MDM4 (MDMX) Overexpression Enhances Stabilization ofStress-induced p53 and Promotes Apoptosis*

Received for publication, October 28, 2003, and in revised form, November 26, 2003Published, JBC Papers in Press, December 1, 2003, DOI 10.1074/jbc.M311793200

Francesca Mancini‡, Francesca Gentiletti‡, Marco D’Angelo‡, Simona Giglio‡, Simona Nanni‡§,Carmen D’Angelo¶, Antonella Farsetti‡�, Gennaro Citro**, Ada Sacchi‡, Alfredo Pontecorvi‡ ‡‡,and Fabiola Moretti‡�§§

From the ‡Laboratory of Molecular Oncogenesis, ¶Laboratory of Experimental Chemotherapy, and **Animal HouseService, Regina Elena Cancer Institute, Via delle Messi D’Oro 156, Rome 00158, Italy, ‡‡Institute of Medical Pathology,Catholic University, Largo Vito 1, Rome 00100, Italy, and �Neurobiology and Molecular Medicine Institute,National Council of Research, Viale Marx 15, Rome 00137, Italy

Rescue of embryonic lethality in MDM4�/� micethrough concomitant loss of p53 has revealed a func-tional partnership between the two proteins. Biochem-ical studies have suggested that MDM4 may act as anegative regulator of p53 levels and activity. On theother hand, MDM4 overexpression has been reported tostabilize p53 levels and to counteract MDM2-degrada-tive activity. We have investigated the functional role ofMDM4 overexpression on cell behavior. In both estab-lished and primary cells cultured under stress condi-tions, overexpression of MDM4 significantly increasedp53-dependent cell death, in correlation with enhancedinduction of the endogenous p53 protein levels. Thisphenomenon was associated with induced p53 tran-scriptional activity and increased levels of the proapo-ptotic protein, Bax. Further, p53 stabilization was ac-companied by decreased association of the protein to itsnegative regulator, MDM2. These findings reveal a novelrole for MDM4 by demonstrating that in non-tumor cellsunder stress conditions it may act as a positive regulatorof p53 activity, mainly by controlling p53 levels. Theyalso indicate a major distinction between the biologicalconsequences of MDM4 and MDM2 overexpression.

p53 is the most frequently inactivated tumor suppressorgene in human cancer. Following different stress conditions,the p53 protein is stabilized and functionally activated, result-ing in two main outcomes: cell cycle arrest or apoptosis (1). Toensure a proper cell growth under physiological conditions, p53function is tightly controlled by maintaining the protein at lowlevels and partially inactive (1–3). A key molecule in the reg-ulation of p53 basal levels and activity is the MDM2 protein(3, 4).

MDM4 (MDMX) was identified in 1996 as a p53-bindingprotein, structurally related to the p53 negative regulator,MDM2 (5). The cross-talk between MDM4 and p53 has been

established by the analysis of knock-out mice; the MDM4�/�

mouse is characterized by embryonic lethality, whereas thedouble knock-out p53�/� MDM4�/� mouse is alive and developsnormally (6–8). The comparison of MDM4�/� and MDM2�/�

mice, both characterized by embryonic lethality and rescued bysimultaneous knock-out of the p53 gene, has revealed a maindifference in the determinants of lethality; MDM4�/� embryosdo not develop beyond 7–11 days due to loss of cell proliferation,whereas MDM2�/� embryos die by massive apoptosis at theblastula stage (6, 9, 10). These results, while confirming a rolefor MDM4 and MDM2 as major regulators of p53 activity,suggest that the two proteins act in nonoverlapping pathways,regulating p53 function in different ways. MDM4�/� embryofibroblasts undergo growth arrest in vivo and in vitro (6–8) andexpress high levels of p53 and of its target gene p21, a wellknown negative regulator of cyclin/cyclin-dependent kinases(11, 12), suggesting a negative control of p53 levels by MDM4,in normal growth conditions. However, a recent report demon-strated that in the absence of MDM4, MDM2 degrades p53 lessefficiently (13), suggesting that the higher p53 levels observedin MDM4�/� mice could be due to impairment of this MDM2function.

