INTRODUCTION

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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^ARF) 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^INK4A) 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  (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.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Plasmids. A BglII-XbaI 5' fragment (172 bp) and a KpnI-BamHI 3' fragment (199 bp) of the modified ER were generated by PCR, using pBSKER as a template (56). The two fragments were ligated to a 603-bp XbaI-KpnI fragment of the ER and cloned into the BamHI site of pBSKHA (32), thereby generating pBSKHAER. Sequencing of the resulting plasmid showed that no mutations were incorporated in the ER fragment. pBSKHAERE2F-1, pBSKHAERE2F-2, pBSKHAERE2F-3, and pBSKHAERE2F-4 were generated by cloning BamHI fragments containing the full open reading frames of the E2Fs into pBSKHAER. Coupled in vitro transcription-translation of the resulting plasmids resulted in ³⁵S-labeled proteins of the expected size.

Generation of cell lines. ERE2F-1 clones were generated by transfecting Rat1 cells with pCMVHAERE2F-1 constructs, using the calcium phosphate method (1). Cells were selected in Dulbecco's modified Eagle medium (DMEM) containing 10% bovine calf serum (BCS) and supplemented with G418 (0.5 mg/ml) and then cloned by limiting dilution. ERE2F pools were generated by infecting Rat1 cells with retroviruses produced in Phoenix cells (kindly provided by Garry Nolan) transfected with pBabeHAERE2F constructs. Briefly, Phoenix cells were plated at a density of 2 million cells per 10-cm-diameter dish and 2 days later were transfected with 10 µg of DNA. Supernatants were collected after 2 days, filtered, and used to infect Rat1 cells. The viral supernatant was left on the cells for 3 h, and the procedure was repeated twice to increase the efficiency of infection. Two days after infection, the Rat1 cell cultures were split and puromycin-resistant cells were selected in medium supplemented with 2.5 µg of puromycin/ml. For starvation conditions, cells were grown in DMEM-0.1% BCS for 48 h and induced with fresh 10% serum or by the addition to starvation medium to which 4-hydroxytamoxifen (OHT) was added to a final concentration of 300 nM. Cycloheximide (CHX) was used at a final concentration of 10 µg/ml. The addition of CHX was shown to reduce protein synthesis, as measured by [³⁵S]methionine incorporation, by more than 99%.

Immunofluorescence. To stain for expression of the E2Fs, cells grown on coverslips were fixed and permeabilized in 20°C cold acetone-methanol (1:1) for 10 min. Coverslips were air dried and stained by two different procedures, depending on the level of protein expression. For transiently transfected U2OS cells, a two-step protocol that included a primary mouse monoclonal antibody (anti-E2F-1 [KH95], anti-E2F-2 [TFE22], or anti-E2F-3 [TFE31]) followed by a goat anti-mouse Cy3-conjugated antibody (Jackson ImmunoResearch Laboratories, Inc.) was used. Stable transfected Rat1 cells expressing a low level of protein were stained with the E2F-1-, E2F-2-, or E2F-3-specific antibodies as described above and developed by using a TSA-Direct kit (NEN Life Science Products) according to the manufacturer's suggestions.

Transactivation assays. U2OS cells were transfected by the calcium phosphate method (1) with 50 ng of pCMVE2F, 2 µg of reporter plasmid, and 0.5 µg of pCMV-gal per 60-mm-diameter dish. Cells were collected 40 h after addition of the precipitate, and lysates were tested for luciferase and -galactosidase activities as described previously (7). Rat1 ERE2F cells were transfected with 2 µg of reporter plasmid and 0.5 µg of pCMV-gal per 60-mm-diameter dish. Fifteen hours after transfection, the cells were induced with OHT for 24 h and lysates were tested for luciferase and -galactosidase activities. To determine the activity of the CDC25A promoter during the cell cycle, Rat1 cells were transfected with 5 µg of reporter plasmid and 1 µg of pCMV-gal per 10-cm-diameter dish. Twenty hours after transfection, the cells were starved for 40 h and subsequently induced with 10% serum. Samples were collected and tested for luciferase and -galactosidase activities as well as protein content. The cell cycle profiles were analyzed with a Becton Dickinson FACScan flow cytometer.

