The RING Domain of Mdm2 Can Inhibit Cell Proliferation1

[CANCER RESEARCH 62, 1222–1230, February 15, 2002] The RING Domain of Mdm2 Can Inhibit Cell Proliferation1

Jinjun Dang, Mei-Ling Kuo, Christine M. Eischen,2 Lilia Stepanova, Charles J. Sherr, and Martine F. Roussel3 Departments of Tumor Cell Biology [J. D., M-L. K., L. S., C. J. S., M. F. R.], Biochemistry [C. M. E.], and Howard Hughes Medical Institute [L. S., C. J. S.], St. Jude Children’s Research Hospital, Memphis, Tennessee 38105

ABSTRACT biquitin chains that are necessary for recognition by the proteasome (20). One possibility is that mono-ubiquitination of p53 is required to Mdm2 is a p53-inducible phosphoprotein that negatively regulates p53 expose a nuclear export signal, and that p53 polyubiquitination and by binding to it and promoting its ubiquitin-mediated degradation. Alter- degradation then proceed in the cytoplasm (21–23). Because Mdm2 is natively spliced variants of Mdm2 have been isolated from human and mouse tumors, but their roles in tumorigenesis, if any, remain elusive. We a direct transcriptional target of p53, its expression acts in a negative cloned six alternatively spliced variants of Mdm2 from E␮-Myc-induced feedback loop to terminate the p53 response (24). However, Mdm2 is itself subject to positive regulation through Ras signaling (25) and to mouse lymphomas, all of which lacked the NH2-terminal p53-binding domain but conserved the remainder of the Mdm2 protein. Enforced negative control by ATM-mediated phosphorylation (26) and through expression of full-length Mdm2 in primary mouse embryo fibroblasts or direct binding of the ARF tumor suppressor protein (27–29). Apart bone marrow-derived, interleukin 7-dependent pre-B cells accelerated from p53, ARF, and CBP/p300, Mdm2 has been found to directly their proliferation, whereas unexpectedly, overexpression of truncated associate with the retinoblastoma protein, E2F1, Numb, MTBP, p73, Mdm2 isoforms inhibited their growth. Truncated variants were active as and ribosomal protein L5 (30, 31). Therefore, it is unlikely that p53 is inhibitors whether they localized predominantly to the nucleus or cyto- the only physiological target of Mdm2. A homologue of Mdm2, plasm. Despite the absence of the p53-binding domain, growth inhibition Mdm4 (MdmX), although not a target of p53 transcriptional regula- remained strictly p53 dependent (but not p19Arf dependent) and could be overcome by full-length Mdm2. The intact RING finger domain at the tion, can also negatively regulate p53-mediated transcription (32–35). Mdm2 COOH terminus (amino acids 399–489) was necessary and suffi- The recent demonstration that loss of Mdm4 in the mouse germ-line, cient for growth inhibition by truncated Mdm2 proteins and could phys- like the disruption of Mdm2, results in embryonic lethality that is ically interact with either the RING finger domain or central acidic region rescued on a p53-null background has led to the conclusion that of full-length Mdm2. However, such interactions do not inhibit Mdm2 E3 Mdm2 and Mdm4 regulate p53 via different pathways (36). ubiquitin ligase activity in vitro using p53 as a substrate. Expression of The integrity of the COOH-terminal RING finger domain of Mdm2 growth-inhibitory Mdm2 isoforms in tumors remains an enigma. is necessary for both its E3 ubiquitin protein ligase activity (15, 37–39) and RNA-binding activity (40). Mutation of cysteine 464 to INTRODUCTION alanine disrupts the integrity of the RING finger and abolishes Hdm2- mediated p53 ubiquitination and nuclear export (14, 21, 22, 37). Mdm2 was discovered as an amplified gene on murine double- Mdm2 also mediates its own ubiquitination in a RING finger-depen- minute chromosomes in a spontaneously transformed 3T3 cell line dent manner (Refs. 16 and 37), and Lysine 444 might be important for (1). Subsequent analysis demonstrated that Mdm2 (Hdm2 in human) Mdm2 E3 ligase activity.5 is overexpressed in 5–10% of human tumors (2, 3), and that its A number of alternatively spliced variants of Mdm2 have been iden- expression is not only able to immortalize primary rodent embryonic tified and isolated from both human and rodent tumor cells (41–49). fibroblasts but also to transform them in cooperation with activated Expression of alternatively spliced Mdm2 transcripts correlates with Ras (4, 5). Embryos lacking Mdm2 die in utero, but lethality is high-grade malignancy in human ovarian tumors, bladder carcinomas, rescued in a p53-null genetic background, indicating that an essential astrocytic tumors, and breast cancer (42, 43, 49), irrespective of their p53 function of Mdm2 is to negatively regulate p53 activity (6, 7). On- status. Of the characterized Mdm2 variants, most sustained deletions of cogenic properties of Mdm2 are conferred, at least in part, by its the p53-binding domain. Some Mdm2 isoforms were reported to trans- ability to inactivate p53. Similar to inactivating p53 mutations, over- form NIH-3T3 cells (42). In E␮-Myc transgenic mice, many of the B-cell expression of Mdm2 also induces chromosome instability, inappro- lymphomas that arise sustain either p53 or Arf loss of function, with or priate centrosome duplication, and changes in ploidy (8). without overexpression of Mdm2 (50). Some lymphomas also expressed Mdm2 binding to the p53 NH terminus antagonizes p53 transcrip- 2 variant Mdm2 isoforms, which coexisted with full-length Mdm2, irre- tional activity (9–11), inhibiting p53 acetylation and transactivation spective of whether the tumors sustained Arf deletions or p53 mutations. by interfering with p300/CBP (12, 13). Mdm2 also functions as an E3 This prompted us to clone and characterize these variants and to examine ligase to ubiquitinate p53 (14–16) and to enforce its export from the their role in tumorigenesis. nucleus to the cytoplasm, where it is degraded in proteasomes (17– 19). It is unlikely that Mdm2 E3 ubiquitin ligase activity alone is MATERIALS AND METHODS sufficient to trigger p53 proteolysis, because Mdm2 mono-ubiquiti- nates p53 at multiple sites but does not catalyze addition of polyu- Isolation of Variant Mdm2 cDNAs. Full-length and alternatively spliced Mdm2 mRNAs were amplified by RT-PCR4 (ProSTAR First-Strand RT-PCR Received 9/5/01; accepted 12/14/01. kit; Stratagene), using 1 ␮g of total RNA isolated from E␮-Myc-induced The costs of publication of this article were defrayed in part by the payment of page lymphomas (Table 1; Ref. 50). Primers were chosen from the noncoding charges. This article must therefore be hereby marked advertisement in accordance with region of the published Mdm2 cDNA sequence [bp 169, sense (ϩ), 5Ј- 18 U.S.C. Section 1734 solely to indicate this fact. Ј 1 This work was supported in part by NIH Grants CA-71907 (to M. F. R.), Cancer CCATCGATCACCGCGCTTCTCCTGCGGCC-3 and bp 1702, antisense Center Core Grant CA-21765, and by the American Lebanese Syrian Associated Charities (Ϫ)5Ј-ATCGATATAAAATTCTATTTTTGTGAGCAGGTC-3Ј], including a of St. Jude Children’s Research Hospital. C. J. S. is an investigator of the Howard Hughes ClaI site (underlined; Ref. 4). The amplified cDNA fragments were cloned into Medical Institute. 2 Present address: Eppley Cancer Institute, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198-6805. 4 The abbreviations used are: RT-PCR, reverse transcription-PCR; HA, hemagglutinin; 3 To whom requests for reprints should be addressed, at Department of Tumor Cell WT, wild type; MEF, mouse embryo fibroblast; FBS, fetal bovine serum; IL, interleukin; Biology, DTRT 5006C, Mail Stop 350, St. Jude Children’s Research Hospital, 332 North NLS, nuclear localization sequence; NES, nuclear export sequence; IRES, internal ribo- Lauderdale, Memphis, TN 38105. Phone: (901) 495-3481/3597; Fax: (901) 495-2381; somal entry site; GFP, green fluorescent protein; Ubc, ubiquitin conjugating. E-mail: [email protected] 5 H. Yasuda, personal communication. 1222