Biochemical studies based on transient overexpression ofMDM4 in different cell types, have revealed two distinct activ-ities of the protein toward p53: (i) inhibition of p53 transactingactivity (5, 14–16) and (ii) antagonism of MDM2-driven degra-dation of the p53 protein (14–19). The apparent contradictionbetween the latter effect and the hypothesis that MDM4 is anegative regulator of p53 levels has been partially solved by Guet al. (13), who have shown that the effects on p53 levels dependon the relative ratio of MDM4 and MDM2. Antagonism of p53degradation prevails when MDM4 levels largely exceed those ofMDM2, whereas in all of the other conditions, the two proteinscooperate in the degradation of p53 (13). Since MDM2 levelsvary within the cell depending upon stress signals of differentintensity (20–22), and MDM4 appears to be preferentially de-graded under conditions that activate p53-induced growth ar-rest (23–25), it is reasonable to hypothesize that MDM4 maydifferentially affect p53 levels in different growth conditions.

The negative regulation of p53 transactivating propertiesappears to depend on the presence of MDM2 (13) and to beaffected by MDM4 subcellular localization (13, 19). In turn,stress conditions as well as p53 activation induce nucleartranslocation of overexpressed MDM4 (26). However, the ef-fects of MDM4 on p53 transactivating function under theseconditions have not been examined.

In order to investigate the biological consequences of MDM4

* This work was supported by research grants from Ministerodell’Istruzione, dell’Universita e della Ricerca (MIUR), Ministero dellaSalute, Progetto “Oncologia” MIUR-CNR, and Associazione ItalianaRicerca sul Cancro. The costs of publication of this article were defrayedin part by the payment of page charges. This article must therefore behereby marked “advertisement” in accordance with 18 U.S.C. Section1734 solely to indicate this fact.

§ Supported by a Fondazione Italiana Ricerca Cancro fellowship.§§ To whom correspondence should be addressed: Laboratory of Mo-

lecular Oncogenesis, Regina Elena Cancer Institute, Via delle MessiD’Oro 156, Rome 00158, Italy. Tel.: 39-06-52662531; Fax: 39-06-4180526; E-mail: moretti@ifo.it.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 9, Issue of February 27, pp. 8169–8180, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 8169

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on p53 function, we overexpressed MDM4 cDNA in differentcells expressing endogenous wild-type (WT)1 p53 and normallevels of MDM2. Our results show that in NIH3T3 cells, stableoverexpression of MDM4 per se does not alter p53 basal levels;nor does it confer a proliferative advantage or increase colony-forming ability. On the contrary, under stress conditions,MDM4 overexpression enhances cell death, a phenomenon thatcorrelates with increased p53 protein levels and transcriptionalactivity and with increased dissociation of p53 from its negativeregulator MDM2. Similarly, overexpression of human MDM4in human primary cells (human fibroblasts (HF) and humanembryo kidney (HEK) cells) causes significant decrease of cellviability correlated with enhanced p53 induction and activityfollowing adriamycin treatment, whereas no effects were ob-served in MEF p53�/�.

These results provide evidence for a potential new role forMDM4 as a positive regulator of p53 function under stressconditions and indicate a major distinction between MDM4 andMDM2 activities.

MATERIALS AND METHODS

Cell Culture, Plasmids, and Transfections—Mouse NIH3T3 fibro-blasts were cultured at 37 °C in F15 medium (minimum essential mediumwith 26 mM NaHCO3, 2 mg/liter biotin, 10 mM glucose, 4 mM glutamine,essential amino acids (50�; Invitrogen) nonessential amino acids (100�;Invitrogen), BME vitamin solution (100�; Invitrogen)) supplementedwith 8% TET system-approved fetal bovine serum (Clontech).

MEF p53�/� (Dr. S. Soddu (CRS-IRE, Rome)) and HF, derived from aforeskin human explant, were cultured in Dulbecco’s modified Eagle’smedium high glucose supplemented with 10% fetal bovine serum (Hy-clone). All experiments were done between passages 8 and 10. HEK cells(27) were cultured in �-minimum essential medium supplemented with10% fetal bovine serum (Invitrogen) and used between passages 3 and 5.

