Association of p14ARF with the p120E4F Transcriptional Repressor Enhances Cell Cycle Inhibition*

Helen RizosDagger §, Eve Diefenbach, Prerna BadhwarDagger , Sarah WoodruffDagger , Therese M. BeckerDagger , Robert J. Rooney||, and Richard F. KeffordDagger

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-1alpha (hypoxia-inducible transcription factor 1alpha ) (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).

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

beta -Galactosidase Assay-- Yeast cells were transformed sequentially with a LexA-DBD-p14ARF construct, a B42-AD-p120E4F construct, and the beta -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. beta -Galactosidase activity was determined using the Pierce yeast beta -galactosidase kit.

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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.

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).


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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. beta -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.

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).


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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.

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).


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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.

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).


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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.

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).


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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%).


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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.

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).


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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

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.


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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.


    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.

    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.

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
8. Rizos, H., Darmanian, A. P., Holland, E. A., Mann, G. J., and Kefford, R. F. (2001) J. Biol. Chem. 276, 41424-41434[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
21. Tao, W., and Levine, A. J. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 3077-3080[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
25. Sherr, C. J. (1998) Genes Dev. 12, 2984-2991[Free Full Text]
26. Khan, S. H., Moritsugu, J., and Wahl, G. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 3266-3271[Abstract/Free Full Text]
27. Esteller, M., Tortola, S., Toyota, M., Capella, G., Peinado, M. A., Baylin, S. B., and Herman, J. (2000) Cancer Res. 60, 129-133[Abstract/Free Full Text]
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[Abstract/Free Full Text]
31. Yarbrough, W. G., Bessho, M., Zanation, A., Bisi, J. E., and Xiong, Y. (2002) Cancer Res. 62, 1171-1177[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
38. Sugihara, T., Kaul, S. C., Kato, J.-Y., Reddel, R. R., Nomura, H., and Wadhwa, R. (2001) J. Biol. Chem. 276, 18649-18652[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
43. Fernandes, E. R., Zhang, J. Y., and Rooney, R. J. (1998) Mol. Cell. Biol. 18, 459-467[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
48. Rooney, R. J. (2001) Cell Growth Differ. 12, 505-516[Abstract/Free Full Text]
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[Abstract/Free Full Text]
61. Korgaonkar, C., Zhao, L., Modestou, M., and Quelle, D. (2002) Mol. Cell. Biol. 22, 196-206[Abstract/Free Full Text]
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]


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