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Molecular and Cellular Biology, September 1999, p. 6379-6395, Vol. 19, No. 9
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
CDC25A Phosphatase Is a Target of E2F and Is
Required for Efficient E2F-Induced S Phase
Elena
Vigo,
Heiko
Müller,
Elena
Prosperini,
Guus
Hateboer,
Peter
Cartwright,
Maria Cristina
Moroni, and
Kristian
Helin*
Department of Experimental Oncology, European
Institute of Oncology, 20141 Milan, Italy
Received 20 January 1999/Returned for modification 28 May
1999/Accepted 14 June 1999
 |
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.
| |
INTRODUCTION |
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 p19ARF) 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 p16INK4A) 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 |
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.
The ER and ERE2F fragments were subsequently cloned as blunt-end
fragments into pBabepuro (62) which had been cut with
BamHI and blunt ended by treatment with the Klenow
fragment of DNA polymerase (1), resulting in
pBabeHAER, pBabeHAERE2F-1, pBabeHAERE2F-2, pBabeHAERE2F-3, and
pBabeHAERE2F-4. pBabeHAERE2F-1/VP-16 was generated by cloning a
BamHI site-containing fragment of E2F-1 (encoding amino
acids 1 to 368) fused to VP-16 (76) from pBSK111/VP16 (62a) into pBabeHAER. pCMVHAER was generated by cloning a
blunt-end EcoRV-SacII fragment from pBSKHAER into
BamHI-cut and blunt-ended pCMVneoBam (2).
Subsequently, pCMVHAERE2F-1 and pCMVHAERE2F-1(E132) were generated by
cloning the wild-type or the DNA binding mutant of E2F-1
(10), respectively, into the unique BamHI site of pCMVHAER.
The luciferase reporter plasmid CDC25A(
755/+423) luc was constructed
by subcloning the 1,178-bp
SacI fragment of the
CDC25A promoter in pGL3 basic (Promega). Mutations in the
E2F DNA binding
sites were generated by PCR-site-directed mutagenesis.
For the
upstream site (m1), the first PCR was performed with primers A5
(5'-CTAGAGCTCCCAGGGGGCTAAG-3') and A7
(5'-CCTAGTTGGCTTCAAACGGAATCC-3'),
the second PCR was done
with primers A6 (5'-CTAGAGCTCCCGCTCCTCTTCC-3')
and A8
(5'-GGATTCCGTTTGAAGCCAACTAGGAA-3'), and the final reaction
was achieved with primers A5 and A6. For the downstream site (m2),
the
first PCR was performed with primers A5 and A9
(5'-CCGGCCTTTCAAGGTAATAGCGGC-3'),
the second PCR was done
with primers A6 and A10 (5'-GCTATTACCTTGAAAGGCCGGCCT-3'),
and the final reaction was achieved with primers A5 and A6. The
double mutant was generated with a three-fragment ligation, cloning
in
the
SacI site of pGL3 basic the 730-bp
SacI-
PvuII fragment
from m1 and the 430-bp
PvuII-
SacI fragment from m2. The 6× E2F
luciferase construct used, pGL3 TATA basic 6× E2F, has been described
previously (
63) and was the kind gift of Ali
Fattaey.
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.
For microinjection of antibodies, the antibodies were first dialyzed
against PBS to remove NaN
3 and then concentrated to 300
ng/µl by using Amicon G10 filters. Rabbit IgG was added as a
microinjection
marker at a concentration of 2 µg/µl. Rat1 ERE2F-1
cells were
grown on glass coverslips to 50% confluency and then
incubated
in DMEM supplemented with 0.1% BCS for 48 h.
Cytoplasmic microinjection
was then performed in this medium, using the
procedure described
above. After a recovery period of several hours
after the microinjection,
BrdU (100 µM) and OHT (300 nM) were added
to the medium. Twelve
hours later, the cells were fixed as described
above. Injected
cells were identified with a goat-anti-rabbit IgG
coupled to Cy3
(Jackson Laboratories), and BrdU staining was performed
with the
fluorescein isothiocyanate-labeled anti-BrdU antibody from
Becton
Dickinson (BD347583). DAPI was used for staining of DNA. The
slides
were analyzed under an Aristoplan fluorescence microscope
(Leitz).
For peptide competition experiments, the antibody was first
incubated
with a 10-fold molar excess of peptide, then dialyzed and
concentrated
as described
above.
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 |
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.