NIH3T3 cells stably transfected with MDM4, MDM2, or pTRE weremaintained in medium containing 400 �g/ml G418 (23).

Transient transfections were performed by the calcium phosphateprecipitation technique or LipofectAMINE for full-length humanMDM4 (hMDM4) and p54. Briefly, in 60-mm plates, a fixed number ofcells were transfected with 8 �g of hMDM4 or p54 or pCMV�gal orpcDNA3.1 plus 0.8 �g of pEGFP plasmid (Invitrogen), as internalcontrol of transfection efficiency. For transcriptional assays, cells weretransfected with 0.5 �g of Bax-Luc (28) or 800 ng of p21-Luc plasmidsplus 0.25 �g of cytomegalovirus �-galactosidase plasmid, as internalcontrol of transfection efficiency. Cells were harvested 48 h after trans-fection, and Luc activity was assayed on whole cell extracts.

Clonogenicity Assay—Different cell numbers (102, 2 � 102, and 5 �102) were plated in quadruplicate in 6-cm dishes in the presence orabsence of 10 �M doxycycline (Dox). Every 3 days, medium waschanged, and the Dox dose was renewed. 10 days after plating, disheswere stained by crystal violet (0.25% in methanol) for 10 min andair-dried, and colonies (�1-mm diameter) were counted.

Proliferation Rate and Cell Cycle Analysis—Cell proliferation ratewas assessed by determining cell number in a Thomas’s hemocytome-ter, using trypan blue exclusion as a cell viability test.

Cell proliferation under growth factor deprivation was determined byplating 105 cells in 6-cm dishes; after cell adhesion, culture medium wasreplaced with medium containing 0.25% fetal calf serum. After 48 h, 10�M Dox was added. As control, the same cells were grown in the absenceof Dox.

Cell cycle profiles were evaluated by fixing 2 � 105 cells in cold ethanol(70%) overnight and staining DNA for 30 min at room temperature with50 �g/ml propidium iodide in phosphate-buffered saline containing 1mg/ml RNase A. Percentages of cells in the different phases of the cyclewere measured by flow cytometric analysis of propidium iodide-stainednuclei using CELLQUEST software FACScalibur (BD Biosciences).

TUNEL Assay—Cells were fixed in paraformaldehyde solution (4%in phosphate-buffered saline, pH 7.4) for 30 min at room temperatureand permeabilized in 0.1% Triton X-100, 0.1% sodium citrate for 2 min

on ice. Apoptotic nuclei were detected using a TUNEL labeling reactionaccording to the manufacturer’s instructions (Roche Applied Science).TUNEL labeling and phase contrast images were analyzed by the AXIOVISION version 3.0 program.

Immunoprecipitation and Western Blot Analysis—For immunopre-cipitation experiments, cells were lysed in Giordano’s buffer (50 mM

Tris-HCl, pH 7.4, 0.25 M NaCl, 0.1% Triton X-100, 5 mM EDTA) con-taining a mixture of protease inhibitors (Roche Applied Science), andwhole cell extracts were centrifuged at 14,000 rpm for 30 min to removecell debris. Protein concentration was determined by a colorimetricassay (Bio-Rad). Immunoprecipitations were performed by incubatingwhole cell extracts with the indicated antibody, preincubated withprotein G-Sepharose (Pierce), under gentle rocking at 4 °C overnight.Immunoprecipitates were washed three times with Giordano’s buffersupplemented with protease inhibitors, resuspended in 40 �l of 2� SDSLaemmli sample buffer, and then resolved by SDS-PAGE. For Westernblot analysis, cells were lysed in radioimmune precipitation buffer (50mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodiumdeoxycholate, 0.1% SDS, 1 mM EDTA) supplemented with a mixture ofprotease inhibitors (Roche Applied Science). Whole lysates were re-solved by SDS-PAGE and subsequently transferred onto polyvinylidenedifluoride membranes (Millipore). Before immunoblotting, membraneswere stained with Ponceau Red to ensure equal protein loading. Afterblocking, membranes were incubated for 2 h using the following pri-mary antibody: anti-MDM4 monoclonal antibody (6B1A, 114FD, and12G11G), anti-p54 rat polyclonal antibody (raised against full-lengthp54, according to Candi et al. (29)), anti-p21 polyclonal antibody specificfor the mouse protein (kindly provided from Dr. C. Schneider), anti-p21F5 monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz,CA), anti-p53 polyclonal antibody FL393 (Santa Cruz Biotechnology),anti-p53 polyclonal antibody Ab 7 (Oncogene Science), anti-Bax poly-clonal antibody, N-20 (Santa Cruz Biotechnology), anti-MDM2 mono-clonal antibody 2A10, anti-�-tubulin monoclonal antibody, DM1A(Sigma), and anti-Hsp70 mouse monoclonal antibody SPA-820. Mem-branes were developed using ECL (Amersham Biosciences).

