CDC25A Phosphatase Is a Target of E2F and Is Required for Efficient E2F-Induced S Phase

Abstract

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 G₀-G₁ 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.

ACKNOWLEDGMENTS

We thank Karin Holm, Cristian Matteucci, Stefania Lupo, and Giuseppina Giardina for technical assistance in plasmid constructions, FACS analyses, and retroviral infections. We thank T. Littlewood for pBSKER, W. G. Kaelin for pSGE2F-1/VP16, and G. P. Nolan for Phoenix cells. We are grateful to the members of the Lattanzio family who donated the microinjection equipment. We thank Giulio Draetta and Nick Dyson for critical reading of the manuscript.

E.V. was supported by a fellowship from the Fondazione Italiana per la Ricerca sul Cancro, and H.M., G.H., and P.C. were supported in part by fellowships from the European Community’s TMR and Biomed 2 programs. This work was supported by grants from the Human Frontiers Science Program, the European Community’s TMR Programme, and the Associazione Italiana per la Ricerca sul Cancro (AIRC).