To test the feasibility of generating cell lines exhibiting
hormone-dependent activation of the E2F transcription factors,
we
constructed expression plasmids in which a modified version
of the ER
was fused to the N terminus of E2F-1. This modified
version of the ER
does not respond to estrogen but rather to OHT
(
56). To test
whether subsequent effects mediated by the ERE2F-1
fusion were a
consequence of DNA binding, we also constructed
plasmids expressing an
ER fusion containing a DNA binding-deficient
mutant of E2F-1 (E132)
(
10). The constructed expression plasmids
were tested by
transient transfection of Rat1 cells. Expression
of ERE2F-1 was shown
to lead to a 15-fold activation of a cotransfected
reporter plasmid
containing six E2F DNA binding sites after addition
of OHT, while no
transactivation was observed in cells expressing
the E132 mutant (data
not shown). Importantly, we did not observe
a higher basal promoter
activity of the reporter plasmid in the
absence of OHT in cells
expressing ERE2F-1 than in cells that
were transfected with the
reporter plasmid alone (data not shown),
suggesting that the ERE2F-1
fusion protein was completely inactive
in the absence of OHT. The
subcellular localizations of the fusion
proteins were tested by
immunofluorescence analysis. U2OS cells
were transiently transfected
with pCMVHAERE2F-1 and stained for
E2F-1 expression by using an
anti-E2F-1 monoclonal antibody (Fig.
1).
In the absence of OHT, the fusion protein is located in the
cytoplasm,
while after addition of OHT, the majority of the fusion
protein is
located in the nucleus (Fig.
1A and B). Identical results
were obtained
with cells expressing the ERE2F-1 E132 fusion protein
(data not shown).
The translocation of the fusion proteins from
the cytosol to the
nucleus strongly suggests that the activity
of the ERE2F fusion
proteins is regulated by their subcellular
localization.
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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).
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To obtain cell lines that stably express ERE2F-1, Rat1 cells were
transfected with pCMVHAERE2F-1 and pCMVHAERE2F-1(E132),
and
several clones were isolated and tested. For the
cytomegalovirus-expressing
clones, the data presented here are the
results obtained with
clone D (wild type) and clone Q (E132), which
express comparable
amounts of protein (data not shown). Similar results
have, however,
been obtained with several different clones. In
addition, pools
of cells were also selected after infection of Rat1
cells with
retroviruses expressing ERE2F-1 or ERE2F-1/VP-16, in which
the
transactivation and pRB binding domain of E2F-1 is replaced by
the
VP-16 transactivation domain (
76). Results for these pools
of infected cells are also presented here. Since we do not have
an
anti-E2F-1 antibody that recognizes rat E2F-1 and human E2F-1
to the
same extent, it is not possible to give the exact levels
of E2F-1
overexpression in the clones. However, by using equal
amounts of cell
lysates from human U20S cells and Rat1 cells expressing
the E2F-1
fusion proteins, we could estimate that the Rat 1 clones
and the pools
express 5- to 10-fold-higher levels of E2F-1 than
do U2OS cells (data
not
shown).
Addition of OHT to the wild-type and E132 clones leads to nuclear
translocation of the fusion proteins (data not shown), and
as shown in
Fig.
1C and D, a similar phenomenon is observed for
the pool of
infected cells expressing ERE2F-1. Our hypothesis
is that the nuclear
translocation is the consequence of OHT binding
to the ER, the
displacement of ER-associated cellular polypeptides,
and the unmasking
of the E2F-1 nuclear localization signal, located
in the N terminus of
E2F-1 (
63). The pools (data not shown)
and the clones (see
Fig.
9D) were shown to transactivate a reporter
plasmid containing six
E2F DNA binding sites in an OHT-dependent
manner. We also tested the
capacity of the ERE2F-1 fusion protein
to induce S phase in
serum-deprived cells. The D and Q clones
were kept in medium containing
0.1% serum for 48 h, after which
either OHT was added to the
starvation medium or fresh medium
containing 10% serum was provided
(Table
1). Addition of OHT
to clone D
(expressing wild-type E2F-1) but not to clone Q (expressing
the E132
mutant) led to S-phase induction. The activation of E2F-1
by addition
of OHT appears to shorten traversal of G
0-G
1 by
approximately
6 to 7 h compared to serum stimulation (Table
1 and
data not
shown). Similarly, the activation of E2F-1 by addition of OHT
to the E2F-1 pool (Table
1) and to the E2F-1/VP-16-expressing
cells
(data not shown) was sufficient to induce S phase in the
treated cells.
We could also detect a high rate of apoptosis in
the D clone after OHT
addition. Between 14 and 18 h, 30% of the
cells were apoptotic,
as indicated by the sub-G
1 population detected
by
fluorescence-activated cell sorter (FACS) analysis (see Fig.