Formaldehyde Cross-linking and Immunoprecipitation of Chroma-tin—Cells grown as previously described were washed twice with phos-phate-buffered saline and cross-linked as described in Boyd et al. (30).The chromatin solution was precleared by the addition of Protein G(Pierce) for 1 h at 4 °C and incubated with 2 �g of �-p53 (Ab 7), anti-IgG,or no antibody overnight at 4 °C with mild shaking. Before use, ProteinG was blocked with 1 �g/�l sonicated salmon sperm DNA and 1 �g/�lbovine serum albumin for 2 h at 4 °C. Chromatin immunoprecipitation,washing, and elution of immune complexes were carried out as previ-ously described (30). DNA fragments were recovered by centrifugationand resuspended in 30 �l of double-distilled H2O and analyzed by PCR.Total input sample was resuspended in 100 �l of double-distilled H2Oand then diluted 1:100 before PCR. Each reaction mixture contained0.5–1 �l of immunoprecipitated chromatin, 70 ng of each primer, 250�M deoxynucleoside triphosphates (Roche Applied Science), 2 mM

MgCl2, 1� Taq reaction buffer, and 1.25 units of Taq polymerase(ABgene House, Epsom, UK) in a final volume of 30 �l. After 32–35cycles of amplification, PCR products were run on a 2% agarose gel andanalyzed by ethidium bromide staining. For PCR analysis of the p21and bax promoters, the following oligonucleotides spanning the p53binding elements (31) were used: p21-up, 5�-GAG TTT GTG TGG AGGTGA CTT CTT C-3�; p21-down, 5�-CTG GTA GTT GGG TAT CAT CAGGTC T-3�; bax-up, 5�-CTG TCC TTG AAC TCA GAG AGA TGG-3�;bax-down, 5�-GGC TAT CCT GGA ACTCAC TTT TGA-3�. For PCRanalysis of the tubulin gene, the following oligonucleotides were used:tub-up, 5�-GCA CTC TGA TTG TGC CTT CA-3�; tub-down, 5�-AGCAGG CAT TGG TGA TCT CT-3�. The linearity of the PCRs was verifiedby analyzing (i) a 10-fold dilution of the DNA samples and (ii) PCRproducts obtained from increasing amplification cycles.

Adenovirus Generation and Infection—The strategy to create recombi-nant adenovirus was as previously described by He et al. (32). The BamHIfragment of cDNA coding for mouse MDM4 (nucleotides 171–1645) wascloned into the pAdShuttle-CMV (Stratagene). The resulting constructand the control construct (pAdTrack-CMV-ATCC, carrying the cDNAsequence of the green fluorescent protein gene) were linearized with PmeIand transfected by electroporation, together with pAdEasy1 vector (Strat-agene) in electrocompetent E. coli BJ5183 cells (Stratagene). Recombi-nant colonies were selected with kanamycin and screened by restrictionendonuclease digestion. The resulting recombinant adenoviral constructswere digested by PacI and transfected into the packaging cell line 293A(Invitrogen) using the LipofectAMINE Plus protocol (Invitrogen). Trans-fected cells were collected 10–12 days after transfection, and the virallysates were obtained as previously described (32).