Table 1 Expression of Mdm2 spliced variants, p53, and ARF in 55 mM ␤-mercaptoethanol, penicillin/streptomycin, in 10% CO humidified ␮ 2 E -Myc-induced tumors incubators, as described previously (50, 54). NIH3T3 cells stably expressing a Mdm2 zinc-inducible p19Arf protein (pMTCB6-HA-Arf) were generated by transfec- p53 ARF tion of a mammalian expression vector (pMTCB6) in which the HA-tagged Arf Tumor Full length Variant protein protein cDNA was cloned downstream of the sheep metallothionein promoter (MT1). CR71 UDa V6 WT UD ϩ Cells were maintained in DMEM containing 10% FBS, 2 mM glutamine, CR135 UD V2, V5 WT ␮ CR156 ϩ V2, V3, V4 WT ϩ penicillin/streptomycin, and 400 g/ml of G418. Production of high titer CR203 ϩ V2, V3, V5 Mutant ϩ ecotropic viruses in 293T cells and infections of MEFs (53) and pre-B cells CR246 UD V1 Mutant ϩ (50, 54) were carried out as described. Spodoptera frugiperda (Sf9) cells were ϩ ϩ CR325 V3 WT grown at 24°C in Grace’s medium (Life Technologies, Inc.) supplemented a UD, undetectable; ϩ, protein present. with 5% FBS and infected with baculoviruses as described previously (54). Protein Extraction, Immunoprecipitation, and Immunoblotting. MEFs were trypsinized, and after two washes with PBS, were lysed in ice-cold the pGEM-T Easy vector (Promega), and their nucleotide sequences were Tween 20 lysis buffer [50 mM HEPES (pH 7.5), 200 mM NaCl, 1 mM EDTA, determined. Mammalian expression vectors were constructed by subcloning 0.1% Tween 20, 1 mM phenylmethylsulfonyl fluoride, 0.4 unit of aprotinin/ml, the cDNAs into pSR␣MSV-tkneo (generously provided by Drs. Charles Saw- 1mM NaF, 10 mM ␤-glycerophosphate, and 0.1 mM sodium orthovanadate] yers and Owen Witte UCLA, CA; Ref. 51) or MSCV-IRES-GFP retroviral and left on ice for 1 h. After sonication at 4°C(7sϫ 2), cellular debris was vectors at an EcoRI site located immediately 3Ј to the long terminal repeat removed by centrifugation in a microcentrifuge at 14,000 rpm for 15 min at (52). 4°C. Proteins (200 ␮g/lane) were electrophoretically separated on denaturing Generation of Mdm2 Mutants and Epitope-tagged UbcH5. Full-length polyacrylamide gels containing SDS and transferred onto membranes (Osmon- Mdm2 in pGEM-T Easy or in the pSR␣MSV-tkneo vector was used to ics, Westborough, MA). Membranes were immunoblotted with affinity-puri- Arf generate deletion and point mutants by site-directed mutagenesis (Quick- fied rabbit polyclonal antibodies to mouse p19 (55), a monoclonal antibody Change, Site-Directed Mutagenesis kit; Stratagene). The sense primers includ- directed to mouse Mdm2 (2A10) generously provided by Dr. Arnold Levine ing EcoRI and BamHI sites (underlined) were used to construct Mdm2 deletion (Rockefeller University, New York, NY); a monoclonal antibody to the HA mutants M1 (amino acids 198–489), M2 (amino acids 299–489), M3 (amino epitope (generously provided by Dr. Albert Reynolds, Vanderbilt University, acids 399–489), and M4 (amino acids 399–463). Primers were as follows: Nashville, TN); a monoclonal antibody (9E10) recognizing the NH2-terminal M1, 198ϩ,5Ј-CGGAATTCGGATCCACCATGTGCAGCGGCGGCACGA- epitope of c-Myc (56); or with commercial antibodies directed to mouse p53 Cip1 GCAGC-3Ј; M2, 299ϩ,5Ј-CAGAATTCGGATCCACCATGGACTATTG- (Ab-7; Oncogene Research Products), mouse p21 (F-5; Santa Cruz Bio- GAAGTGTACCTCATGC-3Ј; M3, 399ϩ,5Ј-CGGAATTCGGATCCAC- technology, Santa Cruz, CA), Mdm2 (SMP14; Santa Cruz Biotechnology), or CATGTCCAGCAGCATTGTTTATAGCAGC-3Ј; the antisense primers were FLAG (anti-FLAG, M2; Sigma Chemical Co.). Sequential immunoprecipita- for M4, 463- 5Ј- CGGAATTCTATGCACACGTGAAACATGACATGAG-3Ј tion and immunoblotting were performed as described (57). (EcoRI site underlined), and for all constructs except M4, 1702 (Ϫ), 5Ј- Immunofluorescence. Procedures were described in detail previously (18). 4 ATCGATATAAAATTCTATTTTTGTGAGCAGGTC-3Ј (ClaI site under- Cells (3 ϫ 10 ) plated on coverslips were fixed and permeabilized in cold lined). Mutations were introduced into the RING domain, substituting arginine acetone:methanol (1:1, v/v) for 15 min at Ϫ20°C. Coverslips were air dried, for lysine 444 (M5) and alanine for cysteine 462 (M6), using the following blocked with 10% FBS in PBS, and stained with the 2A10 antibody to Mdm2, primers: M5, 5Ј-ATCTGCCAGGGGCGGCCTAGAAATGGCTGCATTG- followed by a goat antimouse antibody conjugated with fluorescein (Amer- TTCACG-3Ј (mutation underlined); M6, 5Ј-CACCTCATGTCATGTTTCAC- sham). Nuclei were visualized by 4Ј,6-diamidino-2-phenylindole staining. GGCTGCAAAGAAGCTAAAAAAA-3Ј. Constructs containing a HA tag or In Vitro Ubiquitination Assay. The ubiquitination assay was based on FLAG-tag were created by PCR by fusion of the HA or FLAG sequence to the protocols published previously (14, 16, 58). p53 was ubiquitinated in 50 ␮lof 5Ј end of the amplified Mdm2 cDNA sequences. Primers used to generate total volume reactions containing Sf9 lysates containing E1 (10 ␮g of lysate HA-Mdm2, HA-⌬464–489, and HA-M3 were as follows: HA, 2ϩ,5Ј-GGG- protein), FLAG-UbcH5 (10 ␮g of lysate protein), Mdm2 (10 ␮g of lysate ATCCAGCCATGGGTTACCCATACGACGTCCCAGACTACGCTACCT- protein), and p53 (10 ␮g of lysate protein) in 100 mM Tris-HCl (pH 7.5), 5 mM ␮ GCAATACCAACATGTCTGTGTCTAC-3Ј; HA, 399ϩ,5Ј-GGATCCAGC- MgCl2, 0.6 mM DTT plus 15 M ubiquitin (Sigma Chemical Co.), 2 mM ATP, CATGGGTTACCCATACGACGTCCCAGACTACGCTACCTCCAGCAG- 10 mM ␤-glycerol phosphate, 5 ␮g/ml aprotinin, and 5 ␮g/ml ubiquitin- CATTGTTTATAGCAGC-3Ј (BamHI sites underlined); antisense primers aldehyde (Boston Biochemical). Reactions were allowed to proceed at 25°C 1702(Ϫ) and 463(Ϫ) were the same as those listed above. Primers to generate for 2 h. Products were resolved on 7.5% denaturing polyacrylamide gels, FLAG-M3, FLAG-M4, FLAG-M5, and FLAG-M6 were as follows: FLAG transferred to membranes, and immunoblotted with anti-p53 antibodies (Ab-7) Arf amino acid, 399ϩ,5Ј-GACCATGGACTACAAGGACGACGATGACAAGT- visualized by ECL (Amersham). A His6-tagged, NH2-terminal p19 peptide ϩ CCAGCAGCATTGTTTATAGCAGC-3Ј (NcoI site underlined); antisense bp (N37) expressed in Escherichia coli (57) was purified on a nickel (Ni2 ) 1702(Ϫ) and amino acid 463(Ϫ) were as described above. column according to the manufacturer’s instructions (Qiagen) and added (10 A FLAG-tagged UbcH5 construct was generated by PCR using human ␮g) as an Mdm2 inhibitor. Mdm2 mutants V4, FLAG-M3, or FLAG-M4 from UbcH5 cDNA and the following primers: FLAG-H5–1ϩ,5Ј-GACCATG- Sf9 lysates were also added as indicated. GACTACAAGGACGACGATGACAAGGCGCTGAAGAGGATTCAGAA- AGAA-3Ј (NcoI site underlined); H5(Ϫ), 5Ј-GGATCCTTACATTGAAT- RESULTS ATTTCTGAGTCCATTC-3Ј (BamHI site underlined). The PCR product was subcloned into pGEM-T Easy, excised with EcoRI, and subcloned into the Characterization of Alternatively Spliced Mdm2 Variants. pVL-1393 baculovirus expression vector (PharMingen). Baculovirus con- RNA was extracted from E␮-Myc-induced B-cell tumors, the p53 and structs containing full-length Mdm2 and mutants were generated by excision p19Arf status of which had been determined previously (Ref. 50; Table with EcoRI of the Mdm2 inserts from pGEM-T Easy or the MSCV-IRES-GFP 1). cDNAs encoding various Mdm2 isoforms were amplified from vector and subcloning them into the EcoRI site of pVL1393. total RNA by RT-PCR and subcloned, and their nucleotide sequences Cell Culture and Viral Vector Production. WT, p53-null, Arf-null or were determined. This yielded six alternatively spliced Mdm2 variants Cip1-null MEFs were isolated from 13.5 day midgestation mouse embryos as (designated V1 to V6) in addition to full-length Mdm2 cDNA (Fig. described previously (53) and cultured at early passages in DMEM containing 1A). Single or multiple Mdm2 isoforms were expressed in different 10% FBS, 2 mM glutamine, 0.1 mM nonessential amino acids, 55 mM 2- ␮ B-cell tumors, regardless of whether full-length Mdm2 was overex- mercaptoethanol, and 10 g/ml gentamicin, in 8% CO2 humidified incubators. Primary pre-B cells were derived from mouse bone marrow harvested from pressed or not and without any obvious correlation with WT or mutant femurs and tibias of WT animals, expanded, and maintained on feeders of p53 status (Table 1). Alignment of cDNA sequences of all variants NIH3T3 cells expressing human IL-7 (T220-29) or in liquid culture with with that of Mdm2 predicted that five of the six Mdm2 isoforms (V1 recombinant human IL-7 in RPMI 1640 containing 5% FBS, 2 mM glutamine, to V5) would not encode portions of the p53-binding domain (mapped 1223