5C). There
was no sign of apoptosis in the E132-expressing clones
after addition
of OHT, demonstrating the requirement of a functional
DNA binding
domain for cellular effects mediated by E2F-1 (
10,
31,
39,
57,
74). In summary, the activated ERE2F-1 fusion
protein shows all
the expected properties of wild-type E2F-1,
including DNA-dependent
transactivation of E2F-dependent promoters
and the capacity to induce
S-phase entry and apoptosis in serum-starved
fibroblasts. Moreover,
since activated ERE2F-1/VP16 fusion protein
elicits the same responses
as ERE2F-1, these features are not
due to inactivation of pRB by
titration but rather are a genuine
transactivation function of E2F-1.
Finally, the facts that in
the absence of OHT the fusion proteins are
in the cytoplasm and
that there is no detectable leakiness in the
system make these
inducible cell lines a powerful tool to study the
functional effects
of deregulated E2F-1 activity.
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.
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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.
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To analyze whether other genes containing E2F DNA binding sites in
their promoters could be directly activated by E2F-1, RT-PCR
was
performed on RNA prepared from the ERE2F-1-expressing cells,
using
gene-specific primers. As shown in Fig.
3, activation of
E2F-1 alone was
sufficient to increase the transcription of
cdc6,
B-
myb, the gene encoding cyclin A, and
p107 in
addition to the
gene coding for cyclin E, whereas only minor activation
was observed
for
TK,
TS,
DHFR,
cdc2, and
PCNA. We did not observe any
significant
increase in the level of c-
myc or DNA polymerase
. None of the
genes tested were upregulated by ERE2F-1(E132) when
OHT was added
to the cells (data not shown).
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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.
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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.
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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.
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To test the functional properties of ERE2F-2 and ERE2F-3, we analyzed
the abilities of the proteins to induce S-phase entry
in low-serum
medium and their abilities to transactivate a synthetic
promoter. The
data presented in Fig.
5A demonstrate
that activation
of ERE2F-2 or ERE2F-3 by OHT induce S-phase entry in
serum-starved
cells to a degree similar to activation of ERE2F-1. By
transfection
of a reporter plasmid containing six E2F DNA binding sites
into
ERE2F-2- or ERE2F-3-expressing cells, we also observed an
OHT-dependent
transactivation of this promoter construct (see Fig.
9D).
These
results are consistent with previously published results for
transiently
transfected or microinjected E2F-1, E2F-2, and E2F-3
(
53,
57).
To investigate the effects of constitutive
activation of the E2Fs
on cell proliferation, the ERE2F cell lines were
grown in low-serum
medium for 96 h in the absence or presence of
OHT (Fig.
5B). While
cells kept in medium without OHT proliferated
slowly, there was
a dramatic selection against proliferation of cells
expressing
active E2F-1, E2F-2, or E2F-3. In agreement with this, we
observed
a high level of cell death, as evidenced by floating and
highly
refractile cells (data not shown). No cell death was observed
when the ERE2F-1(E132 cells) were grown in low-serum medium in
the
presence of OHT (data not shown). Since the observed phenotype
is
characteristic for cells undergoing apoptotic death, and it
is known
that ectopic expression of E2F-1 induces apoptosis (see
the
introduction), a flow-cytometric assay was used to test whether
the
cell death was due to apoptosis (
65). As shown in Fig.
5C,
activation of E2F-1, E2F-2, or E2F-3 induced apoptosis in
ERE2F-expressing
Rat1 cells grown in low-serum medium in the presence
of OHT. In
the absence of OHT, no sub-G
1 peak was observed,
nor did we observe
a sub-G
1 peak in
ERE2F-1(E132)-expressing cells when they were
grown in low-serum medium
in the presence of OHT (data not shown).
In agreement with these
results, we have previously observed a
strong selection against E2F-1,
E2F-2, and E2F-3 expression in
U2OS cells both with
tetracycline-regulated expression and in
colony formation assays
(
72a).
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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.
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Since these data show that the selected pools of Rat1 cells
expressing ERE2F-2 or ERE2F-3 contain regulated alleles of E2F-2
or
E2F-3 with the expected properties, we performed RT-PCR as
described
for the ERE2F-1-expressing cells. ERE2F-2- or ERE2F-3-expressing
cells
were serum starved and subsequently grown in the presence
of CHX alone,
CHX plus OHT, or OHT alone for 4 h (Fig.
6A). As
shown in Fig.
6A and summarized
in Fig.
6B,
cdc6, B-
myb,
p107,
E2f-1, and the genes encoding cyclins E and A are all
primary
targets of E2F-3, as was previously shown for E2F-1.
