments have also demonstrated a critical role for both Myc and Ras activities in allowing cell-cycle progression. Here we show that inhibition of Ras activity blocks the normal growth-dependent activation of G1 CDK, prevents activation of the target genes of E2F, and results in cell-cycle arrest in G1. We also show that Ras is essential for entry into the S phase in Rb+/+ fibroblasts but not in Rb-/- fibroblasts, establishing a link between Ras and the G1 CDK/Rb/E2F pathway. However, although expression of Ras alone will not induce G1 CDK activity or S phase, coexpression of Ras with Myc allows the generation of cyclin E-dependent kinase activity and the induction of S phase, coincident with the loss of the p27 cyclin-dependent kinase inhibitor (CKI). These results suggest that Ras, along with the activation of additional pathways, is required for the generation of G1 CDK activity, and that activation of cyclin E-dependent kinase in particular depends on the cooperative action of Ras and Myc.