Fig. 1. Structure, expression, and binding characteristics of Mdm2 variants isolated from E␮-Myc-induced B-cell mouse lymphomas. A, schematic structure of Mdm2 variants isolated from E␮-Myc-induced B-cell lymphomas. p53 binding (amino acids 19–102), p19Arf-binding (amino acids 210–304), and RING finger (RD; amino acids 436–476) domains are designated by unshaded boxes. NLSs, NESs, and sequences required for nucleolar localization (NrLS) are shown as black boxes. Arrows, predicted translation ATG start codons. Deleted sequences within Mdm2 variants are shown as lines. Cellular localization of full-length Mdm2 and each variant was determined by indirect immunofluorescence with a monoclonal antibody against Mdm2 (2A10). ND, undetermined. B, primary MEFs at early passage were infected with a control retrovirus vector (Lane 1), or with vectors expressing full-length Mdm2 (Lane 2), or Mdm2 variants (V1 to V6, Lanes 3–8). Cells were lysed 72 h after infection, and proteins were detected by direct immunoblotting (IB) with a monoclonal antibody to Mdm2 (2A10). Right, protein markers (in thousands). C and D, Sf9 cells were coinfected with baculoviruses encoding p53 (C)orp19Arf (D) together with full-length Mdm2 (Lanes 1–3), Mdm2 variant V2 (Lanes 4–6), or Mdm2 variant V4 (Lanes 7–9). Cell lysates were precipitated with normal rabbit serum (NRS; C, Lanes 1, 4, and 7), a monoclonal antibody against a Myc epitope (9E10; D, Lanes 1, 4, and 7), a monoclonal antibody to Mdm2 (2A10; Lanes 2, 5, and 8), or antibodies against p53 (Ab-1) or p19Arf (Lanes 3, 6, and 9). Proteins electrophoretically resolved on denaturing gels were blotted with monoclonal antibody to Mdm2, 2A10. IB, immunoblot.

