From the Westmead Institute for Cancer Research, and
the ¶ Westmead Millennium Institute, University of Sydney,
Westmead Hospital, Westmead, New South Wales 2145, Australia and
the
Department of Molecular Genetics and Microbiology, Duke
University Medical Center, Durham North Carolina 27710
Received for publication, October 28, 2002
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ABSTRACT |
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The p14ARF tumor suppressor is a key
regulator of cellular proliferation and is frequently inactivated in
human cancer. This tumor suppressor functions in the p53 and pRb cell
cycle regulatory pathways and can effectively activate both pathways to
induce growth arrest or cell death. We now report that
p14ARF forms a complex with the E1A-regulated
transcriptional repressor, p120E4F.
p120E4F contacts p14ARF and p53 to form a
ternary complex in vivo and enhances
p14ARF-induced G2 cell cycle arrest in a
p53-dependent manner. We suggest that the interaction of
p14ARF and p120E4F forms an important link
between the p14ARF and p53 tumor suppressor proteins, both
of which exhibit enhanced cell cycle inhibitory activity in the
presence of this transcriptional repressor.
The INK4a/ARF locus on chromosome band 9p21
is disrupted in a wide variety of human tumors at a frequency
comparable with p53 inactivation. The common loss of this locus
presumably reflects its dual coding capacity; this single genomic
sequence encodes the ARF tumor suppressor and the p16INK4a
cyclin-dependent kinase inhibitor (1, 2). Although
p16INK4a is frequently and specifically inactivated in
tumors and in kindreds with a predisposition to melanoma (3-7), recent
evidence confirms that the
ARF1 tumor suppressor is also
targeted in human cancers and is implicated in melanoma predisposition.
Many mutations inactivating p16INK4a also impair the
function of ARF (8, 9) and alterations specifically affecting ARF have
been detected in familial melanoma kindreds (10-12).
The ARF tumor suppressor can induce potent growth arrest or cell death
in response to hyperproliferative oncogenic stimuli. Mutated Ras,
c-Myc, adenovirus E1A, E2F-1 and v-Abl all stimulate ARF expression in
normal cells and induce G1 and G2 cell cycle arrest or apoptosis (13-16). p14ARF can activate the p53
tumor surveillance pathway by interacting with and inhibiting the
p53-antagonist, Hdm2 (17-22). ARF limits the E3 ubiquitin
ligase activity of Hdm2, thus preventing the polyubiquitination,
nuclear export, and subsequent cytoplasmic degradation of p53 (22-25).
Consequently, the loss of ARF not only diminishes the response of the
p53 network to hyperproliferative signals but also reduces the duration
of p53 activity in response to DNA damaging stimuli. Animals lacking
ARF are highly prone to tumor formation, and their embryonic
fibroblasts do not senesce, continuing to cycle after DNA damage
(26).
ARF can also inhibit cell growth through p53- and Hdm2-independent
mechanisms. Various types of human tumors exhibit simultaneous loss of
p53 and ARF (27, 28), and the inactivation of p53 and ARF was not
mutually exclusive in immortalized mouse embryonic fibroblasts (MEFs) (29). Mice lacking both ARF and p53, with or without
the mouse Hdm2 homologue, Mdm2, developed a wider range of tumor types
than animals lacking either tumor suppressor alone (30). Moreover, the
expression of the murine ARF homologue, p19ARF, strongly
suppressed colony formation of p53-null MEFs (29) and inhibited DNA
synthesis in p53-null/Mdm2-null MEFs (30). Although murine and human
ARF display important differences in structure and function, human
p14ARF also reduced the rate of DNA synthesis when p53
function was compromised (31).
The p53/Hdm2-independent functions of ARF are poorly understood but
presumably involve additional ARF-binding partners. ARF can interact
and destabilize the S-phase-inducing transcription factors E2F-1,
E2F-2, and E2F-3 (32, 33). Consistent with a role for E2F degradation
in the mechanism of ARF growth suppression, p53-null MEFs engineered to
over-express E2F-1 were at least partially insensitive to the growth
inhibitory action of p19ARF (29, 32). Other proteins that
complex with ARF include spinophilin, a regulatory subunit of the
protein phosphatase 1 catalytic protein (34), topoisomerase I (35),
MdmX (36), HIF-1 We now report that p14ARF can associate in vitro
and in vivo with the adenovirus E1A-regulated
p120E4F transcription factor. In humans and mice,
p120E4F is an ubiquitously expressed, low abundance
GLI-Kruppel-related zinc finger phosphoprotein that acts as a
transcriptional repressor of the adenovirus E4 gene and, presumably,
other cellular target genes (40-42). In response to adenovirus E1A or
treatment with phorbol ester, p120E4F becomes
hyperphosphorylated and undergoes a reduction in both DNA binding and
transcriptional repressor activities (43, 44). This contrasts with the
properties of p50E4F, a proteolytically derived 50-kDa
amino-terminal fragment of p120E4F that exhibits increased
DNA binding activity in response to E1A-induced phosphorylation and
stimulates the adenoviral E4 gene (40). Thus, although
p50E4F and p120E4F can recognize the same DNA
motif (RTGACGT(C/A)AY) in vitro, the additional residues
specific to p120E4F confer distinct functional and
regulatory properties.
Expression of p120E4F significantly inhibits
cellular growth and cell cycle progression, whereas p50E4F
has no such effect (42, 43, 45-47). The growth inhibitory activity of
p120E4F has been associated with the post-transcriptional
elevation of several cell cycle regulatory proteins, including the CDK
inhibitors p21Waf1 and p27Kip1, cyclin E, and
cyclin B1, with reduced Cdk2, Cdk4/6, and Cdc2 kinase activities
and with the down-regulation of cyclin A2 gene expression (42, 43, 48).
Moreover, p120E4F-induced cell cycle arrest is enhanced by
its interaction with the p53 transcription factor and
hypophosphorylated pRb (46, 47). Although the cyclin A gene is the only
cellular target thus far demonstrated to be down-regulated by
p120E4F, it is likely that other genes involved in the
control of cell cycle progression are regulated by p120E4F,
as ectopic expression of p120E4F can induce cell cycle
arrest at the G1-S and G2-M transitions even
when cyclin A mRNA and protein levels are not reduced (43, 48).
In this study, we demonstrate that p14ARF forms a complex
with p120E4F to modulate cell cycle progression. The
formation of this complex occurs through residues in
p120E4F that are in close proximity to those required for
p53 binding and growth suppression activity and through residues in the
amino-terminal region of p14ARF, which is also
involved in Hdm2, E2F, and topoisomerase binding. Through this
interaction, p14ARF sequesters ectopically expressed
p120E4F in nucleoli. Importantly, p120E4F
contacts both p14ARF and p53 to form a ternary complex
in vivo and enhances G2 cell cycle arrest in a
p53-dependent manner. We propose that p14ARF
co-operates with p53 to induce growth arrest by interacting with and
regulating the activity of the E1A-regulated transcription factor,
p120E4F.