RT-PCR. Reverse transcription-PCR (RT-PCR) was performed on total RNA prepared by the guanidinium thiocyanate-acid phenol method (1). After DNase treatment, 1 µg of RNA was used for cDNA synthesis. In a 50-µl reaction volume were mixed RNA (denatured 1 min at 95°C), dithiothreitol (10 mM), deoxynucleoside triphosphates (0.25 mM each), RNasin (8 U), Superscript with the provided buffer (Life Technologies; 200 U), and random hexamers (25 µM; Pharmacia). Reaction mixtures were incubated at room temperature for 15 min, at 42°C for 45 min, and finally at 95°C for 5 min to inactivate the reverse transcriptase. PCR was performed on 2 µl of the 50-µl cDNA sample. In addition, a PCR sample contained deoxynucleoside triphosphates 50 µM each, buffer with MgCl₂ (1.5 mM final concentration), primers (0.2 µM each), [³²P]dCTP (0.1 µl of 3,000 Ci/mM; Amersham Life Science), Taq polymerase (1 U; Perkin-Elmer), and water (to a final volume of 30 µl). To perform semiquantitative PCR, for each couple of primers we determined on cDNA from cycling Rat1 RNA (at the best annealing temperature) how many cycles were required to detect a clear signal in the linear range. To do so, the reactions were done in triplicate and the samples were collected at the end of various cycles (i.e., 20, 24, or 28). Each sample was run on a 4% polyacrylamide gel in Howley buffer (40 mM Tris, 20 mM sodium acetate, 1 mM EDTA; pH 7.2), and the gel was dried and exposed to an autoradiographic film. Using a phosphorimager (Fuji Inc.), the intensities of the bands were evaluated, and the numbers were plotted to evaluate which conditions to use to be in a linear range. PCR were performed with a PTC-100 machine (MJ Research Inc.), dividing all of the primers into three groups corresponding to three different initial annealing temperatures, 54, 52, and 50°C. While designing the PCR program, we took advantage of the touchdown technique, decreasing the annealing temperature 0.5°C per cycle for the first 10 cycles. Below are indicated, respectively, the initial annealing temperature, the number of cycles, and the sequences of the upstream and downstream primers used for each of the tested genes: for the gene encoding cyclin E, 54°C, 23 cycles, using 5'-ACATTCTACTTGGCACAGGAC-3' and 5'-TGAGACCTTCTGCATCAACTC-3'; for cdc6, 54°C, 23 cycles, using 5'-TTAAGCCGGATTCTGCAAGAC-3' and 5'-TCTGGTATAAGGTGGGAAGTTC-3'; for the gene encoding cyclin A2, 54°C, 23 cycles, using 5'-ATGAGACCCTGCATTTGGCTG-3' and 5'-TTGAGGTAGGTCTGGTGAAGG-3'; for B-myb, 50°C, 24 cycles, using 5'-TGAGGCAGTTTGGACAGC-3' and 5'-TTGAGGTGGTTGTGCCAG-3'; for p107, 54°C, 23 cycles, using 5'-TCATTTGCACCTTCTACCC-3' and 5'-AGTCTATGTGAGATCCTGG-3'; for E2f-1, 54°C, 23 cycles, using 5'-TCTTGGAGCTGCTGAGCC-3' and 5'-TCTGCAGGGTCTGCAATGC-3'; for TK, 50°C, 23 cycles, using 5'-AGCTGATGAGGAGAGTAAG-3' and 5'-ACAATCACTGTCTTGCCTG-3'; for TS, 50°C, 24 cycles, using 5'-TATGGATTCCAGTGGAGAC-3' and 5'-TGCAATCATGTAGGTCAGC-3'; for DHFR, 50°C, 26 cycles, using 5'-TGGTTCTCCATTCCTGAG-3' and 5'-AGGACGTACTAGGAACAG-3'; for cdc2, 54°C, 26 cycles, using 5'-ATATAGTCAGCCTGCAGGATG-3' and 5'-AAGAGCTGGTCAATCTCTGAG-3'; for PCNA, 52°C, 24 cycles, using 5'-ACGTTGAGCAACTTGGAATCC-3' and 5'-TGTTACTGTAGGAGACAGTGG-3'; for c-myc, 52°C, 24 cycles, using 5'-TAGTGCTGCATGAAGAGACAC-3' and 5'-AGTCCAAGTTCTGTCAGAAGG-3'; for the gene encoding DNA polymerase , 50°C, 28 cycles, using 5'-AGCTGATGGATGGTGAAG-3' and 5'-AATACTCCTCTGCTGAGG-3'; for the gene encoding cyclin D1, 50°C, 24 cycles, using 5'-AGATGAAGGAGACCATTCC-3' and 5'-ttcaatctgttcctggcag-3'; for the gene encoding cyclin D3, 50°C, 24 cycles, using 5'-TCATGCCATATCTGAAGCC-3' and 5'-AGATCCAAATGCAGTGACC-3'; for cdc25A, 54°C, 23 cycles, using 5'-TCCAGTGAAGGCAGATGTTC-3' and 5'-AGAACTCACAGTGGAACACG-3'; for cdc25B, 54°C, 24 cycles, using 5'-AGATGAAGCAGGCTACAGAG-3' and 5'-ACCAGTGGAGCACTAATGAG-3'; and for the gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 54°C, 22 cycles, using 5'-ATCCGTTGTGGATCTGACATGC-3' and 5'-TGTCATTGAGAGCAATGCCAGC-3'.