within the first 120 NH2-terminal amino acids) because of deletions of exon 8, again resulting in disruption of its reading frame, but unlike exons 3, 5, or 4–8. The one exception was the V6 isoform that lacked V1, its expression was undetectable. V2 lacked residues specified by sequences encoded by exon 8 alone (residues 134–165). Variants V2 exon 3 and encoded a Mr 75,000 protein, the translation of which was to V5 retained the entire COOH-terminal portion of Mdm2 from likely initiated from methionine 50 (Fig. 1B, Lane 4). This isoform is amino acids 166 to 489, including nuclear localization (NLS) and identical to that previously identified as p76Mdm2 by others (59). The nuclear export (NES) signals located between amino acids 178 to 195, V4 protein (Fig. 1B, Lane 6) initiates at methionine 1, but it contains the p19Arf-binding domain (amino acids 210 to 304), zinc finger a large in-frame deletion from amino acids 11 to 155, as well as (amino acids 303 to 320), and RING finger domain (amino acids deletion of serine 207. As predicted, both the V2 and V4 isoforms 436–476; Fig. 1A). Isoform V1 contained an additional in-frame were predominantly localized to the nucleus of infected MEFs (Fig. deletion of amino acids 231–240. 1A). Of interest, although endogenous Mdm2 expression was not To characterize the proteins encoded by each Mdm2 isoform in detected in MEFs infected with the naked vector (Fig. 1B, Lane 1), we mammalian cells, the cDNAs were subcloned into retroviral expres- observed a modest but consistent increase in its expression in cells sion vectors, packaged into virions, and used to infect primary, early- expressing the truncated Mdm2 variants (Fig. 1B, Lanes 3–7, and see passage MEF strains. Protein expression was monitored by direct below). immunoblotting with a monoclonal antibody to Mdm2 (2A10). Full- Nucleotide sequence analysis predicted that all Mdm2 variants Arf length Mdm2 encoded the expected Mr 90,000 protein (Fig. 1B, Lane would be unable to bind p53 but would still interact with p19 .To 2), which, although larger than that predicted from its sequence, confirm this, insect Sf9 cells were coinfected with baculoviruses undergoes posttranslational modifications (phosphorylation and encoding either WT Mdm2 or variants V2 or V4, together with sumoylation) in mammalian cells. Mdm2 variants V1 and V6 encoded baculoviruses encoding WT p53 (Fig. 1C)orp19Arf (Fig. 1D). Ly- little or no protein (Fig. 1B, Lanes 3 and 8, respectively), whereas all sates of infected cells were then either precipitated with control others encoded protein levels comparable with that of WT Mdm2 antibodies (NRS or 9E10), monoclonal antibodies to Mdm2 (2A10) or (Lanes 4–7). Similar relative levels of protein expression were ob- p53 (Ab-1), or antibodies to the mouse p19Arf COOH terminus, and served after transcription and translation of the variant cDNAs in vitro proteins were separated on denaturing gels were immunoblotted with (data not shown), implying that V1 and V6 were poorly translated. antibody to Mdm2. As expected, the full-length Mdm2 protein copre- Arf Mdm2 isoforms V3 and V5 encoded Mr 55,000 proteins that were cipitated with either p53 or p19 (Fig. 1, C and D, Lanes 1–3). In predicted to initiate at codon 198 (Fig. 1A). Therefore, these variants contrast, Mdm2 variants V2 and V4 were unable to form complexes lacked the NLS and NES and, in agreement, immunofluorescence with p53 but retained the ability to bind p19Arf (Fig. 1, C and D, Lanes analysis (data not shown) revealed that both proteins were predomi- 4–9). nantly expressed in the cytoplasm (summarized in Fig. 1A). Deletion Overexpression of Truncated Mdm2 Isoforms Inhibits Cell of exon 5 in V1 changes its reading frame, also forcing internal Proliferation. Because the Mdm2 variants were expressed in B-cell initiation at methionine 198 and probably accounting for the low level tumors, we suspected that they might compete with WT Mdm2 for of protein detected (Fig. 1B, Lane 3). In addition, the downstream p19Arf binding, facilitating the ability of endogenous Mdm2 to antag- in-frame deletion resulted in production of a cytoplasmic polypeptide onize p53, and thereby accelerating cell proliferation. To test this, we smaller than the V3 and V5 isoforms (Fig. 1B, Lane 3). V6 lacked used retroviral vectors to introduce either full-length Mdm2 or variant 1224

