Mutations of the retinoblastoma tumour suppressor gene (RB1) or components regulating the RB pathway have been identified in almost every human malignancy. The E2F transcription factors function in cell cycle control and are intimately regulated by RB. Studies of model organisms have revealed conserved functions for E2Fs during development, suggesting that the cancer-related proliferative roles of E2F family members represent a recent evolutionary adaptation. However, given that some human tumours have concurrent RB1 inactivation and E2F amplification and overexpression, we propose that there are alternative tumour-promoting activities for the E2F family, which are independent of cell cycle regulation.

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...

We have used high-density DNA microarrays to provide an analysis of gene regulation during the mammalian cell cycle and the role of E2F in this process. Cell cycle analysis was facilitated by a combined examination of gene control in serum-stimulated fibroblasts and cells synchronized at G 1 /S by hydroxyurea block that were then released to proceed through the cell cycle. The latter approach (G 1 /S synchronization) is critical for rigorously maintaining cell synchrony for unambiguous analysis of gene regulation in later stages of the cell cycle. Analysis of these samples identified seven distinct clusters of genes that exhibit unique patterns of expression. Genes tend to cluster within these groups based on common function and the time during the cell cycle that the activity is required. Placed in this context, the analysis of genes induced by E2F proteins identified genes or expressed sequence tags not previously described as regulated by E2F proteins; surprisingly, many of these encode proteins known to function during mitosis. A comparison of the E2F-induced genes with the patterns of cell growth-regulated gene expression revealed that virtually all of the E2F-induced genes are found in only two of the cell cycle clusters; one group was regulated at G 1 /S, and the second group, which included the mitotic activities, was regulated at G 2 . The activation of the G 2 genes suggests a broader role for E2F in the control of both DNA replication and mitotic activities.Rapid progress has been made in the understanding of regulatory pathways that govern the transition of cells from a quiescent state into a cell cycle. Such studies have highlighted the critical role of the signaling pathway that involves the accumulation of D cyclin/cdk4 activity leading to the phosphorylation of the retinoblastoma protein, which then allows an accumulation of E2F transcription activity (21,24). A variety of experiments have demonstrated the role of E2F proteins in the control of expression of genes important for DNA replication as well as further cell cycle progression (5, 18). In particular, E2F activity is responsible for the activation of genes encoding DNA replication proteins, enzymes responsible for deoxynucleotide biosynthesis, proteins that assemble to form functional origin complexes, and kinases that are involved in the activation of initiation.Although much has been learned from these studies of E2F transcription control, important questions remain. For one, the scope of the gene-regulatory control by E2F proteins has not been addressed. In large part, the identification of target genes has followed from the initial studies of the DNA tumor virus oncoproteins, such as adenovirus E1A and simian virus 40 T antigen; previous work demonstrated that these proteins were capable of inducing quiescent cells to enter S phase, and associated with this induction was an activation of various genes encoding DNA replication activities (17). This activity coincides with an ability to inactivate the Rb tumor suppressor protein and thus...