Immunoprecipitation and Western blotting. Rat1 cells expressing ERE2F-1 were serum starved for 48 h in DMEM containing 0.1% BCS and subsequently stimulated for different lengths of time in medium containing 10% serum or in starvation medium to which 1 µM OHT was added. Cells were lysed in ELB (250 mM NaCl, 50 mM HEPES [pH 7.0], 0.1% Nonidet P-40) to which protease inhibitors and 1 mM dithiothreitol were added. After clearing of the lysate by centrifugation, the protein content in the lysate was determined by the method of Bradford. A 200-µg quantity of protein was used for each immunoprecipitation with a polyclonal rabbit antiserum to CDC25A (product no. 06-571; Upstate Biotechnology) by standard protocols (28). Subsequently, the immunoprecipitated proteins were separated on a sodium dodecyl sulfate-8% polyacrylamide gel and processed for Western blotting as described elsewhere (28). The blot was probed with a mouse monoclonal antibody to CDC25A (product no. sc-7389; Santa Cruz) and developed by using an enhanced chemiluminescence kit (ECL; Amersham).

Isolation of the human CDC25A promoter. The CDC25A gene was cloned from a human placenta genomic library screened with a 166-bp probe obtained by PCR performed on human genomic DNA, using the following primers: an upstream primer, +70 bp from ATG of the published cDNA sequence (5'-TCGTGAAGGCGCTATTTGGC-3'); and a downstream primer, +236 bp from ATG (5'-ATCTGTTGACTCGGAGGAGC-3') (22). Ten positive lambda clones were isolated from 10⁶ plaques. A ³²P-labeled oligonucleotide corresponding to the first 25 nucleotides of the published sequence (459 to 434 of the ATG sequence) hybridized to a 1.2-kb SacI fragment in 9 of the 10 isolated phages. The 1.2-kb SacI fragment was cloned in pBluescript SK (Stratagene) and sequenced. The sequence revealed a 1,178-bp SacI fragment starting 755 bp 5' of the previously published start codon.

RNase protection assay. RNase protection was performed as described elsewhere (1). A probe of 258 bp, containing the nucleotide sequence 558 to 315 relative to the ATG, was used. The probe was transcribed by using T7 RNA polymerase on BamHI-cut pBSK in which a PCR fragment (BamHI-XhoI) generated by PCR performed on the 5' region of CDC25A with primers RNase up/BamHI (5'-ATCGGGATCCCGTAGCTGCCATTCGGTTGAG-3') and RNase down/XhoI (5'-GGCGAGCTCGCAACGGCCCAGGCTCAC-3') was cloned. The RNase protection assay was performed on RNA samples from proliferating HeLa cells and from U20S cells synchronized with a double thymidine block followed by a nocodazole block and released from the block for 14 h (79% of cells were in S phase).

Microinjection. For DNA microinjection experiments, Rat1 cells were grown on glass coverslips to 30% confluency and then incubated in DMEM without serum for 48 h. At the time of microinjection, the cells had reached 60 to 80% confluency. Nuclear microinjections were performed with a Zeiss automated microinjection device connected to an Eppendorf injector, using the following settings: time of injection, 0.0 s, pressure, 50 to 150 hPa; angle, 45°; speed, 20. Glass capillaries (product no. GC120TF-10; Clark Electromedical Instruments) were pulled by using the P-87 puller from Sutter Instruments. The injection time per coverslip did not exceed 30 min. The injection medium was DMEM without serum. The DNA used for microinjection was diluted in filter-sterilized phosphate-buffered saline (PBS) to a final concentration of 100 ng/µl for pCMVEGFP (microinjection marker) or 20 ng/µl for pCMVE2F-1, pCMVCyclin E, and pCMVhCDC25A. After microinjection, the cells were allowed to recover for 3 h, and then bromodeoxyuridine (BrdU; 100 µM) was added to all dishes at the same time. Cells were fixed for immunostaining 16 h after the addition of BrdU. Cells were fixed in 4% paraformaldehyde for 5 min at room temperature, washed briefly with PBS, and then incubated for 30 s in 60 mM NaOH. After three washes in PBS, BrdU was detected with an anti-BrdU antibody from Becton Dickinson (BD347580) as a primary antibody and a Cy3-conjugated anti-mouse immunoglobulin G (IgG) from Jackson Laboratories as a secondary antibody. The DNA was stained with 4',6-diamidino-2-phenylindole (DAPI). Injected cells were identified by the green fluorescence emitted from enhanced green fluorescent protein. For each injection, between 100 and 150 injected cells were counted. The experiments were repeated three times with similar results.