was used as an internal control to monitor infection efficiency. Three to 4 days after infection, GFP-positive cells were seeded at 2 ϫ 104 per dish, and proliferation was monitored by counting cells (triplicate plates/day) for 8 days thereafter. MEFs infected with full-length Mdm2 proliferated considerably more rapidly than cells infected with the empty vector (Fig. 2A), became smaller in size (data not shown), and arrested at confluence by day 8. These results contrast directly with those reported by others who concluded that Hdm2 overexpression inhibited the proliferation of NIH-3T3 cells (60). In contrast, MEFs infected with variants V2, V4, and V5 grew at a much reduced rate and eventually stopped proliferating before becoming confluent (Fig. 2A). Both nuclear (V2 and V4) and cytoplasmic (V5) Mdm2 variants had inhibitory effects on cell proliferation. Growth-arrested MEFs remained viable for at least 4 weeks in culture and were morphologically flat and enlarged (data not shown). Although the rate of growth inhibition by each variant differed, V4 showed the strongest effect and was chosen for subsequent experiments. To confirm that growth inhibition was not limited to fibroblasts, primary bone marrow-derived, IL-7-dependent pre-B cells were infected with the control vector or with those encoding WT Mdm2 or V4 (Fig. 2B). Although full-length Mdm2 accelerated pre-B cell proliferation, expression of V4 not only inhibited pre-B cell growth but triggered apoptosis. Therefore, rather than promoting cell division as we initially supposed, tumor-derived Mdm2 variants hampered the proliferation of established NIH-3T3 cells, as well as that of primary MEFs and pre-B cells. Mdm2 Variants Inhibit Cell Proliferation in a p53-dependent Manner. Expression of Mdm2 variants in a subset of E␮-Myc- induced tumors that retained WT p53, Mdm2, and Arf prompted us to determine whether their ability to inhibit growth might depend on p53, p19Arf,orp21Cip1, the latter a direct transcriptional target of p53. We Fig. 2. Overexpression of Mdm2 variants inhibits cell growth. Primary early-passage, expressed either full-length Mdm2 or V4 in early-passage MEFs WT MEFs (passage 4; A), WT bone marrow-derived pre-B cells (B) or primary MEFs derived from mice lacking p53, both p53 and Mdm2, Cip1,orArf and from mice lacking p53 (C, p53Ϫ/Ϫ), Cip1 (D, p21Ϫ/Ϫ), or Arf (E, ArfϪ/Ϫ) were infected with retroviruses expressing Mdm2 full-length (wt, f) and variants (V2, ϫ; V4, Œ; V5, tested their effects on cell proliferation. The rates of proliferation of E) or an empty vector MSCV-IRES-GFP (Vector, F) and seeded at 2 ϫ 104 in 60-mm p53-null MEFs (Fig. 2C) or of those lacking both p53 and Mdm2 (data diameter culture dishes 72–96 h after infection. Growth rates were determined by counting not shown) were unaffected by introduction of either full-length cell numbers from triplicate cultures every day for 8 days (MEFs) or every other day for 12 days (pre-B cells). F, primary WT MEFs were infected with retroviruses expressing Mdm2 or V4. Similar results were obtained with the V2 and V5 full-length Mdm2 (WT, Lane 2), Mdm2 variant (V4, Lane 3), or empty vector (Vect, Lane isoforms in these cells (data not shown). Therefore, although trun- 1) and lysed 72 h after infection. Proteins were separated on denaturing gels and blotted with antibodies against Mdm2, p53, p21Cip1, and p19Arf as indicated. cated Mdm2 variants cannot interact with p53 directly, their ability to inhibit cell proliferation was still p53 dependent. The effect of V4 was partially compromised in Cip1-null and Arf-null MEFs (compare isoforms into an engineered NIH-3T3 cell line in which p19Arf ex- effects of V4 in Fig. 2, D and E, versus A), indicating that although the Cip1 Arf pression can be conditionally up-regulated by addition of zinc to the absence of p21 or p19 facilitates faster cell proliferation, neither culture medium. Two days after infection, p19Arf expression was is strictly required for V4-mediated inhibition. Cip1 induced by addition of 100 ␮M zinc, and cell proliferation was The strict dependency on p53 and partial contributions of p21 Arf monitored using long-term colony assays. Contrary to our expecta- and p19 for growth inhibition suggested that the variant Mdm2 Arf tions, WT Mdm2 bypassed Arf-induced growth arrest, whereas the isoforms might affect the expression of p53, p19 , Mdm2, and Cip1 Mdm2 variants did not (data not shown), inconsistent with the idea that p21 in infected cells. Indeed, p76 (V2) was found previously truncated Mdm2 variants antagonize p19Arf function in this manner. to antagonize the function of WT Mdm2, increasing the levels and Others reported that Mdm2 variants isolated from human tumor activity of p53 (59). In agreement, we had observed modest increases cells were able to transform NIH-3T3 cells (42). Although the NIH- in endogenous Mdm2 levels in cells coexpressing the truncated Mdm2 3T3 cell line used in our studies lacks the Ink4a/Arf locus (55) and is variants (Fig. 1B). Three days after infection with the control vector or readily transformed by oncogenic Ras, enforced expression of variants those encoding Mdm2 or V4, early-passage, WT MEFs were lysed, V2, V4, or V5 in these cells did not transform them but instead and levels of p53, p21Cip1, and p19Arf were determined by immuno- decreased their growth rate in a manner similar to that seen with blotting (Fig. 2F). The very low levels of p53 expressed in primary primary cell strains (data not shown, but see below). MEFs were not detectably changed in cells infected with WT Mdm2 We next tested the effects of the enforced expression of each Mdm2 (Fig. 2F, Lane 2) but were slightly elevated in cells expressing V4 variant on the proliferation of both primary MEFs and mouse bone (Lane 3) compared with those infected with the control vector (Lane marrow-derived pre-B cells. First, early-passage mouse primary 1). More obviously, p21Cip1 expression was significantly induced by MEFs (Fig. 2A) were infected with retroviruses encoding full-length V4, whereas Wt Mdm2, but not V4, increased p19Arf levels. The latter Mdm2, variants V2, V4, or V5, or the empty control vector. The results are consistent with previous findings that active p53 feeds back MSCV-IRES-GFP vector carries the gene encoding GFP in cis, which to repress Arf expression (61, 62). These data are consistent with the 1225

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2002 American Association for Cancer Research. Mdm2 ISOFORMS INHIBIT PROLIFERATION idea that Mdm2 variants trigger a p53 response that slows cell growth, into early-passage MEFs (Fig. 3B). Expression of each mutant con- and that growth retardation depends in part on p21Cip1. struct was confirmed by immunoblotting with antibodies to Mdm2 or The RING Domain Is Necessary and Sufficient for Growth the epitope tags, revealing variable levels of protein overexpression Inhibition by Mdm2 Variants. To define the domain(s) responsible (Fig. 3C). Similar to the natural Mdm2 variants recovered from B-cell for growth arrest by the Mdm2 variants, we prepared additional tumors, mutants M1, M2, and M3 each inhibited cell growth when

Mdm2 mutants (Fig. 3A). NH2-terminal truncation mutants lacked expressed in WT MEFs (Fig. 3B). However, M4, containing a dis- 197 (M1), 298 (M2), or 398 (M3) residues, leaving an intact RING rupted RING finger domain, was completely devoid of inhibitory domain from amino acids 399 to 489 (M3). The truncation mutant activity. (M4) differed from M3 by codeletion of COOH-terminal residues We therefore used M3 as a backbone to generate two point muta-