Surprisingly,
E2F-2 was only able to upregulate the expression of the
gene coding
for cyclin E and, to some extent, the cyclin A gene and
B-
myb;
however, a high basal level of the mRNA in uninduced
cells made
it difficult to estimate the degree of upregulation. In
summary,
the gene encoding cyclin E appears to be the best tested
target
of E2F-1, E2F-2, and E2F-3, followed by the cyclin A gene and
B-
myb. Comparison of the number of genes upregulated and the
magnitudes
of induction by the different E2Fs showed that E2F-1 appears
to
be a better transactivator than E2F-3, which in turn is a better
transactivator than E2F-2. However, it is important to emphasize
that
this hierarchy may be different for as-yet-unknown, and therefore
nontested, E2F target genes.
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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.
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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).
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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.
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To investigate whether the abundance of the cdc25A protein increased in
parallel with the mRNA level, cell extracts were prepared
from Rat1
ERE2F-1 cells before and after stimulation with OHT
or serum (Fig.
7C).
A slight increase in the amount of cdc25A
protein was observed as early
as 4 h after OHT treatment, and
the level was further increased at
8 and 12 h after OHT stimulation.
In agreement with the slower
kinetics of mRNA accumulation after
serum stimulation, the level of the
cdc25A protein did not increase
until 12 h after addition of
serum.
To analyze whether
cdc25A is also a target for E2F-2 and
E2F-3, RT-PCR analysis was performed on RNA samples from ERE2F-1,
-2, and -3 cells that had been starved and then induced with OHT
and CHX
(Fig.
7D). In contrast to E2F-1, E2F-2 and E2F-3 were
unable to
stimulate the transcription of
cdc25A within 4 h of
OHT
addition, whereas cyclin E mRNA levels were increased to similar
extents by the three transcription factors. However,
cdc25A
mRNA
levels were upregulated after treatment of ERE2F-2- or
ERE2F-3-expressing
cells with OHT for 12 h, suggesting that de
novo synthesis of
other proteins is required for the stimulation of
cdc25A transcription
by E2F-2 and E2F-3 (data not shown). In
summary, our results suggest
that the cell cycle regulation of
cdc25A transcription is dependent
on
E2F.
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.
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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).
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An examination of the promoter region of the
CDC25A gene
revealed the presence of two putative consensus E2F DNA binding sites,
as well as DNA binding sites for basic helix-loop-helix proteins
(E
box), Sp1, CCAAT box binding proteins, bovine papillomavirus
E2, AP-2,
and RBPJ-
(Fig.
8C). As for most of the known genes
containing E2F
DNA binding sites,
CDC25A has a TATA-less
promoter.
To investigate whether the two putative E2F binding sites are
responsible for the upregulation of
CDC25A by E2F-1, the
1,178-bp
SacI fragment was cloned in pGL3basic, resulting in
CDC25A(
755/+423)
luc. This construct was transfected into U2OS cells
together with
expression plasmids for E2F-1, -2, or -3 and
cytomegalovirus
-galactosidase
as a control for transfection
efficiency. As shown in Fig.
9A,
each of
the three E2Fs was able to transactivate the
CDC25A
promoter,
although E2F-1 was three times more efficient than E2F-2 or
E2F-3.
Similar data were obtained when the ERE2F-expressing cell lines
were transfected and the E2Fs were activated by OHT (Fig.
9C).
As
a control for the transactivation activities of the E2Fs, we
used a
synthetic reporter construct containing six E2F DNA binding
sites. As
shown in Fig.
9B and D, E2F-1 and E2F-3 transactivated
this construct
to similar extents whereas E2F-2 was two- to threefold
less efficient.
In summary, our data show that the
CDC25A promoter
is
efficiently transactivated by E2F-1, and to a lesser extent
by E2F-2
and E2F-3, whereas we do not see any short-term effects
of E2F-2 or
E2F-3 on the induction of the endogenous
CDC25A mRNA
levels
in the presence of CHX. Taken together, these data indicate
that E2F-2
and E2F-3 are unable to transactivate the endogenous
CDC25A
promoter, perhaps because of site-specific preferences.
The observed
transactivation of the
CDC25A luciferase construct
by E2F-2
and E2F-3 may be indirect and could be a consequence
of E2F-2- or
E2F-3-induced cell cycle progression that leads to
upregulation of
other transcription factors, including E2F-1.
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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.
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To test whether the transactivation of the
CDC25A promoter
by E2F-1 is dependent on the two identified putative E2F binding
sites,
these sites were mutated by PCR-site-directed mutagenesis
(Fig.
10A). The different constructs were
transfected into U2OS
cells with and without an E2F-1 expression
plasmid and pCMV
-gal
as a control for transfection efficiency. The
transactivation
of both mutants by E2F-1 was reduced compared to that
of the wild-type
promoter, suggesting that both sites can bind E2F-1
(Fig.
10B).