Nucleotide sequence accession number. The nucleotide sequence of the human CDC25A promoter has been submitted to the DDJB-EMBL-GenBank databases under accession no. AJ242714.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Generation of cell lines expressing ERE2F-1 fusion proteins. Previously, our laboratory has generated cell lines with tetracycline-regulated expression of the E2F transcription factors (57). Although the use of inducible expression of the E2F transcription factors or, alternatively, infection with adenoviruses containing the E2Fs is a useful technique for the understanding of the functional consequences of deregulated E2F expression (see, e.g., references 12, 13, 35, 57, and 87), neither technique allows the identification of primary target genes of the E2F transcription factors. As an alternative approach, the activities of several proteins have been made hormone dependent by fusion of these proteins to the hormone binding domain of the estrogen receptor (ER) (see, e.g., references 19, 75, 79, and 84). The generation of ER fusion proteins allows the identification of primary events, since the fusion proteins can be activated without de novo protein synthesis.

View larger version (100K): [in this window] [in a new window]   FIG. 1.   Subcellular localization of ERE2F-1. (A and B) Immunofluorescence of U2OS cells transiently transfected with pCMVHAERE2F-1, detected by using an anti-E2F-1 monoclonal antibody (KH95), in the absence (A) or in the presence (B) of OHT. (C to F) A selected pool of Rat1 clones expressing ERE2F-1, stained with an anti-E2F-1 monoclonal antibody, in the absence (C) or in the presence (D) of OHT and the corresponding DAPI staining (E and F).

Identification of genes as direct targets of E2F-1. To identify the primary target genes that allow E2F-1 to induce S phase in serum-starved cells, Rat1 ERE2F-1 cells were serum starved for 48 h and then induced with serum or OHT for different lengths of time in the absence or presence of CHX. RNA was prepared, and RT-PCR was performed under linear reaction conditions as described in Materials and Methods. To test whether this method was feasible, we first analyzed the expression of the cyclin E gene (Fig. 2). As early as 2 h after addition of OHT, cyclin E mRNA levels were increased dramatically, and this increase was not abolished by simultaneous CHX treatment, demonstrating that E2F-1 activation alone is sufficient for increased transcription of the endogenous cyclin E gene (see also Fig. 7B). Consistent with the cyclin E gene being an important downstream target of E2F and the fact that cyclin E is limiting for progression through G₀-G₁ (69, 80), cyclin E transcription was activated 6 to 7 h earlier in cells treated with OHT than in serum-treated cells. This result is also in good agreement with the demonstration that activation of E2F-1 shortens the G₁ phase of the cell cycle by approximately 6 to 7 h.

View larger version (41K): [in this window] [in a new window]   FIG. 2.   Cyclin E is a direct target of E2F-1. ERE2F-1 clone D cells were made quiescent by growing the cells in medium with 0.1% serum for 48 h. RT-PCR was performed on samples of total RNA from ERE2F-1 clone D that had been kept for 48 h in medium containing 0.1% serum and induced for different lengths of time by addition of OHT, serum, or OHT plus CHX. Primers specific for the amplification of cyclin E mRNA were used as described in Materials and Methods.

View larger version (36K): [in this window] [in a new window]   FIG. 3.   Genes activated by E2F-1. (A and B) RT-PCR was performed on RNA samples prepared from ERE2F-1 clone D (A) and from an ERE2F-1 pool (B). Cells were kept for 48 h in medium containing 0.1% serum and induced for different lengths of time with OHT or serum (A) or for 4 h in the presence of CHX, CHX plus OHT, or OHT alone (B). Primers specific for the cDNA amplification of the indicated genes were used as described in Materials and Methods. (C) Genes activated directly by E2F-1 expression. ++, upregulation over 10-fold; +, upregulation between 5- and 10-fold; +/, upregulation between 1- and 5-fold; , no upregulation observed; *, data not shown.