464–489. Each construct was tagged at its NH2 terminus with a tions that should affect RING activities (Fig. 3A). In M5 and M6, FLAG or HA epitope preceded by an initiator methionine codon, lysine 444 (446 in Hdm2) and cysteine 462 (464 in Hdm2) were cloned into the MSCV-IRES-GFP retroviral vector, and introduced replaced by alanine and arginine, respectively; these residues are required to maintain the functional integrity of the RING domain, and its mutation cancels the E3 ubiquitin protein ligase activity of Mdm2, both toward itself and toward p535 (15). When expressed in MEFs, M5 inhibited cell growth, whereas M6 lacked inhibitory activity (Fig. 3B). Together, these results indicated that inhibition of cell proliferation by Mdm2 variants required an intact RING finger, although lysine 444 was likely dispensable for this function. Interaction of RING-containing, Truncated Mdm2 Isoforms with Full-length Mdm2. Although growth inhibition by Mdm2 variants is strictly p53 dependent, all such isoforms lacked a functional p53-binding domain, implying that Mdm2 variants interact with reg- ulators of p53 rather than p53 itself. Given that p19Arf was not required for growth inhibition, endogenous Mdm2 was the most obvious candidate. Indeed, MEFs coinfected with the smallest RING- containing construct M3 together with full-length Mdm2 grew at almost the same rate as MEFs infected with full-length Mdm2 alone (Fig. 4A), indicating that M3 and Mdm2 functionally compete with one another in this regard. The integrity of the Mdm2 RING domain was essential for full-length Mdm2 to override the inhibitory effects of M3, because Mdm2 mutants lacking the complete COOH terminus (⌬464–489) or containing an alanine for cysteine mutation at codon 462 were unable to reverse M3-induced growth inhibition (Fig. 4A). Because all inhibitory Mdm2 variants retained an intact RING finger domain, we determined whether these variants could interact with full-length Mdm2 through their respective RING domains. In preliminary experiments, we found that tagged truncated Mdm2 isoforms isolated from B-cell tumors could coprecipitate with full-length Mdm2 after their coexpression in insect Sf9 cells (data not shown). In the simplest and most informative iteration of these experiments, we infected Sf9 insect cells with baculoviruses expressing FLAG-M3 or HA-tagged full-length Mdm2 (WT) and precipitated with either anti- FLAG, with a control antibody to a Myc epitope (9E10), or with SMP14, a monoclonal antibody recognizing an epitope in the mid portion of Mdm2 that is absent in FLAG-M3 (Fig. 4C). Precipitated proteins electrophoretically separated on denaturing gels were then coimmunoblotted with monoclonal antibody 2A10 to detect Mdm2 and with anti-FLAG to detect FLAG-M3 (Fig. 4C). As expected, anti-FLAG specifically precipitated FLAG-tagged M3, SMP14 reacted only with Mdm2, and 9E10 reacted with neither. By contrast, in lysates of coinfected cells, SMP14 precipitated M3 (Fig. 4C, Lane 2), and anti-FLAG coprecipitated Mdm2 (Fig. 4C, Lane 3). A COOH- Fig. 3. The RING finger domain of Mdm2 inhibits cell growth. A, schematic structure of Mdm2 deletion mutants (M1–M4) and M3 RING domain point mutants (M5 and M6). terminal deletion (FLAG-tagged M4) abolished the interaction of the p53-binding, p19Arf-binding, and RING finger (RD) domains are designated by unshaded RING with full-length Mdm2 (Fig. 4C, Lanes 7–9). However, coex- boxes. The NLS, NES, and sequences required for nucleolar localization (NrLS) are shown pression of full-length Mdm2 deleted in its RING domain (⌬464– as black boxes. Epitopes recognized by Mdm2 antibodies, 2A10 and SMP14, are indicated as black bars above the top schematic. Arrows, predicted ATG translation start codons. 489) together with FLAG-M3 still allowed complexes to form (Fig. RING finger domain mutations generated by PCR are indicated for M5 and M6. B, 4C, Lanes 4–6). Despite the ability of these two proteins to interact, f primary WT MEFs infected with retroviruses expressing full-length Mdm2 (WT, ), the Mdm2 COOH-terminal truncation mutant was unable to override mutants (M1, ‚; M2, E; M3, Œ; M4, ࡗ; M5, ϫ; M6, Ⅺ), or an empty vector (F) were grown and counted as described in Fig. 2. Bars, SE. C, protein expression was confirmed the inhibitory effects of M3 (Fig. 4A). Finally, when we coexpressed in lysates made from WT MEFs after infection with retroviruses encoding Mdm2 mutants FLAG-tagged M3 with HA-tagged M3, the two RING domains were (M1–M6, Lanes 1–6). Immunoblotting (IB) was performed with antibody 2A10 to Mdm2 for Mdm2 mutants M1 and M2 or with an antibody to the FLAG tag for FLAG-M3 to observed to strongly bind to one another (Fig. 4D). These results FLAG-M6. indicated that the RING domains of truncated Mdm2 isoforms were 1226

required for their interactions with full-length Mdm2. However, the intact RING domain (M3) can also recognize a second binding site retained in the COOH terminally truncated Mdm2 ⌬464–489. To identify the second interaction site outside of the Mdm2 RING domain, two additional Mdm2 deletion mutants were coexpressed with FLAG-tagged M3 in Sf9 cells (Fig. 4E). One of these contained deletions in both the p53-binding and RING domains (⌬1–50, 464– 489), and the other lacked residues 305–489, leaving the p53-binding and acidic domains intact. Coprecipitation experiments revealed that both mutants interacted with FLAG-M3 (Fig. 4E), suggesting that, in addition to RING-RING interactions, FLAG-M3 can also bind to the acidic domain of Mdm2. To confirm that similar interactions could occur in mammalian cells, we introduced M3 alone into the NIH-3T3 cell line that conditionally expressed zinc-inducible p19Arf. Induction of p19Arf strongly increased p53 levels and led to Mdm2 expression. Precipitation with the anti-FLAG antibody demonstrated that endogenous Mdm2 protein coprecipitated with M3 under these conditions (Fig. 4B, Lane 2). Similar results were obtained using HA-tagged M3 (data not shown). Mdm2 Variants Do Not Affect Ubiquitination of p53 Mediated by Mdm2. If inhibition of cell proliferation by the overexpressed Mdm2 RING domain depends upon direct binding to endogenous Mdm2, a potential consequence might be disruption of Mdm2 E3 ubiquitin protein ligase activity, leading, in turn, to p53-dependent growth retardation. We therefore tested whether Mdm2 variants could inhibit Mdm2-directed p53 ubiquitination in reconstituted in vitro enzyme reactions containing E1 and E2 (UbcH5) enzymes plus recombinant full-length Mdm2. Multiple ubiquitinated forms of p53 were resolved on denaturing gels and detected with an antibody to p53 (Fig. 5A, Lane 1). This assay does not distinguish between forms of p53 mono-ubiquitinated on multiple lysine acceptor sites from forms containing tandem ubiquitin chains at single sites (polyubiquitination). However, only mono-ubiquitinated forms have been docu- mented in such assays (20). Expression of Mdm2, V4, M3 and M4 proteins in Sf9 cells was confirmed by immunoblotting (Fig. 5B). Addition of full-length Mdm2 to the reaction (10 ␮g of Sf9 lysate as shown in Fig. 5B) was essential for ubiquitination of p53 (Fig. 5A, Lane 1), which was completely inhibited by addition of 1 or 10 ␮gof