In agreement with these results, gel retardation assays
have shown
that oligonucleotides containing either of the two sites can
specifically
bind E2F-containing complexes in vitro (data not shown).
In contrast,
the mutations introduced in m1 and m2 abolished the
binding of
E2F-containing complexes (data not shown). A construct with
both
E2F sites mutated was transfected into cells, and even though
this
construct does not contain any functional E2F DNA binding
sites, its
activity was slightly elevated after E2F-1 expression.
These data
suggest that there are other sites in the 1,178-bp
construct which
respond to cell cycle progression.
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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.
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Since we found that E2F can activate transcription from the
CDC25A promoter, it was deemed interesting to determine the
role
of the E2F sites in the
CDC25A promoter during the cell
cycle.
To this end, Rat1 cells were transiently transfected with the
wild type or one of the two mutant constructs. Cotransfection
was
performed with a
-galactosidase-expressing plasmid. Next,
cells were
starved for 48 h in low-serum medium and subsequently
stimulated
with high-serum medium for different lengths of time
before lysates
were prepared for luciferase and
-galactosidase
assays. Figure
10C
shows that there was a significant upregulation
of luciferase activity
from the wild-type construct 10 to 15 h
after serum stimulation
(late G
1-early S) (Fig.
10D), which corresponds
to the time
of
CDC25A mRNA upregulation observed after serum stimulation
(Fig.
7A) (
43). A less-pronounced upregulation was detected
for the two mutant constructs. Our results suggest that the 5'
E2F site
plays a crucial role in the negative regulation of the
CDC25A promoter in arrested cells while the 3' E2F site is
more
important for the transactivation of the promoter. Based on these
data, we conclude that the two E2F DNA binding sites participate
in the
cell cycle-regulated expression of the
CDC25A promoter.
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):
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|
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 |
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.
By employing a sensitive semiquantitative RT-PCR technique, we tested
whether a series of genes containing E2F DNA binding
sites in their
promoters are transactivated by the E2Fs in the
absence of de novo
protein synthesis. Indeed, we found that several
of the genes
containing E2F DNA binding sites are very good direct
targets for the
E2F transcription factors. These genes encode
proteins that are
directly involved in regulating the initiation
of DNA replication
(CDC6), cell cycle control (cyclin E, cyclin
A, and CDC25A), and growth
control (B-myb, p107, and E2F-1). Several
results indicate that these
genes are direct targets of the E2Fs.
First, all of these genes contain
E2F DNA binding sites in their
promoters, and transient-transfection
experiments have shown that
they respond to E2F overexpression
(references
6,
26,
29,
40,
50,
67,
85,
100, and
102 and this report). Second,
these genes are all
induced in mid- to late G
1 phase of the cell
cycle (Fig.
3A), corresponding to the time at which pRB is phosphorylated
(
3,
88). Third, the kinetics of induction of these genes
is very
rapid after OHT addition (Fig.
3A and
7B). Fourth, the
activation of
these genes occurs in the absence of protein
synthesis.
Interestingly, we also found that several genes that had been described
as being regulated by the E2Fs were only mildly affected
or not
affected at all by (direct) E2F activity (
5,
11,
14,
34,
60,
72,
90,
92,
93). There are numerous possible
reasons for the lack of
an effect of E2F on these genes, but one
consideration is of particular
interest. The experiments demonstrating
the functional role of the E2F
DNA binding site in these genes
have all involved transient-expression
assays that take the promoter
out of its chromosomal context. Since the
accessibility of DNA
binding sites for transcription factors is to a
large degree regulated
by chromatin structure (see, e.g., reference
71), it is likely
that the untimely expression of
the E2Fs does not allow binding
to a consensus E2F DNA binding element.
Moreover, despite the
presence of an E2F DNA binding consensus site, it
may not be occupied
due to interference by, for instance, other
transcription factors.
In agreement with such a notion is the finding
that a previously
identified E2F consensus site in the
CDC2
promoter is never occupied
during the mammalian cell cycle, as
determined by in vivo genomic
footprinting (
11,
93).
Previously, two alternative strategies for analyzing the effects of
deregulated E2F activity on genes containing E2F DNA binding
sites have
been described. One approach involved infecting serum-starved
REF52 rat
cells with recombinant adenoviruses expressing the various
members of
the E2F family (
12,
13,
54). The expression of
genes
containing E2F DNA binding sites was analyzed 10 to 20 h
after
infection of the REF52 cells, and therefore it was not possible
to
analyze the primary effects of E2F expression on target genes
with this
assay. Thus, in agreement with results demonstrating
that
adenovirus-expressed E2Fs induce S-phase entry, it was observed
that
all cell cycle-regulated genes containing E2F DNA binding
sites are
indeed induced by E2F expression (see Fig. 2A in reference
13 and Fig. 1B in reference
54).