Mammalian Expression Constructs
The p14ARF-FLAG
(49), p16INK4a-FLAG (8),
pCMVs-E4F2.5K (encoding full-length p120E4F,
amino acids 1-784) (40) and pCMVs-E4FPstI (encoding
p75E4F, amino acids 1-551) (46) constructs have been
described elsewhere. The mutant p53 expression plasmid encoding human
p53 cDNA with mutations altering amino acids 22, 23 and 281 has
also been described previously (50). Truncated p14ARF
cDNAs were isolated from the pCMV-FLAG5b clones and
fused in-frame with the LexA DNA-binding domain in the
displayBAIT vector (Display Systems Biotech). The
p50E4F-FLAG plasmid (encoding amino acids 1-262) was
amplified from pCMVs-E4F2.5K using the E4F sense
(5'-CTCGAATTCGCCACCATGGAGGGCGAGATG-3') and antisense oligonucleotides
(5'-CCGGGATCCGCACCCGACTCCCGGAAG-3'). The
E4F-(369-566)-displayTARGET and
E4F-(567-784)-displayTARGET clones were each
amplified from pCMVs-E4F2.5K with the following primers:
E4F-(369-566) sense (5'-AGCGAATTCAACCTGCTGCACCAG-3') and antisense
(5'-CTTCTCGAGCTACACGTGCCGCACCAG-3'); E4F-(567-784) sense
(5'-AGCGAATTCCGACACCACACAGGC-3') and antisense
(5'-CTTCTCGAGCTAGACGATGACCGT-3'). All inserts were amplified using
Pfx polymerase (Invitrogen) and verified by automated sequencing.
Cell Culture and Transfections
Human osteosarcoma U2OS (ARF-null, p53 and pRb wild type) and
Saos-2 (ARF wild type, pRb-null and p53-null) cells were grown in
Dulbecco's modified Eagle's medium (Trace Scientific, Sydney, Australia) supplemented with 10% fetal bovine serum and
glutamine. All cells were cultured in a 37 °C incubator with 5%
CO2.
For immunofluorescence studies, cultured cells (1 × 105) were seeded on coverslips in six-well plates and
transfected with 1-3 µg of purified plasmid using LipofectAMINE 2000 (Invitrogen). For cell cycle distribution analysis, cultured cells
(2.5-3 × 105) were seeded in T25 flasks and
co-transfected with the expression plasmids (3-3.5 µg) and
pCMVEGFP-spectrin (1 µg).
Colony Formation Assays
U2OS cells (1 × 105) seeded in triplicate in
six-well plates were transfected with a total of 1.6 µg of DNA:500 ng
of each expression plasmid and 100 ng of pEGFP-N1
(Clontech) as a measure of transfection efficiency.
Cells were harvested 24 h post-transfection, and the percentage of
GFP-positive cells (less than 10% variation among all transfections)
was determined using FACS analysis. The remaining cells were expanded
to a 10-cm2 plate and maintained in medium
containing 300 µg of hygromycin/ml for 15 days, with
antibiotic-containing-media refreshed every 4 days. Colonies were
stained with 0.1% crystal violet in phosphate-buffered saline,
and colonies equal to or larger than 2 mm were scored visually.
Yeast Two-hybrid System
Screening--
Full-length p14ARF cDNA was fused
in-frame with the yeast LexA DNA-binding domain (LexA-DBD) in the
displayBAIT vector (Display Systems Biotech) to create the
hybrid bait protein. A randomly primed human fetal brain cDNA
library constructed in the displayTARGET vector was obtained
from Display Systems Biotech. The displayTARGET vector
encodes proteins fused to the B42-activation domain (B42-AD) and the
hemagglutinin (HA) epitope. BAIT and TARGET
plasmids were co-transformed into Yeast-H yeast cells (Display
Biosystems Biotech) as detailed in the Matchmaker LexA two-hybrid
protocol (Clontech). Colonies with plasmid DNA
encoding target proteins, which interact with the bait protein,
were identified by transcription of the LEU2 reporter gene
integrated into the yeast genome and the green fluorescence protein
encoded by the co-transformed displayREPORTER vector.
Plasmid DNA was rescued from positive colonies, introduced into
Escherichia coli KC8 (Clontech) and
partially sequenced to characterize the target DNA.
Indirect Immunofluorescence--
Approximately 40 h after
transfection, cells were washed in phosphate-buffered saline and fixed
in 3.7% formaldehyde. Cells were immunostained for 50 min with either
monoclonal mouse anti-FLAG M2 antibody (Sigma) or rabbit anti-E4F-Nterm
(40) followed by a 50-min exposure to either anti-fluorescein
isothiocyanate-conjugated anti-mouse secondary IgG (Roche Molecular
Biochemicals, Molecular Probes) or a Texas Red-conjugated anti-rabbit
secondary IgG (Jackson ImmunoResearch). Subcellular distribution of E4F
was determined from a total of at least 250 fluorescent cells from two
independent transfection experiments unless otherwise indicated. The
standard deviation obtained within transfection experiments was always less than ±5%.
Cell Cycle Analysis of Transfected U2OS Cells--
Forty hours
post-transfection, cells were fixed in 70% ethanol at 4 °C for at
least 1 h, washed in phosphate-buffered saline, and stained with
propidium iodide (50 ng/µl) containing ribonuclease A (50 ng/µl).
EGFP-spectrin was used as a marker for analysis of transfected cells.
DNA content from at least 2000 EGFP-spectrin-positive cells was
analyzed using ModFIT software. The percentage of S-phase inhibition
was calculated as described previously (46).
Immunoprecipitations and Western Blotting--
Expression of
endogenous and ectopically expressed proteins was determined 24-40 h
after transfecting U2OS cells with the indicated expression plasmids.
Proteins were extracted for 20 min at 4 °C using RIPA lysis buffer
(1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS in
phosphate-buffered saline) containing protease inhibitors (Roche
Molecular Biochemicals). The protein concentration was determined with
the Dc protein assay kit (Bio-Rad). Proteins were resolved on 12% SDS
polyacrylamide gels and transferred to Immobilon-P membranes
(Millipore). Western blots were probed with antibodies against p53
(D0-1, Santa Cruz Biotechnology), p21Waf1 (C-19, Santa
Cruz), cyclin A (C-19, Santa Cruz), CDK4 (DCS-35, NeoMarkers), FLAG
(M2, Sigma), and E4F-Nterm (40).
For immunoprecipitation analysis, 1 × 106 cells were
transfected with 4-6 µg of expression plasmid and 500 ng of
pEGFP-N1 (Clontech) as a measure of
transfection efficiency. Cells were harvested 24 h
post-transfection and the percentage of GFP-positive cells (~40% of
the total cell population) was determined using FACS analysis. Cells
were lysed in RIPA lysis buffer containing protease inhibitors (Roche
Molecular Biochemicals) for 20 min at 4 °C and precleared using 1 µg of normal IgG for 1 h. Immunoprecipitation was performed
overnight at 4 °C with polyclonal E4F antibody and 50 µl protein
A-agarose in a total volume of 1.5 ml. Immunoprecipitates were then
washed four times with RIPA lysis buffer containing protease
inhibitors, resolved using SDS-PAGE, and detected by immunoblot
analysis. For immunoprecipitations using M450-tosylactivated DYNALbeads
(DYNAL), 2 µg of antibody was adsorbed chemically as described by the
manufacturer. Cell lysates were not precleared, and
immunoprecipitations were performed at 4 °C for 90 min.