E2F-2 and E2F-3 induce S phase and apoptosis. To extend the analyses of the regulation of E2F-dependent genes to the other members of the E2F family, we infected Rat1 cells with retroviruses expressing ERE2F-2, ERE2F-3, or ERE2F-4. Data concerning ERE2F-4 will not be presented here, since we were not able to show any transactivating potential of the fusion protein before or after addition of OHT. The lack of regulation of this protein may be due to the fact that the majority of ERE2F-4 remains in the cytoplasm after addition of OHT (data not shown). In contrast, the ERE2F-2 and ERE2F-3 proteins are localized in the cytoplasm in the absence of OHT, while nuclear translocation is observed after addition of OHT (Fig. 4). We noticed a slight nuclear staining in some of the ERE2F-2-expressing cells even in the absence of OHT, indicating that the activity of the ERE2F-2 fusion is not as tightly regulated as that of ERE2F-1 or ERE2F-3.

View larger version (40K): [in this window] [in a new window]   FIG. 4.   Subcellular localization of ERE2F-2 and ERE2F-3 in the presence or absence of OHT. (A and B) A pool of puromycin-resistant Rat1 cells infected with retroviruses expressing ERE2F-2, stained with an anti-E2F-2 monoclonal antibody (TFE22), in the absence (A) or in the presence (B) of OHT. (C and D) A pool of puromycin-resistant Rat1 cells infected with retroviruses expressing ERE2F-3, stained with an anti-E2F-3 monoclonal antibody (TFE31), in the absence (C) or in the presence (D) of OHT.

View larger version (32K): [in this window] [in a new window]   FIG. 5.   Activation of E2F-1, E2F-2, or E2F-3 results in S-phase entry and apoptosis. (A) E2F-1, E2F-2, or E2F-3 activation is sufficient for S-phase induction. Rat1 cells expressing ERE2F-1, ERE2F-2, or ERE2F-3 were grown in medium containing 0.1% serum for 48 h and subsequently induced with OHT. Cells were harvested for FACS analysis at the indicated times after addition of OHT, and the cell cycle profile was determined. (B) E2F-1, E2F-2, and E2F-3 induce cell death. Cells were serum starved for 48 h in medium with 0.1% serum, and OHT was subsequently added to the starvation medium. At the indicated times, cells were harvested and living cells were counted after being stained with Trypan blue. The percentage of surviving cells was calculated by using the number serum-starved cells in the absence of OHT as the reference point. (C) Activation of E2F-1, E2F-2, or E2F-3 induces apoptosis. FACS analysis of Rat1 cells expressing ERE2F-1, ERE2F-2, or ERE2F-3, showing the percentage of apoptotic cells (sub-G1 fraction) at 0 and 24 h after OHT addition. The cells were grown in medium with 0.1% serum for 48 h prior to addition of OHT.

View larger version (31K): [in this window] [in a new window]   FIG. 6.   Genes activated by E2F-2 and E2F-3. (A) RT-PCR was performed on RNA samples prepared from ERE2F-2 and ERE2F-3. The cells were grown in medium containing 0.1% serum for 48 h, and OHT was subsequently added for 4 h in the presence of CHX, CHX plus OHT, or OHT alone. (B) Summary of genes activated directly by E2F-2 and E2F-3 expression. ++, upregulation over 10-fold; +, upregulation between 5- and 10-fold; +/, upregulation between 1- and 5-fold; , no upregulation observed; ?, increased levels in serum-starved cells.

CDC25A as a novel E2F target. Since the use of ERE2F cell lines in combination with RT-PCR gave encouraging results when testing whether a gene can be a direct target of E2F regulation, we decided to use the system to identify other genes regulated by the E2Fs. For this purpose, we tested four genes (those encoding cyclins D1 and D3, as well as cdc25A and cdc25B) whose expression previously was shown to be cell cycle regulated (43, 46, 59) but with no indication that E2F activity participates in the regulation. Of the four tested genes, only cdc25A was shown to be affected by E2F expression (Fig. 7 and data not shown). As shown in Fig. 7A, a strong increase in the cdc25A mRNA level was observed 4 h after OHT addition, while serum stimulation of the same cell line led to the highest level of accumulation at 12 h. To confirm that the upregulation of cdc25A by E2F-1 was a primary effect, the level of cdc25A mRNA was measured shortly after OHT addition (Fig. 7B). An increase in cdc25A mRNA levels was observed as early as 1 h after OHT addition, and this increase became more pronounced at 2 to 3 h after stimulation. Moreover, cdc25A upregulation was sustained even in the presence of CHX, demonstrating that the transactivation of the cdc25A promoter by E2F-1 does not require de novo protein synthesis (Fig. 7B and D).