In agreement with this result,
we also found that addition of OHT to
the ERE2F-expressing Rat1
cell lines induced upregulation of all cell
cycle-regulated genes
tested at later time points (Fig.
3A; compare 0 and 16
h).
In the other published approach, which more indirectly evaluates the
effect of deregulated E2F expression, the cell cycle-regulated
expression of genes containing E2F binding sites in mouse embryo
fibroblasts (MEFs) derived from mice with targeted disruption
of
Rb or both
p107 and
p130 was analyzed
(
33,
41,
73). In
accordance with our results, the loss of
pRb in the established
MEFs led to deregulated expression of the genes
encoding cyclins
E and A and of p107, whereas the loss of p107 and
p130 led to
deregulated expression of B-
myb,
cdc2, the cyclin A gene,
E2f-1,
and
TS. Furthermore, in agreement with our results, no change
in
expression was observed for
DHFR,
TK, the DNA
polymerase
gene, c-
myc,
PCNA, the cyclin D1
gene, or
cdc25A. Although these
results are surprisingly
similar to ours, there are some unexpected
differences. It is widely
believed that E2F-1, E2F-2, and E2F-3
are specifically regulated by pRB
(
18,
53), and although our
data show that B-
myb
is upregulated after expression of E2F-1,
E2F-2, or E2F-3, such
deregulation is not observed in
Rb/ MEFs. In
addition, the loss of pRB is expected to lead to deregulation
of E2F-1,
E2F-2, and E2F-3 activities, which we and others have
shown is
sufficient to induce S-phase entry in quiescent cells
(see the
introduction). However, it is clear from the analyses
of the
Rb/ MEFs that they can be made quiescent by
serum starvation. We
don't know the reasons for these unexpected
differences, but apart
from the different cell lines used and E2F
expression levels obtained,
there are other possible explanations for
the discrepancies. Many
cell divisions are required for the
establishment of MEFs, and
the adaptive processes during early
development may modulate the
effects of any genetic mutation introduced
into the embryo. Although
it is clearly difficult to analyze for all
possible genetic alterations
and differences in gene expression
profiles, it is evident that
the
Rb/ MEFs
express higher levels of p107 than wild-type MEFs. It is
very likely
that this higher level of p107 is sufficient to suppress
the expression
of B-
myb (
50), and it has been shown that p107
blocks E2F-1-induced S-phase entry (
101).
Our results also demonstrate that E2F-1 is more active in inducing
transcription of genes containing E2F DNA binding sites
than is E2F-3,
which is again more active than E2F-2. This hierarchy
of activity was
unexpected since E2F-1, E2F-2, and E2F-3 induce
S-phase entry and
apoptosis in Rat1 cells to similar extents and
since E2F-1 and E2F-3
are equally good at inducing transcription
from a synthetic promoter.
Moreover, we have shown that the newly
identified target for E2F-1,
CDC25A (see further below), is not
transactivated by E2F-2
or E2F-3 and that
CDC6 is not a direct
target of E2F-2. It
is noteworthy, however, that
CDC25A and
CDC6 expression is increased in ERE2F-2 and ERE2F-3 OHT-treated cells
before
entry into S phase of the cell cycle, suggesting that the
expression of
other transcription factors as a consequence of
E2F-2 or E2F-3
activation transactivates
CDC25A and
CDC6. A
possible
explanation for this observation is that the
E2F-1
promoter contains
two E2F DNA binding sites and is transactivated by
E2F-1, E2F-2,
and E2F-3 (
38,
44,
64). Moreover, we have
observed that
the activation of E2F-3 is sufficient to transactivate
the E2F-1
promoter in the absence of protein synthesis (Fig.
6) and
that
the activation of E2F-1, E2F-2, and E2F-3 by OHT leads to higher
levels of the E2F-1 transcript shortly after addition of OHT
(
75a).
Since CDC25A and CDC6 are both essential for entry
into S phase
(
29,
36,
43,
100), these data may suggest that
E2F-2- or
E2F-3-induced S-phase entry is dependent on functional E2F-1.
However, it is important to emphasize that at this stage we are
only in
the position to make conclusions about what happens in
our Rat1 cell
lines, and that the regulation of CDC25A by the
different members of
the E2F family should be studied in other
cell lines and by other
methods before firm conclusions are made.
Moreover, there exists the
formal possibility that CDC25A transcription
is elevated due to
derepression of the promoter, by a mechanism
in which E2F-1 replaces a
preexisting E2F-repressive complex on
the promoter. In support of such
a model is a recent report (published
while this paper was undergoing
review) that independently identified
CDC25A as an
E2F-responsive promoter and in which it was found
that E2F-4/p130 is
bound to the 5' repressive element (
41a) (Fig.