Immunoprecipitation washes and protein detection were carried out as
described above.
Yeast proteins were extracted as described in the manufacturer's yeast
protocols handbook (Clontech). Electrophoresis was performed on SDS polyacrylamide gels, and proteins were transferred to
Immobilon-P membranes (Bio-Rad). Western immunoblotting of proteins
tagged with the LexA DNA-binding domain or the HA epitope was carried
out using the monoclonal mouse anti-LexA antibody (2-12, Santa Cruz)
and monoclonal anti-HA antibody (F-7, Santa Cruz), respectively.
Isolation of E4F as a Candidate p14ARF-interacting
Protein--
The yeast two-hybrid assay was employed to identify
proteins capable of interacting with the p14ARF tumor
suppressor. Full-length p14ARF fused to the LexA
DNA-binding domain was used as bait to screen a human fetal brain
cDNA expression library fused to the B42 activation domain (Display
Systems Biotech). Sequence analysis of the 30 positive clones revealed
that a single clone corresponded to the carboxyl-terminal half (amino
acids 369-784) of the E1A-regulated transcription factor,
p120E4F. This region is specific to full-length
p120E4F and contains a central zinc finger domain with
residues that are involved in the contact of p120E4F with
p53 and pRb (46, 47).
p120E4F Interacts Physically with p14ARF in
Vivo--
To verify that E4F interacts with p14ARF
in vivo, the full-length p120E4F cDNA cloned
in pCMV5 (40) was overexpressed transiently in human Saos-2 cells
with either FLAG-tagged p14ARF or FLAG-tagged
p16INK4a. Approximately 24 h post-transfection, cell
lysates were immunoprecipitated with anti-E4F polyclonal antibody and
blotted with anti-FLAG monoclonal antibody. As shown in Fig.
1, p14ARF-FLAG and
p120E4F were efficiently co-precipitated, indicating that
these two proteins are capable of interacting in vivo.
p120E4F was also co-precipitated with
p14ARF-FLAG using anti-FLAG or anti-ARF monoclonal
antibodies (data not shown). The control immunoprecipitations performed
with p16INK4a-FLAG-transfected cells were negative.
Binding to p120E4F Requires the Amino-terminal Half of
p14ARF--
p14ARF interacts with Hdm2,
topoisomerase I, and the E2F transcription factors via its
amino-terminal half (amino acids 1-64). This region also contains an
arginine-rich nucleolar localization sequence (NoLS), which forms part
of the Hdm2-binding domain (Fig. 2) (24,
49, 51). Not surprisingly these amino-terminal 64 amino acids are
necessary for the growth inhibitory activity of p14ARF. The
carboxyl terminus of p14ARF (amino acids 65-132) is not
well conserved but is required for full p14ARF activity;
this region also contains an arginine-rich nucleolar localization
sequence, a fragment that was shown to interact weakly with Hdm2
using surface plasmon resonance (52).
To define the region of p14ARF that associates with
p120E4F, we generated deletion constructs of
p14ARF fused to the LexA DNA-binding domain and
quantitatively assayed their ability to interact in yeast with
full-length p120E4F fused to the B42 activation domain. The
amino-terminal half of p14ARF was found to interact with
p120E4F, whereas no E4F binding was detected with the
carboxyl-terminal p14ARF fragment (Fig. 2). Partial
deletion of the amino-terminal Hdm2 binding site
(p14ARF-(14-132)) disrupted Hdm2 binding but did
not affect its interaction with p120E4F (Fig. 2).
The central p120E4F Zinc Finger Domain Is Required for
p14ARF Binding--
p120E4F contains six
C2H2 zinc finger motifs clustered in two
separate regions. Two motifs occur within the amino-terminal domain and
are present in the proteolytically derived p50E4F, whereas
the remaining four are grouped within a central region (amino acids
493-569) found only in the full-length protein. The interaction of
p120E4F with p53 requires residues within the last two of
these central zinc finger motifs (amino acids 521-580) and is somewhat
impaired by deletion of the last motif (carboxyl-terminal to amino acid 551) (46). The interaction of p120E4F with pRb occurs
through two independent regions; one is in the amino-terminal half that
is common to both p120E4F and p50E4F, and the
other involves residues in the carboxyl-terminal half (amino acids
486-551) that are distinct from the p53-binding region (46,
47).2
To further delineate the region within the C-terminal half of
p120E4F that associates with p14ARF, we
engineered two E4F deletion constructs from the C terminus of
p120E4F. In a yeast two-hybrid assay, the E4F fragment,
containing three of the central zinc finger domains (E4F-(369-566)),
interacted strongly with full-length p14ARF (Fig.
3) and even more potently with the
p14ARF-(14-132) fragment (data not shown). The C-terminal
p120E4F amino acids (amino acids 567-784) did not interact
directly with p14ARF (Fig. 3).
p120E4F Interacts with p14ARF and p53 to
Form a Ternary Complex--
p14ARF does not interact
directly with p53 in vivo, but it can associate with p53 via
Hdm2 (53). However, because the p120E4F-binding domains for
p14ARF and p53 may be distinct, it is possible that
p14ARF can also associate with p53 via p120E4F.
To investigate the formation of
p14ARF-p120E4F-p53 complexes, full-length p53
and p14ARF-FLAG were transiently overexpressed with or
without p120E4F in the U2OS cell line. At 24 h
post-transfection, cell lysates were immunoprecipitated with a
polyclonal p53 antibody and blotted with anti-FLAG, anti-p53 monoclonal
antibodies, and the polyclonal anti-E4F antibody. As expected, in the
absence of ectopic p120E4F, p53 did not
co-immunoprecipitate with p14ARF-FLAG (Fig.
4). In contrast, when p120E4F
was expressed with p53 and p14ARF, p53 was capable of
complexing with p14ARF in vivo (Fig. 4).
p14ARF Relocalizes p120E4F to the
Nucleolus--
p14ARF is predominantly a nucleolar protein
that can sequester its binding partners, in particular Hdm2 and E2F1,
to this region. The effect of transiently expressed p14ARF
on the subcellular distribution of p120E4F and an E4F
truncation mutant containing amino acids 1-551 (p75E4F)
was analyzed in U2OS cells. These cells accumulate functional p53 and
pRb but lack endogenous p14ARF. U2OS cells transiently
overexpressing E4F, with or without ectopic full-length FLAG-tagged
p14ARF expression, were stained with antibodies against E4F
and the FLAG epitope. When ectopically expressed, both
p120E4F and p75E4F were distributed throughout
the nucleus with some cytoplasmic staining in the majority of
transfected U2OS cells and were always excluded from the nucleoli (Fig.
5). Likewise, when co-expressed with
p14ARF, p75E4F remained nucleoplasmic and was
not detected in nucleoli. In contrast, there was a clear and
quantitative relocalization of full-length p120E4F to
nucleoli when it was co-expressed with p14ARF-FLAG (Fig.
5). Not surprisingly, the nucleolar sequestration of
p120E4F was associated with the localization of
p120E4F exclusively to the nucleus. Over-expressed
p120E4F displayed heterogeneous subcellular distribution in
U2OS cells; 62% of transfected cells accumulated predominantly nuclear
E4F, whereas 31% of cells showed nuclear and cytoplasmic
p120E4F distribution. When p120E4F was
co-expressed with p14ARF-FLAG, the proportion of U2OS cells
accumulating only nuclear p120E4F increased to 93%.