View larger version (53K): [in this window] [in a new window]   FIG. 7.   CDC25A is a direct target of E2F-1. (A and B) RT-PCR performed on RNA samples from ERE2F-1 clone D cells that were starved and induced with OHT or serum (A) or with OHT alone, CHX plus OHT, CHX alone, or serum alone (B). Primers for specific amplification of cdc25A, the cyclin E gene, and GAPDH were used as indicated and as described in Materials and Methods. (C) Activation of E2F-1 leads to accumulation of the cdc25A protein. Immunoprecipitation followed by Western blotting with cdc25A-specific antibodies was performed with 200-µg quantities of cell lysate at the indicated time points after addition of OHT or serum. The masses of molecular size markers are indicated to the left in kilodaltons. (D) Activation of E2F-2 and E2F-3 is not sufficient to induce transcription of CDC25A. RT-PCR was performed on RNA prepared from ERE2F-1, ERE2F-2, and ERE2F-3 pools as described above. Cyclin E served as an internal positive control. The cells were starved and induced for 4 h in the presence of CHX, CHX plus OHT, or OHT alone.

E2F-dependent cell cycle regulation of CDC25A transcription. To understand the role played by E2Fs in the regulation of CDC25A transcription, we cloned the CDC25A gene from a human placenta genomic library. Ten positive lambda clones containing the 5' end of the CDC25A cDNA were isolated, and hybridization with an oligonucleotide corresponding to the first 25 nucleotides of the published cDNA sequence (22) identified an approximately 1.2-kb SacI genomic fragment. This fragment was cloned and sequenced, and it was shown to contain an 1,178-bp insert (Fig. 8A). The 3' SacI site is located 20 bp upstream of the initiation codon of the previously published cDNA sequence (22). To determine the position of transcription initiation in the CDC25A gene, RNase protection experiments were performed. As shown in Fig. 8B, an RNA probe of 258 nucleotides containing 237 nucleotides of the CDC25A gene yielded several protected fragments in RNAs prepared from both U2OS and HeLa cells. The longest of these protected fragments was approximately 120 nucleotides, corresponding to a position at 440 nucleotides 5' of the CDC25A start codon. The 5' end of the human CDC25A gene isolated by Galaktionov and Beach (22) corresponds to a position 442 nucleotides 5' of the CDC25A start codon, and we have therefore decided to use this as the +1 reference point.

View larger version (64K): [in this window] [in a new window]   FIG. 8.   Sequence and schematic representation of the human CDC25A promoter. (A) Nucleotide sequence of the 1,178-bp CDC25A promoter SacI fragment containing 755 nucleotides upstream of the transcription initiation site and 423 nucleotides of the 5' untranslated region of the human CDC25A cDNA. Two E2F DNA binding sites are underlined. The transcription initiation site, which coincides with the longest cDNA clone isolated, is indicated with an arrow. (B) RNase protection assay. RNA was prepared from HeLa and U2OS cells and processed for RNase protection, using a 258-nucleotide probe containing 237 nucleotides of the CDC25A gene surrounding the putative transcription initiation site. The longest protected fragment, corresponding to approximately 120 nucleotides, is indicated with an arrow. The length of the probe is indicated to the right, and the number of nucleotides in a double-stranded DNA molecular size marker is indicated to the left. RNA has a slower mobility than DNA, and it is estimated to be 5 to 10% different from DNA. (C) Schematic representation of transcription factor binding sites in the 1,178-bp human CDC25A promoter. The transcription start site is depicted with an arrow. The two E2F sites are indicated, as are DNA consensus binding sites for AP-2, RBPJ-, bovine papillomavirus E2 (BPVE2), CCAAT-box binding proteins, Sp-1, and bHLH (E-box).

View larger version (24K): [in this window] [in a new window]   FIG. 9.   E2F transactivation of the CDC25A promoter. (A and B) Activation of the CDC25A promoter by transient transfection of pCMVE2F-1, pCMVE2F-2, and pCMVE2F-3. U2OS cells were transiently transfected with pCMVE2F-1, pCMVE2F-2, or pCMVE2F-3 together with CDC25A(755/+423) luc (A) or with a synthetic E2F-responsive promoter, 6× E2Fluc (B). , transfected with empty pcnv expression vector. (C and D) Activation of the CDC25A promoter in the ERE2F-expressing cell lines. Rat1 cells expressing ERE2F-1, ERE2F-2, or ERE2F-3 were transfected with CDC25A(755/+423) luc (C) or with 6× E2Fluc (D). +, addition of OHT; , without OHT. pCMV-gal was cotransfected in all experiments, and -galactosidase activity served as a control for transfection efficiency. Numbers indicate fold induction relative to the sample transfected with empty pCMV (A and B) or fold induction after addition of OHT (C and D). The luciferase counts are normalized for -galactosidase activity. The presented data are representative of at least three different experiments.