a peptide representing the NH2-terminal 37 amino acids (N37) of p19Arf (Fig. 5A, Lanes 2 and 3). Mutant Mdm2 proteins failed to induce ubiquitination of p53 (Fig. 5A, Lanes 6, 9, and 11) and did not Fig. 4. Mdm2 rescues RING finger domain-mediated growth inhibition. A, primary inhibit p53 ubiquitination mediated by full-length Mdm2 (Fig. 5A, f WT MEFs were infected with retroviruses expressing full-length Mdm2 (WT, )or Lanes 4, 5, 7, 8, and 10). It should be noted that lysates containing FLAG-tagged M3 (Œ), or were coinfected with both (ϫ), with M3 plus Mdm2 (⌬464– 489; F), or with M3 plus Mdm2 (C462A; ‚). Growth rates were determined by counting equal quantities of total protein (10 ␮g) expressed far more V4 than cell numbers from triplicate cultures every day for 8 days. B, NIH3T3 cells containing a full-length Mdm2 (Fig. 5B). Moreover, the mutant Mdm2 proteins zinc-inducible Arf protein under the control of the metallothionein promoter (pMTCB6HA-ARF) were infected with a retrovirus encoding FLAG-tagged M3 (Lane 2). were unable to reverse inhibition of Mdm2-dependent ubiquitination Arf Uninfected cells were used as controls (WT, Lane 1). Zinc (100 ␮M) was added to the by p19 N37 (Fig. 5, C and D). Therefore, induction of p53 in cells cultures for an additional 24 h to induce p19Arf, after which cell lysates were prepared. overexpressing the growth-inhibitory Mdm2 variants is not likely to Proteins precipitated with anti-FLAG (M2, FLAG) were separated on denaturing gels and blotted with antibody to Mdm2 (2A10) and anti-FLAG. IP, immunoprecipitation; IB, be attributable to their ability to directly inhibit p53 ubiquitination by immunoblot. C, insect Sf9 cells were coinfected with baculoviruses expressing FLAG- full-length Mdm2. tagged M3 and HA-tagged full-length Mdm2 (HA-WT), FLAG-M3 and HA-tagged Mdm2 with a truncation of the RING domain (HA-⌬464–489), or HA-tagged full-length Mdm2 and FLAG-tagged M4 (FLAG-M4). Cell lysates were precipitated with anti-Myc (9E10) DISCUSSION used as a negative control (Lanes 1, 4, and 7), antibodies to Mdm2 (SMP14, Lanes 2 and 5; 2A10, Lane 9), or with anti-FLAG (M2, Lanes 3, 6, and 8). Electrophoretically separated proteins were immunoblotted with anti-Mdm2 (2A10) and anti-FLAG (M2). IP, Despite the frequent disruption of the ARF-Mdm2-p53 pathway in immunoprecipitation; IB, immunoblot. D, insect Sf9 cells were coinfected with baculo- both human and mouse cancers, the contribution, if any, of Mdm2 viruses expressing HA-tagged M3 (HA-M3) and FLAG-tagged M3 (FLAG-M3, Lanes variants to tumorigenesis has remained puzzling. Among six alterna- 1–3) or infected with individual baculoviruses (Lanes 4–7) and lysed 48 h after infection. ␮ Cell lysates were precipitated with anti-Myc (9E10, Lane 1), anti-HA (HA, Lanes 2, 4, and tively spliced Mdm2 variants that we isolated from E -Myc-induced 6), and anti-FLAG (Lanes 3, 5, and 7), and immunoblotted with anti-FLAG (Lanes 1–5) B-cell lymphomas, five failed to bind p53. Variant V6 was the only or anti-HA (Lanes 6 and 7). IP, immunoprecipitation; IB, immunoblot. E, insect Sf9 cells isoform that contained an intact p53-binding domain, but because of were coinfected with baculoviruses expressing HA-tagged Mdm2 mutants, HA ⌬1–50, 469–489 (Lanes 1–3), or HA ⌬305–489 (Lanes 4–6), together with FLAG-M3. Cells a frame shifting deletion, it did not encode a detectable protein were lysed 48 h after infection. Lysates were precipitated with anti-Myc (9E10, Lanes 1 product. Each variant retained the central p19Arf-binding domain and and 4), anti-HA (Lanes 2 and 5), and anti-FLAG (Lanes 3 and 6), and separated proteins were immunoblotted with anti-FLAG and anti-HA. IB, immunoblot. an intact COOH-terminal RING finger domain. Counterintuitively, the enforced expression of these Mdm2 variants in primary MEFs and 1227

single point mutation at cysteine 462 that prevents Mdm2 from ubiquitinating both p53 and itself (16, 37) as well as by a partial COOH-terminal deletion (amino acids 464–489), both of which dis- rupt the structure of the RING finger domain. Therefore, the integrity of the RING domain was required for a functional interaction. More- over, the isolated RING domain, when expressed alone, was found to be growth inhibitory. In contrast, elimination of lysine 444 was without effect, implying that such modification is not required for growth inhibition. Mutation of the cryptic nucleolar localization sequence (amino acids 464–471) within the RING finger domain of Mdm2, which is required for p19Arf-mediated sequestration in the nucleolus, did not affect growth inhibition, suggesting that nucleolar translocation of Mdm2 must not be necessary either (data not shown; Refs. 18, 57, and 63). Deletion mapping studies indicated that the isolated RING domain of Mdm2 was capable of binding the full-length Mdm2 protein. Binding was mediated by direct RING-RING interactions as well as by an association of the isolated RING domain with the acidic domain of Mdm2. Other investigators reported that full-length Hdm2 was growth inhibitory when expressed in NIH-3T3 cells (60), whereas we obtained the opposite result, i.e., overexpression of Mdm2 accelerated proliferation. In their studies, the inhibitory domain of Hdm2 was mapped to residues 155 to 324, which includes the central Mdm2 acidic domain but not the COOH-terminal RING. Again, our results do not agree, and the basis for these discrepancies remains unresolved. Because RING fingers appear to have no intrinsic E3 protein ligase activity of their own, the simplest interpretation is that an interaction between the full-length and variant Mdm2 proteins might impair the E3 ligase activity of Mdm2. However, this now seems unlikely, because neither the expression of variant Mdm2 V4 nor expression of the RING domain alone inhibited Mdm2-mediated p53 ubiquitination in an in vitro assay. Another possibility is that overexpression of Mdm2 RING domains in cells can sequester the E2 Ubc enzymes that are required for Mdm2 E3 ligase activity. We do not favor this for several reasons: (a) E2 enzymes interact with many E3s and are not Fig. 5. In vitro ubiquitination of p53 is unaffected by truncated Mdm2 isoforms. A, in vitro ubiquitination of p53 was performed with recombinant proteins expressed in Sf9 generally thought to be rate-limiting in vivo; and (b) the crystal cells, including full-length Mdm2, the V4 variant, the RING domain (M3), or a COOH- structure of the Cbl E3 ligase indicates that E2 binding is coordinated terminally truncated RING domain mutant (M4). N37, representing the first 37 NH2- terminal amino acids of p19Arf, was prepared in bacteria using a chemically synthesized both by residues within the RING domain and through additional minigene template (58). Ladders of ubiquitinated forms of p53 were visualized with contacts elsewhere in the protein (64). In addition, overexpression of antibody to p53. Sf9 lysates containing Mdm2 (10 ␮g of total protein) or lysates UbcH5 or UbcH7 together with Mdm2 variants failed to rescue their expressing M3, M4, or V4 (1 or 10 ␮g of total protein as indicated at the top of the panel) were used. B, expression of proteins in Sf9 cells infected with full-length Mdm2 and growth inhibitory effects (data not shown). Therefore, the mechanism variant V4 was confirmed by immunoblotting (IB) with antibody 2A10 to Mdm2, whereas by which truncated Mdm2 isoforms inhibit cell growth remains un- protein expression from Sf9 cells infected with FLAG-tagged M3 and FLAG-tagged M4 was confirmed by immunoblotting with anti-FLAG (M2). Uninfected cells were used as defined. controls. C, in vitro p53 ubiquitination assay with full-length Mdm2 alone (Lanes 3–5)or Despite the ability of truncated Mdm2 isoforms to associate directly together with variant Mdm2 V4 (Lanes 8–10) with increasing concentration of N37- with Mdm2, an important caveat is a lack of formal proof that Mdm2 p19Arf (Lanes 4, 5 and 9, 10). D, p53 ubiquitination assay with full-length Mdm2 together with the RING domain (M3, Lanes 3–5) or a RING domain mutant (M4, Lanes 7–9) with is, in fact, their critical target. There is no simple way to test whether increasing quantity of N37-p19Arf (Lanes 4, 5 and 7–9). Mdm2 is required, because cells from Mdm2-null mice cannot be propagated unless they also lack p53 (6, 7). It may well prove that bone marrow-derived pre-B cells and in immortalized NIH-3T3 fi- Mdm2-related Mdm4 (MdmX) or other proteins that interact with broblasts inhibited rather than enhanced cell proliferation. Isoforms Mdm2 are responsible for the observed effects. In agreement with data that predominantly localized to the cell nucleus (e.g., V2 and V4) as of others (65), we confirmed that the RING finger domain of Mdm2 well as those that remained cytoplasmic (e.g., V5) were active when could interact with Mdm4 and vice versa (data not shown). The exact overexpressed. Introduction of Mdm2 variants into cells appreciably role of Mdm4 in inhibiting p53 function remains unclear, but unlike stabilized p53 and induced both endogenous Mdm2 and p21Cip1. Mdm2, Mdm4 is not a p53-inducible gene, does not seem to be Growth inhibition was strictly p53 dependent and was only partially expressed at particularly high levels during p53 stress responses, and alleviated in cells lacking either p21Cip1 or p19Arf. although it inhibits p53-dependent transcription, Mdm4 is not thought