Over-expression of p120E4F did not alter the subcellular
distribution of the predominantly nucleolar p14ARF in
transiently transfected U2OS cells (data not shown).
Co-expression of p120E4F and p14ARF Induces
Enhanced Cell Cycle Arrest--
The expression of either
p120E4F or p14ARF can suppress cell
proliferation at the G1-S and G2-M-phase
transitions; the growth arrest induced by either protein is enhanced in
the presence of functional pRb and p53 pathways (20, 29, 46, 47, 54).
To evaluate whether the interaction of p14ARF and
p120E4F also influences growth arrest, U2OS cells, which
retain wild type p53, Hdm2 and pRb, were transiently transfected with
p14ARF and/or p120E4F. At 40 h
post-transfection, p120E4F over-expression had a minor
effect on the cell cycle distribution of U2OS cells, inhibiting S-phase
by ~10% while increasing the percentage of cells in G1
and G2-M each by only 4-5% (Fig.
6). p14ARF expression had a
more potent effect, inhibiting S-phase by 40%, with a
concomitant increase in the percentage of G2-M cells but no
increase in G1 cells (Fig. 6). The simultaneous
co-expression of p14ARF and p120E4F caused
S-phase inhibition to a consistently greater extent than the expression
of p14ARF alone, inhibiting S-phase by more than 65% while
increasing the percentage of G2-M cells and to a lesser
extent G1 cells. As expected, p14ARF-induced
growth arrest was associated with increased p53 protein levels in the
U2OS cell line, but the enhanced growth arrest induced when
p14ARF was co-expressed with p120E4F was not
associated with further increases in p53 or with any detectable changes
in cyclin A protein levels (Fig. 7).
The ability of p14ARF and p120E4F to induce
growth arrest in a cooperative manner was further analyzed by a colony
formation assay. U2OS cells were transfected with
p14ARF-FLAG or pCMVs-E4F2.5K alone,
with the combination of p14ARF-FLAG and
pCMVs-E4F2.5K, or with pCMV-FLAG5b vector (as a
control). After 2 weeks of selection in hygromycin-containing medium,
the number of drug-resistant colonies obtained was determined relative to the vector control. As expected, p14ARF-FLAG (14 ± 3% of the vector control) and p120E4F (32 ± 7% of
the vector control) individually inhibited U2OS colony formation (Fig.
6). But again, the co-expression of p14ARF-FLAG and
p120E4F in the U2OS cell line produced a stronger
inhibitory effect, reducing the number of colonies to 6 ± 3%
relative to the vector control (Fig. 6).
p53 Is Required for Cell Cycle Arrest Induced by p14ARF
and p120E4F Co-expression--
The
growth-suppressive function of ARF is enhanced in the presence of
p120E4F and p53, and p120E4F can interact with
p53 to promote cell cycle inhibition. We therefore investigated the
influence of p53 on the cell cycle inhibition induced by the
co-expression of p14ARF and p120E4F. In initial
experiments, p14ARF-FLAG and
pCMVs-E4F2.5K constructs were transiently expressed in the
p53-null, pRb+ WMM1175 melanoma cell line. Enforced expression of
p14ARF alone in the WMM1175 cell line induced a
dose-dependent reduction in S-phase at 40 h
post-transfection (data not shown). In contrast, at this time point,
overexpression of p120E4F did not alter the cell cycle
distribution of WMM1175 cells. Likewise, co-expression of
p120E4F and p14ARF did not significantly
enhance the ability of p14ARF to inhibit S-phase entry
(data not shown).
To determine the requirement of functional p53 for
p14ARF/p120E4F-induced cell cycle arrest, we
co-expressed a dominant-negative mutant form of p53
(p5322,23,281) in U2OS cells. This p53 mutant lacks
a functional transcriptional activation domain and is catalytically
inactive but can oligomerize with and inactivate wild type p53 (50). As
expected, p14ARF-induced cell cycle arrest in U2OS cells
was severely diminished when p14ARF was co-expressed with
p5322,23,281. The p53 mutant also abolished the cooperative
cell cycle inhibitory effect of p14ARF and
p120E4F in U2OS cells. When p5322,23,281 was
transiently co-expressed with p14ARF and
p120E4F in U2OS cells, there was no detectable S-phase
inhibition 40 h post-transfection (Fig.
8).
The p14ARF tumor suppressor is frequently altered in a
wide variety of human tumors and is implicated in the genetic
predisposition to melanoma. This protein is activated in response to
hyperproliferative signals emanating from oncoproteins and inducers of
S-phase entry, such as Myc, E1A, Ras, and E2F1. Once activated,
p14ARF induces G1- and G2-phase
growth arrest or sensitizes cells to apoptosis. The activity of
p14ARF involves an elaborate series of protein
interactions, including binding to the p53-antagonist, Hdm2. In
p53-positive cells, it is proposed that ARF sequesters Hdm2 in
nucleoli, thereby stabilizing p53 and activating
p53-dependent G1- and G2-phase cell
cycle arrest (reviewed in Ref. 55). In cells lacking p53, ARF
suppresses growth through various protein interactions, e.g.
ARF associates with and destabilizes the activating E2F transcription
factors (32, 33). We now report that p14ARF can also
modulate cell cycle progression by interacting with the E1A-regulated
transcription factor, p120E4F.
In searching for p14ARF-interacting molecules, we isolated
a carboxyl-terminal fragment specific to full-length
p120E4F and localized the p14ARF-binding site
in p120E4F to amino acids 369-566, a region that contains
both p53- and pRb-binding domains (46, 47). Although the E4F binding
sites for p53 and p14ARF are in close proximity, p53 and
p14ARF can simultaneously bind to p120E4F (Fig.
4). Thus p120E4F, like Hdm2, can interact with
p14ARF and p53 in binary (as shown in the p53-null Saos 2 cells; Fig. 1) or ternary complexes (Fig. 4). Not surprisingly,
p14ARF interacts with p120E4F via its
amino-terminal domain, a highly basic region that is involved in the
p14ARF interactions with Hdm2, E2F-1, and topoisomerase I. Although it is likely that some of these proteins compete for ARF, it
appears that the amino-terminal 13 amino acids of p14ARF,
which are critical for Hdm2 binding, are less important for E2F and
p120E4F interactions (Fig. 2) (24, 49, 51, 52, 56-59).
To establish the biological consequence of the
p14ARF-p120E4F interaction, we analyzed the
effect of co-expressing these proteins in the U2OS osteosarcoma cell
line. The accumulation of exogenous p14ARF sequestered
exogenous p120E4F into the nucleoli (Fig. 5). The
significance of nucleolar import remains to be defined; Mdm2 nucleolar
targeting is not required for ARF function in response to E2F or Ras
(56, 60) but Mdm2 did accumulate with ARF in nucleoli in MEFs
undergoing cellular senescence (22). It has been suggested that protein
expression levels are the key determinant of nucleolar sequestration by
ARF (61), and consistent with this observation, we found that exogenous ARF did not import endogenous Hdm2 or endogenous p120E4F
into U2OS nucleoli (data not shown). Although the significance of
ARF-driven nucleolar sequestration remains unresolved, it is a useful
indicator to assess ARF-p120E4F binding, which was also
confirmed in co-immunoprecipitation assays (Fig. 1) and as such helped
to further delineate residues required for p14ARF
interaction to amino acids 551-566 in p120E4F.
