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

E Vigo

H Müller

E Prosperini

G Hateboer

P Cartwright

M C Moroni

K Helin

Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy

^{Corresponding author. Mailing address: Department of Experimental Oncology, European Institute of Oncology, Via Ripamonti 435, 20141 Milan, Italy. Phone: 39 02 5748 9860. Fax: 39 02 5748 9851. E-mail:}ti.aelic.oei@nilehk.

Received 1999 Jan 20; Revisions requested 1999 May 28; Accepted 1999 Jun 14.

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.

Abstract

Deregulation of cell cycle control mechanisms is a hallmark of human cancer. In particular, there is ample evidence for the deregulation of two control pathways containing the two prototypic tumor suppressor proteins, p53 and the retinoblastoma protein, pRB (88). p53 is believed to be a surveillance factor that can induce apoptosis or growth arrest under specific circumstances, such as DNA damage, hypoxia, or deregulated growth induced by oncogenes (for a review, see reference 55). The importance of p53 in the regulation of cell proliferation is illustrated by the frequent inactivation of the TP53 gene or mutations of the upstream regulators of p53 (e.g., MDM2 and p19^{) in human tumors.}

pRB occupies a central role in regulating the G₁-S transition of the mammalian cell cycle, a very important moment of the cell cycle at which the cell decides whether it should proliferate, differentiate, or die (for reviews, see references 3 and 96). The importance of the pRB pathway to normal growth control is emphasized by the frequent inactivation of the RB-1 gene or mutation of upstream regulators of pRB (e.g., cyclin D1, CDK4, or p16^{) in human tumors. Of the numerous cellular proteins that interact with pRB, the best characterized are the E2F transcription factors, and it is widely believed that pRB, to a large extent, exerts its control of cell proliferation by binding to and inhibiting the activity of these transcription factors (see, e.g., references}57, 76, 99, and 101).

Mice with targeted disruptions of Rb have an increased number of cells in S phase in the central and peripheral nervous systems compared to wild-type mice, and the Rb^−/− neuronal cells fail to undergo differentiation (8, 42, 51, 52, 58). Subsequently, the Rb^−/− mice die between days 13.5 and 14.5 of gestation, exhibiting profound apoptotic cell death in the hemopoietic and nervous systems (8, 42, 51). Consistent with the E2Fs being key downstream targets of pRB, several similarities between the effects of E2F overexpression in tissue culture cells and the loss of Rb function in mice have been observed. For instance, ectopic expression of E2F-1, E2F-2, E2F-3, and, to a lesser extent, E2F-4 is sufficient to induce S phase in quiescent immortalized rat fibroblasts (13, 45, 57), whereas E2F-5 and E2F-6 are unable to do so (7, 13, 25, 57). Moreover, overexpression of E2F-1, but not other E2Fs, has been shown to induce apoptosis in tissue culture cells (13, 49, 77, 87, 98) and transgenic mice (27, 37). Recently, genetic evidence of E2F-1 being a critical downstream target for pRB in vivo was provided by two sets of data showing that Rb^E2f-1^−/− mice live longer, that their incidence of pituitary tumors is reduced compared to that of Rb^+/− mice, and that Rb^E2f-1^−/− embryos survive longer than Rb^−/− embryos (94, 99). Although the Rb^+/− and Rb^−/− mice survive longer in an E2f-1^−/− genetic background, it is noteworthy that they still die, demonstrating (as expected) that E2F-1 is not the only target for pRB.

Thus, the E2Fs can be described as key downstream effectors in a pathway that is very frequently deregulated in human cancer and whose functional integrity is essential for normal cell proliferation. Therefore, it becomes important to understand how these transcription factors are regulated and to know which genes are regulated by the E2Fs (for reviews, see references 18, 30, and 91). The majority of E2F-regulated genes encode proteins that are involved in DNA replication and/or in cell cycle progression. These genes include those encoding DNA polymerase α (72), thymidine kinase (TK) (14), HsORC1 (66), dihydrofolate reductase (DHFR) (5, 60, 90), CDC6 (29, 68, 100), MCM2 to MCM7 (54), cyclin A (40, 85), and cyclin E (6, 26, 67), p107 (102), B-myb (50), c-myc (34, 92), CDC2 (11, 93), E2F-1 (38, 44, 64), and E2F-2 (86). Although the E2Fs have been reported to be essential for the proper cell cycle regulation of several of these genes, it is evident that deregulated E2F activity leads to only a marginal increase in the level of expression of most of these genes (12, 41). Moreover, since several of the proteins participating in DNA replication are very stable, it is not clear why the transcription of these genes needs to be cell cycle regulated. None of the known gene products whose expression is regulated by the E2Fs is able to induce S phase by itself, suggesting that combinations of two or more products are required or that the responsible and limiting targets which can regulate S-phase entry have not yet been identified.

In an effort to better understand how deregulation of the pRB pathway can result in hyperproliferation, we have generated cell lines in which is expressed E2F-1, E2F-2, or E2F-3 fused to a modified version of the estrogen receptor ligand binding domain (ER) (56). The use of ER fusion proteins allows the identification of primary genetic targets for these proteins, since the activation can occur in the absence of de novo protein synthesis. Moreover, the rapid activation of the ER fusion protein after addition of the ligand is another feature that should allow the identification of genes that are required for S-phase induction by the E2Fs. By using the ERE2F cell lines, we demonstrate that in the absence of de novo protein synthesis the E2Fs are sufficient to induce transcription of the genes encoding cyclin E and cyclin A, as well as cdc6, p107, E2f-1, and B-myb, whereas E2F activation has little effect on the transcription of TK, the thymidine synthase gene (TS), PCNA, DHFR, cdc2, or c-myc.

Finally, we have used the ERE2F cell lines to identify cdc25A as a novel E2F target gene. We show that the cell cycle-regulated expression of cdc25A is dependent on E2F and that CDC25A is required for E2F-induced S-phase entry. In addition, we show that CDC25A can cooperate with cyclin E, another target of the E2Fs, to induce S-phase entry in quiescent cells.

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).

ACKNOWLEDGMENTS

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