Because the ability of NH2-terminally truncated Mdm2 isoforms to to catalyze p53 degradation (34, 39). Disruption of Mdm4 in the inhibit cell proliferation required p53 but could not be mediated mouse germ-line leads to embryonic lethality accompanied by growth through a direct interaction with p53 itself, we reasoned that their arrest, but not apoptosis, and these effects are rescued on a p53-null interaction with full-length Mdm2 might be required. In agreement background (36). Intriguingly, disruption of the Mdm4 gene resulted with this idea, Mdm2 variants were observed to associate directly with in production of a truncated product that likely encodes the RING full-length Mdm2. Growth inhibition was completely abrogated by a domain. This formally leaves open the possibility that embryonic 1228

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2002 American Association for Cancer Research. Mdm2 ISOFORMS INHIBIT PROLIFERATION lethality results from a gain of function (overexpression of the Mdm4 6. Montes de Oca Luna, R., Wagner, D. S., and Lozano, G. Rescue of early embryonic RING) versus Mdm4 loss. lethality in mdm2-deficient mice by deletion of p53. Nature (Lond.), 378: 203–206, 1995. Whatever the exact mechanisms, enforced overexpression of the 7. Jones, S. N., Roe, A. E., Donehower, L. A., and Bradley, A. Rescue of embryonic truncated Mdm2 variants led to p53 activation, which could be re- lethality in Mdm-2-deficient mice by absence of p53. Nature (Lond.), 378: 206–208, 1995. versed by simultaneous overexpression of full-length Mdm2. Simi- 8. Carroll, P. E., Okuda, M., Horn, H. F., Biddinger, P., Stambrook, P. J., Gleich, L. L., larly, a recent study showed that Mdm2 rescues cell growth arrest Li, Y-Q., Tarapore, P., and Fukasawa, K. Centrosome hyperamplification in human mediated by another Mdm2 binding protein, MTBP (66). MEFs cancer: chromosome instability induced by p53 mutation and/or Mdm2 overexpression. Oncogene, 18: 1935–1944, 1999. transduced by Mdm2 variants alone arrested irreversibly after several 9. Momand, J., Zambetti, G. P., Olson, D. C., George, D., and Levine, A. J. The mdm-2 days, remained viable for as long as 4 weeks in culture, and assumed oncogene product forms a complex with the p53 protein and inhibits p53-mediated an enlarged and flat morphology reminiscent of senescent fibroblasts. transactivation. Cell, 69: 1237–1245, 1992. Cip1 10. Oliner, J. D., Pietenpol, J. A., Thiagalingam, S., Gyuris, J., Kinzler, K. W., and Interestingly, although p21 induction in response to Mdm2 vari- Vogelstein, B. Oncoprotein MDM2 conceals the activation domain of tumour sup- ants was relatively robust, the induced levels of endogenous Mdm2 pressor p53. Nature (Lond.), 362: 857–860, 1993. 11. 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Oncoprotein MDM2 is a ubiquitin ligase E3 variants are found in many types of human tumors, including invasive for tumor suppressor p53. FEBS Lett., 420: 25–27, 1997. breast cancer (47), late-stage and high-grade ovarian and bladder 15. Honda, R., and Yasuda, H. Association of p19ARF with Mdm2 inhibits ubiquitin carcinomas (42), and liposarcomas (48). Although the role of these ligase activity of MDM2 for tumor suppressor p53. EMBO J., 18: 22–27, 1999. 16. Honda, R., and Yasuda, H. Activity of MDM2, a ubiquitin ligase, toward p53 or itself Mdm2 variants in the onset or late stages of tumors remains elusive, is dependent on the RING finger domain of the ligase. Oncogene, 19: 1473–1476, their occurrence and persistence in both human and mouse tumors 2000. 17. Tao, W., and Levine, A. J. Nucleocytoplasmic shuttling of oncoprotein Hdm2 is suggest that they somehow contribute to tumorigenicity. Our results required for Hdm2-mediated degradation of p53. Proc. Natl. Acad. Sci. USA, 96: reveal that when overexpressed in primary MEFs or pre-B cells, these 3077–3080, 1999. Mdm2 variants paradoxically inhibit rather than promote cell growth. 18. Weber, J. D., Taylor, L. J., Roussel, M. F., Sherr, C. J., and Bar-Sagi, D. Nucleolar Arf sequesters Mdm2 and activates p53. Nat. Cell Biol., 1: 20–26, 1999. We have considered several possibilities to rationalize these results: 19. Lu, W., Pochampally, R., Chen, L., Traidej, M., Wang, Y., and Chen, J. Nuclear (a) it may prove that, similar to activated Ras (67), the enforced exclusion of p53 in a subset of tumors requires MDM2 function. Oncogene, 19: expression of these Mdm2 variants triggers growth arrest in primary 232–240, 2000. 20. Lai, Z., Ferry, K. V., Diamond, M. A., Wee, K. E., Kim, Y. B., Ma, J., Yang, T., cells, but in collaboration with Myc, promotes proliferation; and (b) Benfield, P. A., Copeland, R. A., and Auger, K. R. Human Mdm2 mediates multiple alternatively, expression of truncated Mdm2 variants might allow monoubiquitination of p53 by a mechanism requiring enzyme isomerization. J. Biol. ␮ Chem., 276: 31357–31367, 2001. certain cells to escape E -Myc-induced apoptosis during early stages 21. Boyd, S. D., Tsai, K. Y., and Jacks, T. An intact HDM2 RING-finger domain is of lymphomagenesis, after which subsequent genetic changes then required for nuclear exclusion of p53. Nat. Cell Biol., 2: 563–568, 2000. allow the expansion of this resistant population. However, we found 22. Geyer, R. K., Yu, Z. K., and Maki, C. G. The MDM2 RING-finger domain is required to promote p53 nuclear export. Nat. Cell Biol., 2: 569–573, 2000. that enforced expression of Mdm2 variants in early-passage MEFs did 23. Stommel, J. M., Marchenko, N. D., Jimenez, G. S., Moll, U. M., Hope, T. J., and not inhibit Myc-ER-induced apoptosis in response to tamoxifen (data Wahl, G. M. 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Cancer Res 2002;62:1222-1230.

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