At 40 h post-transfection, ectopic expression of
p14ARF in U2OS cells induced a minor but consistent
decrease in S-phase cells, with a concomitant increase in the
proportion of cells in the G2-phase. Over-expression of
p120E4F had a lesser effect on the cell cycle distribution
of these cells, but because we do not know the nature of the genes
regulated by p120E4F, a greater effect might have required
a longer period of expression. Nevertheless, at this time point, U2OS
cells over-expressing both p120E4F and p14ARF
showed a much greater reduction in the percentage of S-phase cells,
with a consistent increase in the proportion of cells in G2-phase. Increases in G1-phase cells were
observed occasionally (Fig. 6). S-phase reduction was also observed by
others in Balb/c and NIH3T3 cells over-expressing p120E4F
and either p53 or pRb, respectively (46, 47), although those reports
attribute this reduction solely to G1-phase arrest.
However, it is clear that p120E4F also effects
G2-phase progression (48), although the mechanisms underlying this effect, as well as p14ARF-induced
G2 arrest, are not yet well defined. With
p120E4F, the most relevant changes in cell cycle regulatory
proteins would seem to be the post-transcriptional elevation of cyclin B1, p21Waf1, and p27Kip1 protein levels, the
reduced levels of cdk2 and cdc2 kinase activities, and the reduced
expression of cyclin A2 (42, 43, 48). With p14ARF, both
G1 and G2 arrest have been associated with
elevated p53 and p21Waf1 levels (20). Not surprisingly,
then, U2OS cells over-expressing p14ARF had increased
levels of p53 and p21Waf1 (Fig. 6). There was, however, no
further increase in the amount of p53 or p21Waf1 in U2OS
cells co-expressing p14ARF and p120E4F (Fig.
7), and thus the cooperative effect of p120E4F must be due
to the down-regulation of other genes involved in cell cycle
progression. To date, only cyclin A has been identified as target for
p120E4F, but we observed no significant change in the
levels of cyclin A in U2OS arrested cells (Fig. 7). Therefore,
clarification of this mechanism will require identification of other
p120E4F cellular target genes.
Defining the role of p53 in the tumor suppressor functions of
p14ARF is critical because most human cancers do not
accumulate functional p53. As such, we investigated whether
p120E4F could enhance the growth -suppressive function of
p14ARF in a p53-null background. Although ectopic
p14ARF expression induced a dose-dependent
S-phase reduction in the p53-null WMM1175 melanoma cell line, this was
not enhanced when p120E4F was co-expressed. Further,
expression of the dominant-negative mutant p5322,23,281
abolished the cooperative growth arrest induced by
p14ARF-p120E4F in the U2OS cell line (Fig. 8).
This data should be interpreted with caution, however, because the
WMM1175 cell line also carries alterations affecting other cell cycle
regulatory genes, which may be required for ARF function (62), and this
catalytically inactive p53 mutant may diminish the function of the p53
homologues, p63 and p73. The influence of p63 and p73 on ARF function
has not been thoroughly investigated. We attempted to clarify the influence of p53 on p120E4F transcriptional activity in the
presence and absence of ARF using transcriptional assays with an
E4F-responsive cyclin A promoter construct. Unfortunately, because of
p14ARF-induced cell cycle inhibition, the results generated
from these experiments were ambiguous.
The interaction of p120E4F with p14ARF, p53,
and pRb appears to create a complex but effective network for growth
control that is disrupted during human tumorigenesis and by viral
infection. In vivo, the hypophosphorylated form of pRb
complexes with p120E4F in growth-arrested cells (47). In
this active state, pRb also associates with and suppresses the activity
of the E2F transcription factors. Progression into S-phase is
accelerated when the ability to suppress E2F and to function with
p120E4F is disrupted through pRb hyperphosphorylation. The
activation of E2F in the presence of oncogenic stimuli induces
p14ARF (63), which interacts with Hdm2 and
p120E4F, to promote the assembly of
p14ARF-Hdm2-p53 (53) and
p14ARF-p120E4F-p53 complexes (Fig.
9). The p14ARF-Hdm2-p53
complex has been detected in vivo and may actively stimulate the transcription of genes involved in G1 and
G2 cell cycle arrest or apoptosis. In contrast, we propose
that the E4F complex may function to suppress the expression of genes
required to stimulate G2-phase progression (Fig. 9). The
formation and transcriptional activity of these multimeric complexes is
being investigated, but regardless of the protein components, their
assembly would be effectively circumvented during tumorigenesis. Most
human tumors carry alterations that inactivate the pRb,
p14ARF, and p53 pathways, and we have shown that the
frequent loss of p14ARF would also diminish the activity of
p120E4F.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(hypoxia-inducible transcription factor 1
) (37),
the cytoplasmic peroxisomal protein, pex19p (38), and CARF (novel
collaborator of ARF), a novel
serine-rich protein (39). Although the extent to which these ARF
complexes modulate the cell cycle inhibitory action of ARF is not well
established, spinophilin and CARF appear to cooperate with ARF in
promoting growth arrest, whereas pex19p retains p19ARF in
the cytoplasm and down-regulates the p19ARF-p53 pathway
(38, 39).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Galactosidase Assay--
Yeast cells were transformed
sequentially with a LexA-DBD-p14ARF construct, a
B42-AD-p120E4F construct, and the
-galactosidase reporter plasmid, pSH18-34 (Clontech). Transformants were grown on
dextrose
ura
his
trp
plates, and five colonies for each
transformant type were picked and grown overnight in dextrose
ura
his
trp
liquid medium.
-Galactosidase activity was determined
using the Pierce yeast
-galactosidase kit.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (15K):
[in a new window]
Fig. 1.
p14ARF associates with
p120E4F in mammalian cells. Saos-2 cells
were transfected with 3 µg of p14ARF-FLAG or
p16INK4a-FLAG and 3 µg of
pCMVs-E4F2.5K. Approximately 24 h post-transfection,
cell extracts were prepared and immunoprecipitated using protein
A-agarose with a polyclonal antibody to E4F. Western analysis of the
immunoprecipitates was performed with FLAG-specific M2 antibody (Sigma)
to detect FLAG-tagged proteins. IP, immunoprecipitation;
WB, Western blot.
View larger version (12K):
[in a new window]
Fig. 2.
p14ARF amino acids 1-64 are
sufficient for binding p120E4F. A, a yeast
two-hybrid binding assay was performed using truncations of
p14ARF to identify the region required for binding of
p120E4F, and this was compared with p14ARF-Hdm2
binding affinity. Yeast cells were transformed with the indicated
truncated p14ARF constructs cloned in-frame with the LexA
DNA-binding domain. Nucleolar localization sequences (NoLS)
and Hdm2-binding domains are highlighted in the
p14ARF construct diagrams. Proteins encoded by these
expression plasmids were tested for binding to either full-length
p120E4F or Hdm2 cloned in-frame with the B42 activation
domain. -Galactosidase activity was determined from five independent
yeast colonies, and the binding affinities were expressed relative to
full-length p14ARF. B, analysis of
p14ARF-expression levels in yeast by Western
immunostaining. Total yeast proteins were separated using SDS-PAGE and
probed with an anti-LexA (2-12, Santa Cruz Biotechnology) monoclonal
antibody. The p14ARF-(65-132)-LexA-DBD protein expressed
at high levels and was loaded at half the concentration of the other
yeast protein extracts.