1 The abbreviations used are: WT, wild-type; HF, human fibro-blast(s); HEK, human embryonic kidney cell(s); MEF, mouse embryofibroblast(s); Dox, doxycycline; Ab, antibody; Adr, adriamycin; hMDM4,human MDM4; CREB, cAMP-response element-binding protein;TUNEL, terminal dUTP nick end labeling.

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FIG. 1. Effects of MDM4 overexpression on cell growth. A, NIHpTRE, MX-18, and MX-24 cells were cultured in the presence of Dox for the indicatedtime. Cell lysates were then collected and analyzed by Western blot using the anti-MDM4 monoclonal antibody 6B1A. p53 levels were detected byimmunoprecipitating (Ip) 200 �g of total cell lysate with anti-p53 monoclonal antibody 421 and further Western blot analysis (WS) with anti-p53 polyclonalantibody Ab 7. B and C, 104 cells for NIH-MDM4 clones MX-18 and MX-24 or control cells (NIHpTRE) were plated in 6-cm dishes and cultured in thepresence (B) or in the absence (C) of 10 �M Dox. Every 3 days, medium was changed, and the Dox dose was renewed. Viable cells were determined by trypanblue exclusion. D, 102 cells for MX-18, MX-24, and NIHpTRE control cells were plated in quadruplicate in 6-cm dishes in the presence or absence of Dox 10�M. Every 3 days, medium was changed, and the Dox dose was renewed. 10 days after plating, dishes were stained by crystal violet, and the colonies (�1mm diameter) were counted. The histogram represents the mean of the ratio of colonies in the presence and absence of Dox. The significance of the data wasstatistically evaluated by a paired one-sided t test.

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FIG. 2. Effects of MDM4 on p53 levels and activity. A, 200 �g of whole cell extract, from NIHpTRE and MX-18 cells, treated with Adr asdescribed under “Results,” were immunoprecipitated (Ip) with anti-p53 monoclonal antibody 421 and analyzed by Western blot (WS) with theanti�p53 polyclonal antibody, Ab 7. B, NIHpTRE, MX-18, MX-24, and M2–23 cells were plated in quadruplicate and cultured in the absence orpresence of Dox, and 8 h later, they were transiently transfected by the calcium phosphate method. After removal of calcium phosphateprecipitates, cells were cultured for 48 h. Adr was added 24 h before lysis. The histogram shows the ratio of -fold induction of luciferase activity

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To generate high titer viral stocks, the packaging cells 293A (Invitro-gen) were infected, and viral titers were determined as plaque-formingunits/ml by a plaque test assay as previously described (33).

Adenoviral infections were carried out on cell monolayers (in 60-mmPetri dishes) at the indicated multiplicities of infection by 1-h incuba-tion at 37 °C in the presence of 1 ml of medium. Fresh culture mediumwas then added, and cells were subjected to the treatments as indicatedin Fig. 7.

RESULTS

Stable MDM4 Overexpression Does Not Affect p53 Levels orChange the Proliferative Capability of NIH3T3 Cells—In orderto analyze the functional effects of MDM4 overexpression, weused immortalized NIH3T3 mouse fibroblasts, expressing WTp53, stably transfected with MDM4 cDNA under control of atetracycline-inducible promoter (Tet-ON). Following 24-htreatment with 10 �M Dox, several clones were isolated andscreened for MDM4 expression (NIH-MDM4), as previouslyreported (23). As a control, a mixed population of NIH3T3 cellsstably transfected with the pTet-ON coding plasmid and thepTRE empty vector, was used (NIHpTRE) (23).

First, we investigated the effects of stable MDM4 overex-pression on the endogenous p53 levels. Analysis of p53 proteinin NIH-MDM4 clones (MX-18 and MX-24) at different timepoints following Dox addition did not show changes in p53levels during cell growth or differences in comparison withNIHpTRE cells (Fig. 1A), indicating that MDM4 overexpres-sion does not alter p53 basal levels in NIH3T3.