View larger version (22K): [in this window] [in a new window]   FIG. 10.   E2F-dependent cell cycle regulation of the CDC25A promoter. (A) Sequences of the putative E2F DNA binding sites in the CDC25A promoter. These sites were mutated to the indicated sequences by PCR-site-directed mutagenesis as described in Materials and Methods. (B) Both E2F DNA binding sites respond to E2F transactivation. Transfection of U2OS cells with CDC25A(755/+423) luc (wild type) and mutants thereof, with (+) or without () pCMVE2F-1. The adjusted luciferase activities are the relative luciferase activities after normalization for the activity of cotransfected pCMV-gal. (C and D) The activity of the CDC25A promoter during the cell cycle. Rat1 cells transfected with CDC25A(755/+422) luc wild type (wt), m1, or m2 were starved for 48 h and subsequently induced with serum. Samples were collected at different time points to evaluate the luciferase activity and the cell cycle profile as determined by FACS analysis. The adjusted luciferase activity in panel C indicates fold induction relative to the activity of the wild-type promoter at time 0 h, obtained by first normalizing the luciferase counts for the -galactosidase activity of cotransfected pCMV-gal. In panel D are shown the percentages of S-phase cells for each time point. The data presented are representative of at least three different experiments.

CDC25A cooperates with cyclin E to induce S phase. E2F activity can bypass the mitogen requirement for S-phase entry in rat fibroblasts. Since expression of none of the known E2F-regulated genes is sufficient to relieve the mitogen requirement for S-phase entry in serum-starved cells, it is not known how the E2Fs induce entry into S phase. Previous data have demonstrated that CDC25A is essential for entry into S phase (36, 43). CDC25A is a tyrosine phosphatase, and one of its activities is the removal of an inhibitory phosphate molecule from the G₁ cyclin-dependent kinases (reviewed in reference 15). Studies of Drosophila genetics have demonstrated the requirement for cyclin E for E2F-induced S-phase entry (16, 17), and it has been shown that cyclin E is also required for entry into S phase (47, 70). Thus, it is very likely that cyclin E is also required for E2F-induced S-phase entry in mammalian cells. Previous publications have shown that cyclin E alone is not sufficient to induce S-phase entry in quiescent fibroblasts (69, 80). Cyclin E associates with CDK2, and the complex formed needs several modifications before it is active. Since CDK2 is present throughout the cell cycle, including quiescence (20, 83, 95), it is conceivable that the cyclin E-CDK2 complex is inactive as a result of CDC25A's absence. To test whether CDC25A, alone or in combination with cyclin E, is sufficient to induce S-phase entry, serum-starved Rat1 fibroblasts were microinjected with plasmids expressing cyclin E or CDC25A, or with control plasmids (Fig. 11A). A plasmid expressing the green fluorescent protein was included in all microinjections to facilitate the identification of injected cells. After injection, the cells were cultured in medium containing BrdU, and cells that entered S phase were identified by using anti-BrdU antibodies. Injection of E2F-1 resulted in a significant number of cells undergoing DNA synthesis compared to control-injected cells. Expression of cyclin E or CDC25A alone was not sufficient to induce an increase in the number of S-phase cells compared to the control. However, when cyclin E and CDC25A were coexpressed, a significant increase in the number of S-phase cells was observed (Fig. 11A). This result is in agreement with recently published data demonstrating that performed active cyclin E-CDK2 or cyclin D1-CDK4 is sufficient to induce DNA synthesis when microinjected into quiescent cells (9). In contrast to the previously published data, our data demonstrate that two E2F targets, when coexpressed, are sufficient to initiate DNA synthesis in quiescent fibroblasts. Our data also demonstrate that coexpression of CDC25A and cyclin E is not as efficient at inducing S-phase entry as expression of E2F-1 alone, suggesting that other targets of E2F-1 are also limiting for progression through the G₁ phase of the cell cycle.