View larger version (13K):
[in a new window]
Fig. 3.
p120E4F central zinc
finger motifs are required for binding p14ARF.
A, yeast two-hybrid binding assay was performed using
truncated mutants of p120E4F to identify the region
required for binding p14ARF. Yeast cells were transformed
with the indicated truncated p120E4F constructs cloned
in-frame with the B42-DNA activation domain. Zinc finger motifs and
p53-binding domain are highlighted in the E4F construct
diagrams. Proteins encoded by these expression plasmids were tested for
binding to full-length p14ARF fused to LexA DNA-binding
domain. Binding affinity was determined from five independent yeast
colonies. B, analysis of E4F expression levels in yeast by
Western immunostaining. Total yeast proteins were separated using
SDS-PAGE and probed with an anti-HA (F-7, Santa Cruz) monoclonal
antibody. The amounts of yeast protein extracts separated using
SDS-PAGE were not equivalent, as the expression of the
p120E4F-B42 fusion proteins varied significantly. As a
result, the binding properties were not quantitatively expressed.
View larger version (25K):
[in a new window]
Fig. 4.
p120E4F contacts
p14ARF and p53 to form a ternary complex. U2OS cells
were transfected with 2 µg of p14ARF-FLAG, 2 µg of pRc/CMV hp53, and either 2 µg of
pCMVs-E4F2.5K or 2 µg of pCMV-FLAG5b vector.
Approximately 24 h post-transfection, cell extracts were prepared
and immunoprecipitated using DYNAL beads coated with a polyclonal
antibody to p53 (FL393, Santa Cruz). Western analysis of the
immunoprecipitates was performed with FLAG-specific M2 antibody
(Sigma), monoclonal p53 antibody (DO-1, Santa Cruz), and a polyclonal
antibody against E4F. IP, immunoprecipitation.
View larger version (21K):
[in a new window]
Fig. 5.
p14ARF induces the nucleolar
sequestration of p120E4F. A, U2OS
cells were transiently transfected with either p120E4F or
p120E4F and p14ARF-FLAG. At ~40 h
post-transfection cells were fixed, permeabilized, and immunostained
sequentially with a polyclonal anti-E4F antibody and a monoclonal mouse
anti-FLAG M2 antibody (Sigma) followed by a 50-min exposure to a
Texas Red (TXR)-conjugated anti-rabbit IgG antibody
and a fluorescein isothiocyanate (FITC)-conjugated
anti-mouse IgG antibody. LM, light microscopy. B,
U2OS cells were transiently transfected to express (i)
p120E4F and pCMV-FLAG5b, (ii) p120E4F and
p14ARF-FLAG, (iii) p75E4F and pCMV-FLAG5b, or
(iv) p75E4F and p14ARF-FLAG. Approximately
40 h post-transfection, cells were immunostained as described
above, and the number (n) of transfected cells accumulating
nucleolar E4F was determined.
View larger version (14K):
[in a new window]
Fig. 6.
p14ARF and
p120E4F cooperate to inhibit S-phase entry.
A. U2OS osteosarcoma cells (2.5 × 105) were
transfected with the p14ARF-FLAG plasmid (1.5 µg), the indicated E4F plasmids (1.5 µg), and
pCMVEGFP-spectrin (1 µg). The cell cycle distribution of
green fluorescent cells was determined at 40 h post-transfection
using propidium iodide staining. B, the percentage of
S-phase inhibition was calculated from at least four independent
transfection experiments using the following formula: ((percentage of
cells in S-phase in the vector transfected cells percentage of
cells in S-phase in cells transfected with ARF/E4F expression
plasmids)/(percentage of cells in S-phase in the vector-transfected
cells)) × 100. C, colony formation assay in human U2OS
cells transfected with the indicated plasmid DNA and grown in the
presence of 300 µg/ml hygromycin for 2 weeks. After drug selection
the surviving colonies were stained with crystal violet, and colonies
of 2 mm or more in size were scored. The percentage of colonies
represents the result of three experiments calculated as
compared with the number of colonies obtained by transfection with
empty vector (pCMV-FLAG5b, 100%).
View larger version (38K):
[in a new window]
Fig. 7.
Effect of p14ARF and/or
p120E4F expression on p53,
p21Waf1, and cyclin A accumulation. U2OS
osteosarcoma cells (2.5 × 105) were transfected with
the p14ARF-FLAG plasmid (1.5 µg), the
indicated E4F plasmid (1.5 µg), and pEGFP-N1 (1 µg). At
40 h post-transfection, ~1 × 105 cells
expressing GFP were collected using the FACS Vantage cell sorter (BD
Biosciences). The sorted populations were tested using flow cytometry
and contained at least 95% GFP-positive cells. The sorted cells were
lysed, and equivalent amounts of lysates were separated using SDS-PAGE.
E4F, cyclin A, p53, CDK4, p21Waf1, and
p14ARF-FLAG levels were determined by Western blotting with
polyclonal E4F, C-19, DO-1, DCS-35, C-19, and M2, respectively.
View larger version (21K):
[in a new window]
Fig. 8.
p14ARF and
p120E4F require functional p53 to inhibit
S-phase entry. U2OS osteosarcoma cells (2.5 × 105) were transfected with the
p14ARF-FLAG plasmid (1.5 µg), the indicated
E4F plasmid (1.5 µg), and either 500 ng of pCMV-FLAG5b or
500 ng of pCMV-p5322,23,281 and
pCMVEGFP-spectrin (1 µg). The cell cycle distribution of
green fluorescent cells was determined at 40 h post-transfection
using propidium iodide staining. The DNA distribution of
pCMV-FLAG5b-transfected U2OS cells was not altered by the
co-expression of the p5322,23,281 mutant (data not
shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (15K):
[in a new window]
Fig. 9.
p53-dependent cell cycle control
by p14ARF. We propose that the activation of ARF by
hyperproliferative stimuli promotes the assembly of multimeric
transcriptional regulatory complexes. In particular, the formation of a
ternary complex involving p14ARF-Hdm2-p53 acts as a
transcriptional activator to stimulate the expression of genes involved
in growth suppression or cell death. In contrast, the assembly of
p14ARF-p120E4F-p53 acts as a potent
transcriptional repressor inhibiting the transcription of genes
involved in G2-phase progression. The net effect of
ARF stimulation in cells expressing functional p53 is potent
G1 and G2 cell cycle arrest. DP,
DRTF (differentiation-regulated transcription factor)
protein.
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ACKNOWLEDGEMENTS |
---|
We thank Mary Sartor and Carol Devine for expert technical assistance, Dr. Tom Shenk for supplying the pCMVEGFP-spectrin plasmid, and Dr. Arnold Levine for supplying the pRc/CMV hp53 plasmid.