We had previously shown that under normal growth condi-tions, MDM4 inhibits p53 transcriptional activity in this sys-tem (23). Accordingly, it has been proposed that MDM4 couldbe a negative regulator of p53 function (7, 8, 19, 34, 35). Sincecultured fibroblasts deficient for p53 function have an in-creased proliferation rate (36–38), the growth of MDM4-ex-pressing clones was compared with that of NIHpTRE controlcells. In the presence of Dox, all cells had a similar proliferationrate (Fig. 1B) and fraction of viable cells (data not shown),indicating that overexpression of MDM4 does not alter theseparameters. At confluence, whereas NIHpTRE cells remainedviable, the NIH-MDM4 clones (MX-18 and MX-24) exhibited areproducible reduction of viability, a phenomenon not observedin the absence of Dox (Fig. 1, compare B with C). It has alsobeen reported that p53 inactivation correlates with increasedability of cells to survive and proliferate when plated at lowdensities (37, 38). Therefore, a colony-forming assay with dif-ferent NIH-MDM4 clones was performed. Cells at differentdensities were plated and, every 3 days, refed with fresh me-dium with Dox. Surprisingly, at the lowest density (100 cells/6-cm dish), MDM4 overexpression significantly reduced colonyformation in comparison with NIHpTRE control cells (Fig. 1D).

All of these data show that MDM4 overexpression per se doesnot alter the growth properties of NIH3T3 cells or affect en-dogenous p53 protein basal levels and rather suggest that itmight increase cell susceptibility to stress conditions.

MDM4 Overexpression Stabilizes Transcriptionally Activep53—To ascertain whether MDM4 does indeed affect cell via-bility under stress conditions, we analyzed the cell responsefollowing treatment with Adriamycin (Adr), a DNA damage-

inducing drug that activates the tumor suppressor p53 (39) andarrests NIH3T3 cells in the G1/G2 phase of cell cycle (23). Sinceenhancement of p53 levels is a common finding following Adrstimulus (39), we first tested the effects of MDM4 overexpres-sion on p53 levels.

Adr (0.9 �M) was added 16 h after induction of MDM4 byDox, and p53 protein levels were evaluated. Indeed, Adr treat-ment increased p53 levels in both NIHpTRE and MX-18 cells(Fig. 2A), but more substantially and for a longer period of timein the latter, indicating that MDM4 overexpression enhancesp53 levels under these conditions. We then tested whether theinduced p53 protein retained transcription activating compe-tence. We performed transient transfection assays using thep53-responsive promoters of the bax and p21 genes. MDM4expression was induced 8 h before transfection, cells were thentransfected and treated with adriamycin for 24 h before lysis.In all NIH-MDM4 clones, the addition of doxycycline caused areproducible increase of p21 and more strongly of bax promoteractivity (Fig. 2B), whereas in NIHpTRE control cells, it did notcause further induction of either promoter. Overexpression ofMDM2, achieved through the same inducible system (M2–23clone) (23), caused inhibition of both promoters, as expected(40), indicating that the induction observed in NIH-MDM4clones is specifically related to MDM4 overexpression.

These observations lead us to conclude that stable MDM4overexpression differentially affects p53 levels under normal orstress conditions of growth in NIH3T3 cells, by increasing thelevels of activated p53. Further, the induced p53 appears toretain its transactivating capability and particularly is func-tional in the activation of its proapoptotic target, Bax.

To confirm this, the binding of p53 to endogenous p21 andbax promoters was tested by chromatin immunoprecipitationassay. Under normal growth conditions and at different timepoints after adriamycin treatment, cells were formaldehyde-cross-linked, and chromatin was immunoprecipitated by p53antibody Ab 7 or an unrelated antibody as control. PCR ampli-fication of the immunoprecipitated DNAs with specific primersfor the p53-binding regions, revealed an increasing signal ofthe p21 promoter in both NIH-MDM4 clones and in controlcells although delayed in the first (Fig. 2C). On the contrary,analysis of the bax promoter fragment showed increased signalonly in the MDM4-expressing cells (Fig. 2D). Thus, followingAdr treatment, p53 was indeed recruited onto the endogenousp21 promoter in both NIH-MDM4 clones and in control cells,whereas it was recruited onto the bax promoter exclusively inNIH-MDM4-expressing clones, in agreement with results onp53 transcriptional activity (Fig. 2B).