10A).
However, we do not favor such a model, since E2F-2 and E2F-3
apparently
do not lead to transactivation of the promoter and
since we have not
seen any measurable effects on cell cycle progression
by using a
protein in which a transactivation-deficient, but DNA-binding-capable,
mutant of E2F-1 (amino acids 1 to 374) was fused to the estrogen
receptor ligand binding domain (data not
shown).
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.
The identification of
CDC25A as an E2F target is interesting
for several reasons. First, CDC25A is a tyrosine phosphatase
whose
activity is believed to be essential for the activation
of the
G
1 cyclin-dependent kinases (
15). Second, CDC25A
is essential
for entry into the S phase of the cell cycle (
36,
43). Third,
CDC25A has oncogenic properties, and it is
overexpressed in human
primary cancers (
24,
97). Fourth,
CDC25A has been described
as an essential downstream target
of c-Myc (
23). In agreement
with this proposed central role
for CDC25A in the regulation of
cell proliferation, we have shown that
CDC25A activity is required
for E2F-1-induced S-phase entry and that
CDC25A can cooperate
with cyclin E (another target of E2F-1) to induce
entry into S
phase.
In contrast to the modest
CDC25A induction as a consequence
of c-Myc activation (
23), we observe a robust immediate
transactivation
by E2F-1. Indeed, a direct comparison of levels of
CDC25A induction
after c-Myc activation and after E2F-1
activation showed that
CDC25A is only slightly elevated by
c-Myc compared with E2F-1
(
95a). Moreover,
CDC25A
expression is elevated in mid- to late
G
1 after serum
stimulation, with kinetics that are in good agreement
with it being a
target gene for the E2F transcription factors
(
43) (Fig.
7A). In contrast, c-
myc expression is elevated in
early
G
1, and it is therefore expected that c-Myc target genes
are induced in that period of the cell cycle. Finally, we show
that the
cell cycle-regulated expression of
CDC25A is dependent
on
E2F, and we suggest that
CDC25A is a bona fide E2F target
gene
which may, to a certain extent, also be regulated by c-Myc.
E2F-induced S-phase entry.
Deregulated expression of E2F-1,
E2F-2, or E2F-3 can relieve the mitogen requirement for entry into S
phase in rodent fibroblasts. The expression of the E2Fs shortens the
traversal of the G0-G1 phases by 6 to 7 h
compared to that of serum-stimulated Rat1 fibroblasts. In agreement
with this result, we find that the direct targets of E2F transcription
factors are induced 6 to 7 h earlier by E2F activation than by
serum activation (Fig. 2 and 7B). Interestingly, previous results have
shown that expression of cyclin E or cyclin A, two of the downstream
targets for E2F, can accelerate the entry into S phase by 2 to 3 h
(69, 80, 81), suggesting that these proteins are implicated
in the biological effects mediated by the E2Fs. Although cyclins E and
A have been shown to shorten the traversal of G1 by 2 to
3 h, they are unable to relieve the mitogen requirement for entry
into S phase (69, 80, 81). However, it was recently
demonstrated that the injection of physiological levels of active
G1 kinases into serum-starved fibroblasts is sufficient to
induce S-phase entry (9). In agreement with and as an
extension of these data, we have shown that coexpression of two
downstream targets of the E2Fs, cyclin E and CDC25A, is sufficient to
induce S phase in quiescent fibroblasts, suggesting that both these
targets are limiting for entry into the S phase of the cell cycle. It
is important to note, however, that the coexpression of CDC25A and
cyclin E is not as efficient at inducing S phase as is the expression
of the E2Fs, suggesting that other direct targets of the E2Fs are
limiting for the entry into S phase. Recent data from our laboratory
have shown that another E2F target, CDC6, is limiting for fast
progression through G1 (29). Unfortunately, it
has not been possible to detect CDC6 after its microinjection into
quiescent cells (63a). Therefore, we have been unable to test whether coexpression of CDC6 with cyclin E and CDC25A will relieve
the mitogen requirement for entry into S phase as efficiently as the E2Fs.
E2F-induced apoptosis.