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FOOTNOTES |
---|
* This work was supported by the National Health and Medical Research Council of Australia, the New South Wales Cancer Council, University of Sydney, and the New South Wales Health Department.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 61-2-9845-9509; Fax: 61-2-9845-9102; E-mail: helen_rizos@wmi.usyd.edu.au.
Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.M210978200
2 G. Del Sal, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are: ARF, alternative reading frame; MEF, mouse embryonic fibroblast; CDK, cyclin-dependent kinase; GFP, green fluorescent protein; EGFP, enhanced GFP; FACS, fluorescence-activated cell sorter; HA, hemagglutinin; DBD, DNA-binding domain.
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---|
1. | Serrano, M., Hannon, G. J., and Beach, D. (1993) Nature 366, 704-707[CrossRef][Medline] [Order article via Infotrieve] |
2. | Duro, D., Bernard, O., Della Valle, V., Berger, R., and Larsen, C. J. (1995) Oncogene 11, 21-29[Medline] [Order article via Infotrieve] |
3. | Kamb, A., Shattuck-Eidens, D., Eeles, R., Liu, Q., Gruis, N. A., Ding, W., Hussey, C., Tran, T., Miki, Y., Weaver-Feldhaus, J., McClure, M., Aitken, J. F., Anderson, D. E., Bergman, W., Frants, R., Goldgar, D. E., Green, A., MacLennan, R., Martin, N. G., Meyer, L. J., Youl, P., Zone, J. J., Skolnick, M. H., and Cannon-Albright, L. A. (1994) Nat. Genet. 8, 23-26[Medline] [Order article via Infotrieve] |
4. | Hussussian, C. J., Struewing, J. P., Goldstein, A. M., Higgins, P. A. T., Ally, D. S., Sheahan, M. D., Clark, W. H., Jr., Tucker, M. A., and Dracopoli, N. C. (1994) Nat. Genet. 8, 15-21[Medline] [Order article via Infotrieve] |
5. | Holland, E. A., Schmid, H., Kefford, R. F., and Mann, G. J. (1999) Genes Chromosomes Cancer 25, 339-348[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Harland, M.,
Meloni, R.,
Gruis, N.,
Pinney, E.,
Brookes, S.,
Spurr, N. K.,
Frischauf, A.-M.,
Bataille, V.,
Peters, G.,
Cuzick, J.,
Selby, P.,
Bishop, D. T.,
and Bishop, J. N.
(1997)
Hum. Mol. Genet.
6,
2061-2067 |
7. |
Soufir, N.,
Avril, M.-F.,
Chompret, A.,
Demenais, F.,
Bombled, J.,
Spatz, A.,
Stoppa-Lyonnet, D.,
the French Familial Melanoma Group,
Bénard, J.,
and Bressac-de Paillerets, B.
(1998)
Hum. Mol. Genet.
7,
209-216 |
8. |
Rizos, H.,
Darmanian, A. P.,
Holland, E. A.,
Mann, G. J.,
and Kefford, R. F.
(2001)
J. Biol. Chem.
276,
41424-41434 |
9. | Hashemi, J., Bendahl, P. O., Sandberg, T., Platz, A., Linder, S., Stierner, U., Olsson, H., Ingvar, C., Hansson, J., and Borg, A. (2001) Genes Chromosomes Cancer 31, 107-116[CrossRef][Medline] [Order article via Infotrieve] |
10. | Rizos, H., Puig, S., Badenas, C., Malvehy, J., Darmanian, A. P., Jimenez, L., Mila, M., and Kefford, R. F. (2001) Oncogene 20, 5543-5547[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Randerson-Moor, J. A.,
Harland, M.,
Williams, S.,
Cuthbert-Heavens, D.,
Sheridan, E.,
Aveyard, J.,
Sibley, K.,
Whitaker, L.,
Knowles, M.,
Newton Bishop, J.,
and Bishop, D. T.
(2001)
Hum. Mol. Genet.
10,
55-62 |
12. |
Hewitt, C., Wu, C. L.,
Evan, G.,
Howell, A.,
Elles, R. G.,
Jordan, R.,
Sloan, P.,
Read, A. P.,
and Thakker, N.
(2002)
Hum. Mol. Genet.
11,
1273-1279 |
13. |
de Stanchina, E.,
McCurrach, M. E.,
Zindy, F.,
Shieh, S.-Y.,
Ferbeyre, G.,
Samuelson, A. V.,
Prives, C.,
Roussel, M. F.,
Sherr, C. J.,
and Lowe, S. W.
(1998)
Genes Dev.
12,
2434-2442 |
14. |
Zindy, F.,
Eischen, C. M.,
Randle, D. H.,
Kamijo, T.,
Cleveland, J. L.,
Sherr, C. J.,
and Roussel, M. F.
(1998)
Genes Dev.
12,
2424-2433 |
15. | Bates, S., Phillips, A. C., Clark, P. A., Stott, F., Peters, G., Ludwig, R. L., and Vousden, K. H. (1998) Nature 395, 124-125[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Radfar, A.,
Unnikrishnan, I.,
Lee, H. W.,
DePinho, R. A.,
and Rosenberg, N.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
13194-13199 |
17. | Zhang, Y., Xiong, Y., and Yarbrough, W. G. (1998) Cell 92, 725-734[Medline] [Order article via Infotrieve] |
18. | Pomerantz, J., Schreiber-Agus, N., Liégeois, N. J., Silverman, A., Alland, L., Chin, L., Potes, J., Chen, K., Orlow, I., Lee, H.-W., Cordon-Cardo, C., and DePinho, R. A. (1998) Cell 92, 713-723[Medline] [Order article via Infotrieve] |
19. |
Kamijo, T.,
Weber, J. D.,
Zambetti, G.,
Zindy, F.,
Roussel, M. F.,
and Sherr, C. J.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
8292-8297 |
20. |
Stott, F. J.,
Bates, S.,
James, M. C.,
McConnell, B. B.,
Starborg, M.,
Brookes, S.,
Palmero, I.,
Ryan, K.,
Hara, E.,
Vousden, K. H.,
and Peters, G.
(1998)
EMBO J.
17,
5001-5014 |
21. |
Tao, W.,
and Levine, A. J.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
3077-3080 |
22. | Weber, J. D., Taylor, L. J., Roussel, M. F., Sherr, C. J., and Bar-Sagi, D. (1999) Nat. Cell Biol. 1, 20-26[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Honda, R.,
and Yasuda, H.
(1999)
EMBO J.
18,
22-27 |
24. |
Weber, J. D.,
Kuo, M. L.,
Bothner, B.,
DiGiammarino, E. L.,
Kriwacki, R. W.,
Roussel, M. F.,
and Sherr, C. J.
(2000)
Mol. Cell. Biol.
20,
2517-2528 |
25. |
Sherr, C. J.
(1998)
Genes Dev.
12,
2984-2991 |
26. |
Khan, S. H.,
Moritsugu, J.,
and Wahl, G. M.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
3266-3271 |
27. |
Esteller, M.,
Tortola, S.,
Toyota, M.,
Capella, G.,
Peinado, M. A.,
Baylin, S. B.,
and Herman, J.