View larger version (55K): [in this window] [in a new window]   FIG. 11.   CDC25A cooperates with cyclin E in inducing S phase and is required for efficient E2F-1-induced S-phase entry. (A) Cyclin E and CDC25A cooperate to induce S phase in serum-starved Rat1 fibroblasts. E2F-1, cyclin E, CDC25A, or cyclin E plus CDC25A expression vectors (20 ng/µl each) were injected into serum-starved Rat1 fibroblasts. pCMVEGFP (100 ng/µl) was coinjected as an injection marker. At 16 h after microinjection, the percentage of BrdU-positive microinjected cells was determined. GFP, green fluorescent protein. (B) CDC25A is necessary for efficient E2F-1-induced S-phase entry. Rat1 ERE2F-1 cells were incubated in DMEM containing 0.1% serum for 48 h. The indicated antibodies were then microinjected (300 ng/ml) along with rabbit IgG as a microinjection marker (2 µg/µl). At 3 h after microinjection, OHT was added (300 nM) to induce E2F-1 activity. At 12 h after the induction of E2F-1, cells were harvested and processed for immunofluorescence analysis, and the percentage of BrdU in microinjected cells was determined. rIgG, rabbit IgG; pept., peptide. (C) An example of an antibody microinjection experiment. The percentage of BrdU in injected cells is lower in samples injected with antibodies against CDC25A.

CDC25A is required for efficient E2F-induced S-phase entry. Since the results obtained so far suggest that CDC25A is a critical target for E2F-1 and that coexpression of CDC25A with another E2F target gene is sufficient to induce entry into S phase, we wanted to assess whether CDC25A is required for E2F-1-induced S-phase entry. Quiescent Rat1 cells expressing ERE2F-1 were microinjected with two affinity-purified polyclonal antibodies to CDC25A and the appropriate controls (Fig. 11B). Rabbit IgG was included in all injected samples to facilitate the identification of injected cells. After injection, cells were incubated in medium containing OHT and BrdU for 12 h. Cells undergoing DNA synthesis were identified with an anti-BrdU antibody (Fig. 11C). As shown in Fig. 11B, 72% of control-injected ERE2F-1-expressing cells entered S phase within the first 12 h after addition of OHT, whereas microinjection of the two affinity-purified antibodies to CDC25A significantly decreased (by 50%) the ability of E2F-1 to induce DNA synthesis. Moreover, preincubation of one of these antibodies with the antigenic peptide blocked the ability of the antibody to prevent E2F-1-induced S-phase entry. These data demonstrate that CDC25A is required for efficient induction of S-phase entry by E2F-1.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Elimination of cell cycle control mechanisms is one of the key features of human cancer. In particular, genes in the pRB pathway are very frequently found to be mutated. Several data have identified the E2F transcription factors as key downstream effectors in the pRB pathway. In agreement with this, ectopic expression of members of the E2F family recapitulates the phenotypes associated with the loss of function of the pRB pathway. To understand the underlying molecular mechanisms by which the E2Fs influence the cell phenotype, it is essential to identify the genes that are directly regulated by the E2Fs, i.e., what the primary targets are and how their deregulated expression results in hyperproliferation.

E2F target genes. To identify the primary targets of the E2F transcription factors, we have developed cell lines expressing E2F-1, E2F-2, or E2F-3 fused to a modified version of the estrogen receptor ligand binding domain (ER). The activation of the E2Fs by OHT demonstrated that the ERE2F fusion proteins possess the expected biochemical features, including the abilities to transactive E2F-dependent synthetic promoters and to induce S-phase entry and apoptosis. Moreover, these changes were found to be dependent on the ability of E2F to bind to DNA but independent of pRB binding. Based on these results, we concluded that activation of the ERE2F fusion proteins by OHT is a reliable system to study the effects of deregulated E2F expression on gene activation and cell proliferation.

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CDC25A is an E2F target. To date, no systematic screen for E2F-regulated genes has been published. We believe that the ERE2F-expressing cell lines are excellent tools for performing such a screen. Meanwhile, however, we have used the ERE2F-expressing cell lines to test whether known cell cycle-regulated genes are induced by the E2Fs in the absence of protein synthesis. By performing such assays, we identified CDC25A as a direct target of E2F-1. The cloning and sequencing of the promoter identified two E2F DNA binding sites that can be transactivated by E2F-1 expression. Mutational analyses showed that the upstream-most E2F DNA binding site is essential for proper cell cycle regulation of the promoter whereas the downstream site appears to be important for the overall activity of the CDC25A promoter. To our surprise, our data suggest that CDC25A is a direct target of E2F-1 but not of E2F-2 or E2F-3. The reason for this apparent specificity is presently unknown. No data showing differences in the binding site specificities of E2F-1, E2F-2, and E2F-3 have so far been published. Moreover, our unpublished results suggest that the two E2F DNA binding sites can bind to E2F-1, E2F-2, and E2F-3 in vitro (95a), indicating that other factors implicated in regulating CDC25A may be important for the specificity of the transactivation. Future work will be required to investigate the molecular and cellular basis for the lack of CDC25A transactivation by E2F-2 and E2F-3 in our system. <