Several experiments have suggested a
role for E2F-1 in apoptosis. Ectopic expression of E2F-1 has been shown
to lead to p53-dependent and -independent apoptosis in tissue culture
cells and transgenic mice (37, 39, 49, 74, 77, 87, 98). Lack
of E2f-1 in the developing mouse leads to a decrease in
thymocyte apoptosis (21). E2F-1 expression has been shown to
lead to increased levels of p53 (35, 48), which may be a
result of induced expression of p19ARF (4, 13,
82). In agreement with a role for p53 in E2F-1 induced apoptosis,
recent results have shown that the apoptotic effect of E2F-1 can be
overcome by overexpression of MDM2 (48). Rb/ mice undergo p53-dependent apoptosis in
the central nervous system and in the developing eye lens and
p53-independent apoptosis in the peripheral nervous system (58,
61). The induction of apoptosis in pRb-deficient mice appears to
be mediated by E2f-1, since Rb/ mice
survive longer in an E2f-1/ genetic
background than in a wild-type genetic background, most likely due to a
decrease in apoptosis in the central nervous system and the developing
lens (94).
In contrast to the current assumption that it is only E2F-1 and not the
other members of the E2F family that can induce apoptosis
(
13,
48; see also Discussion in reference
94),
we have demonstrated
in this article that E2F-2 and E2F-3 are also
efficient inducers
of apoptosis. Consistent with these results, we
observed a high
level of cell death and selection against cell
proliferation in
cells expressing active E2F-1, E2F-2, or E2F-3. We
have observed
that even in high-serum medium, induced expression of
E2F-1, E2F-2,
or E2F-3 in clonal U2OS cells leads to cell death
(
72a). The
reason for the discrepancy between our results
and those of others
(
13,
48) is presently unknown. However,
it may be ascribed
to different technical aspects, such as the use of
different cell
lines and assays. It should be noted, however, that in
the assays
described by others, apoptosis as measured by a
sub-G
1 peak is
not observed prior to 3 to 4 days after
infection with recombinant
adenoviruses expressing E2F-1 (
13,
48). Therefore, it appears
that our assay conditions are more
sensitive than those previously
described, since we detected an
efficient apoptotic response less
than 24 h after E2F activation.
Moreover, E2F-1 may be a more
efficient inducer of apoptosis in some
cells, and E2F-2 and E2F-3
may in fact be dependent on E2F-1 function
to induce apoptosis
(as they may be dependent on E2F-1 to induce
S-phase entry). Such
a model may, in fact, also provide an explanation
for the efficient
suppression of apoptosis in pRb-deficient mice
lacking E2f-1 (
94).
Conclusions.
In Fig. 12, a
model for the central role of the E2F transcription factors in
regulating cell proliferation and apoptosis is presented. In this
article we have identified several direct targets of the E2F
transcription factors, including genes that are important for DNA
replication, cell cycle control, and growth control. We have
demonstrated that two of these genes (those encoding CDC25A and cyclin
E) can cooperate in inducing S-phase entry in serum-starved cells.
Furthermore, we have provided evidence that not only E2F-1 but also
E2F-2 and E2F-3 expression leads to apoptosis. E2F-1 has been
demonstrated to induce p53-dependent and -independent apoptosis, and it
has been suggested that transactivation of ARF by the E2Fs
provides the connection from the pRB pathway to the p53 pathway and the
induction of apoptosis. The question, however, is what is mediating
E2F-induced apoptosis. It is known that overexpression of
ARF alone is not sufficient for the induction of apoptosis (78), suggesting that other targets of the E2F transcription factors are implicated in this event. Furthermore, several recent results have demonstrated that the activity of ARF is dependent on
functional p53 (for a review, see reference 89), and
it is therefore unclear how the E2Fs induce p53-independent apoptosis. The establishment of ERE2F-expressing cell lines provides an excellent tool for addressing these and other questions related to the role of
the E2F transcription factors in cell cycle control.
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FIG. 12.
Model for the central role of the E2F transcription
factors in cell proliferation and apoptosis. The stimulation of resting
cells with growth factors results in the induction of a signal cascade
leading to the phosphorylation of the retinoblastoma protein (pRB) by
D-type cyclins in association with CDK4 or CDK6, as well as the
activation of the E2F transcription factors. The E2F transcription
factors are essential for controlling several genes whose products are
required for DNA replication, cell cycle progression, and/or growth.
Deregulation of E2F activity as a result of upstream mutations in the
pathway leads to premature entry into S phase of the cell cycle
and depending on the genetic status of the cellto hyperproliferation
or apoptosis. E2F-induced apoptosis occurs via p53-dependent and
-independent pathways. One of the connections between the pRB and p53
pathways is provided by ARF, and the transactivation of ARF
by the E2Fs may be involved in p53-dependent apoptosis. Presently we do
not know which gene products are implicated in p53-independent
E2F-induced apoptosis. PIGs, p53-induced genes; IGF-BP3, insulin growth
factor-binding protein 3.
|
|
| |
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).
| |
FOOTNOTES |
*
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: khelin{at}ieo.cilea.it.
| |
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Molecular and Cellular Biology, September 1999, p. 6379-6395, Vol. 19, No. 9
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Abstract
Introduction