(2000)
Cancer Res.
60,
129-133 |
28. | Sanchez-Cespedes, M., Reed, A. L., Buta, M., Wu, L., Westra, W. H., Herman, J. G., Yang, S. C., Jen, J., and Sidransky, D. (1999) Oncogene 18, 5843-5849[CrossRef][Medline] [Order article via Infotrieve] |
29. | Carnero, A., Hudson, J. D., Price, C. M., and Beach, D. H. (2000) Nat. Cell Biol. 2, 148-155[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Weber, J. D.,
Jeffers, J. R.,
Rehg, J. E.,
Randle, D. H.,
Lozano, G.,
Roussel, M. F.,
Sherr, C. J.,
and Zambetti, G. P.
(2000)
Genes Dev.
14,
2358-2365 |
31. |
Yarbrough, W. G.,
Bessho, M.,
Zanation, A.,
Bisi, J. E.,
and Xiong, Y.
(2002)
Cancer Res.
62,
1171-1177 |
32. |
Martelli, F.,
Hamilton, T.,
Silver, D. P.,
Sharpless, N. E.,
Bardeesy, N.,
Rokas, M.,
DePinho, R. A.,
Livingston, D. M.,
and Grossman, S. R.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
4455-4460 |
33. | Eymin, B., Karayan, L., Seite, P., Brambilla, C., Brambilla, E., Larson, C. J., and Gazzeri, S. (2001) Oncogene 20, 1033-1041[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Vivo, M.,
Calogero, R. A.,
Sansone, F.,
Calabro, V.,
Parisi, T.,
Borrelli, L.,
Saviozzi, S.,
and La Mantia, G.
(2001)
J. Biol. Chem.
276,
14161-14169 |
35. | Karayan, L., Riou, J. F., Seite, P., Migeon, J., Cantereau, A., and Larson, C. J. (2001) Oncogene 20, 836-848[CrossRef][Medline] [Order article via Infotrieve] |
36. | Jackson, M. W., Lindström, M. S., and Berberich, S. J. (2001) Oncogene 276, 25336-25341 |
37. |
Fatyol, K.,
and Szalay, A. A.
(2001)
J. Biol. Chem.
276,
28421-28429 |
38. |
Sugihara, T.,
Kaul, S. C.,
Kato, J.-Y.,
Reddel, R. R.,
Nomura, H.,
and Wadhwa, R.
(2001)
J. Biol. Chem.
276,
18649-18652 |
39. |
Hasan, K.,
Yaguch, T.,
Sugihara, T.,
Kumar, P. K. R.,
Taira, K.,
Reddel, R.,
Kaul, S. C.,
and Wadhwa, R.
(2002)
J. Biol. Chem.
277,
37765-37770 |
40. | Fernandes, E. R., and Rooney, R. J. (1997) Mol. Cell. Biol. 17, 1890-1903[Abstract] |
41. |
Rooney, R. J.,
Rothammer, K.,
and Fernandes, E. R.
(1998)
Nucleic Acids Res.
26,
1681-1688 |
42. |
Fajas, L.,
Paul, C.,
Vie, A.,
Estrach, S.,
Medema, R.,
Blanchard, J. M.,
Sardet, C.,
and Vignais, M. L.
(2001)
Mol. Cell. Biol.
21,
2956-2966 |
43. |
Fernandes, E. R.,
Zhang, J. Y.,
and Rooney, R. J.
(1998)
Mol. Cell. Biol.
18,
459-467 |
44. | Fognani, C., Della Valle, G., and Babiss, L. E. (1993) EMBO J. 12, 4985-4992[Abstract] |
45. |
Fernandes, E. R.,
and Rooney, R. J.
(1999)
Mol. Cell. Biol.
19,
4739-4749 |
46. | Sandy, P., Gostissa, M., Fogal, V., Cecco, L. D., Szalay, K., Rooney, R. J., Schneider, C., and Del Sal, G. (2000) Oncogene 19, 188-199[CrossRef][Medline] [Order article via Infotrieve] |
47. |
Fajas, L.,
Paul, C.,
Zugasti, O., Le,
Cam, L.,
Polanowska, J.,
Fabbrizio, E.,
Medema, R.,
Vignais, M. L.,
and Sardet, C.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
7738-7743 |
48. |
Rooney, R. J.
(2001)
Cell Growth Differ.
12,
505-516 |
49. | Rizos, H., Darmanian, A. P., Mann, G. J., and Kefford, R. F. (2000) Oncogene 19, 2978-2985[CrossRef][Medline] [Order article via Infotrieve] |
50. | Lin, J., Teresky, A. K., and Levine, A. J. (1995) Oncogene 10, 2387-2390[Medline] [Order article via Infotrieve] |
51. | Lohrum, M. A., Ashcroft, M., Kubbutat, M. H., and Vousden, K. H. (2000) Curr. Biol. 10, 539-542[CrossRef][Medline] [Order article via Infotrieve] |
52. | Bothner, B., Lewis, W. S., DiGiammarino, E. L., Weber, J. D., Bothner, S. J., and Kriwacki, R. W. (2001) J. Mol. Biol. 314, 263-277[CrossRef][Medline] [Order article via Infotrieve] |
53. | Zhang, Y., and Xiong, Y. (1999) Mol. Cell 3, 579-591[Medline] [Order article via Infotrieve] |
54. | Quelle, D. E., Zindy, F., Ashmun, R. A., and Sherr, C. J. (1995) Cell 83, 993-1000[Medline] [Order article via Infotrieve] |
55. | Sherr, C. J. (2001) Nat. Rev. Mol. Cell. Biol. 2, 731-737[CrossRef][Medline] [Order article via Infotrieve] |
56. | Llanos, S., Clark, P. A., Rowe, J., and Peters, G. (2001) Nat. Cell Biol. 3, 445-452[CrossRef][Medline] [Order article via Infotrieve] |
57. | Midgley, C. A., Desterro, J. M. P., Saville, M. K., Howard, S., Sparks, A., Hay, R. T., and Lane, D. P. (2000) Oncogene 19, 2312-2323[CrossRef][Medline] [Order article via Infotrieve] |
58. | Clark, P. A., Llanos, S., and Peters, G. (2002) Oncogene 21, 4498-4507[CrossRef][Medline] [Order article via Infotrieve] |
59. | Mason, S. L., Loughran, O., and La Thangue, N. B. (2002) Oncogene 21, 4220-4230[CrossRef][Medline] [Order article via Infotrieve] |
60. |
Lin, A. W.,
and Lowe, S. W.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
5025-5030 |
61. |
Korgaonkar, C.,
Zhao, L.,
Modestou, M.,
and Quelle, D.
(2002)
Mol. Cell. Biol.
22,
196-206 |
62. | Rizos, H., Darmanian, A. P., Indsto, J. O., Shannon, J. A., Kefford, R. F., and Mann, G. J. (1999) Melanoma Res. 9, 10-19[Medline] [Order article via Infotrieve] |
63. | Parisi, T., Pollice, A., Cristofano, A. D., Calabró, V., and La Mantia, G. (2002) Biochem. Biophys. Res. Commun. 291, 1138-1145[CrossRef][Medline] [Order article via Infotrieve] |