To definitely confirm these data, the protein levels of p21 andBax were analyzed by Western blot. Adriamycin treatmentcaused a progressive increase in Bax levels in NIH-MDM4 clonesover 24 h, whereas in control cells, Bax levels were stable (Fig.2E), in good agreement with previous results (see Fig. 2D). Levelsof p21 analyzed in the same cell lysates were increased in allpopulations (Fig. 2E) and were not increased further by p53stabilization. Analysis of MDM4 levels under the same conditionsdid not show evident modifications (Fig. 2E).

(corrected for �-galactosidase) from cells cultured in the presence or absence of Dox. The values represent the mean of two different experimentsin duplicate, and lines indicate S.D. values. C and D, cells cultured as described in A were collected at different times of Adr treatment as indicatedand formaldehyde-cross-linked, and the chromatin was immunoprecipitated with anti-p53 Ab 7 antibody or an anti-IgG unspecific antibody. Thehistograms represent the relative signal intensities of the PCR products using specific primers for p21 (C) or bax (D) promoters. PCR productsresolved by electrophoresis were stained with ethidium bromide and quantified by densitometry. All signal intensities were corrected for thecorresponding signal obtained from PCR of the tubulin gene. The signal intensity obtained from PCR of chromatin from cells grown under normalconditions was set to 1 (white bars). The graphs are representative of at least three independent PCR amplifications of two independent sets of DNApreparations. E, cell lysates from NIHpTRE, MX-18, and MX-24 cells cultured in the presence of Adr were collected at 4, 8, and 24 h and analyzedby Western blot using the anti-Bax polyclonal antibody N-20, anti-p21 polyclonal antibody, anti-MDM4 monoclonal antibody 6B1A, or anti-tubulinDM1A as loading control (LC).

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Thus, following Adr treatment, MDM4 overexpression en-hances p53 stabilization that correlates with up-regulation ofBax, without altering the p21/Waf1/Cip1 protein levels. Inter-estingly, in the absence of Adr, the basal levels of p21 and Baxin NIH-MDM4 clones are lower than in NIHpTRE control cells.Since these levels are affected by the p53 status (31, 36), theseresults would confirm that under normal growth conditions,MDM4 overexpression negatively affects p53 transactivatingfunction, in agreement with what was previously reported (23).

MDM4 Overexpression Causes Cell Apoptosis under StressConditions—Since following Adr treatment, MDM4 stabilizesfunctional p53 and causes Bax induction, we tested whethercell growth of NIH-MDM4 was affected under these conditions.A decrease in cell viability was observed in MDM4-overexpress-ing clones after as little as 4 h of treatment and became morepronounced at later times (Fig. 3A). In comparison, NIHpTREcells did not show a significant decrease in viability. A TUNELassay further showed the presence of apoptotic cells only inMDM4-expressing clones (Fig. 3B). These data clearly indicatethat MDM4 overexpression causes an apoptotic response, fol-lowing Adr treatment.

MDM4�/� fibroblasts are blocked at the G0/G1 phase of cellcycle and express high levels of p21 (6–8). This phenotype isrescued by concomitant knock-out of the p53 gene, leading tothe hypothesis that MDM4 interferes with p53-mediatedgrowth arrest (8). We had previously shown that MDM4 over-expression down-regulates basal levels of p21 (see Fig. 2E). Toascertain whether the apoptosis associated with MDM4 over-expression is not related to inhibition of growth arrest, weanalyzed cell cycle profiles following growth factor withdrawal,a condition that causes a reversible growth arrest in the G0

phase of the cell cycle in many cell types, including fibroblasts(41, 42), and is mediated by p53 activity too (41, 43).

Both NIH-pTRE and NIH-MDM4 cells cultured in mediumcontaining 0.25% fetal calf serum had a reduced proliferationrate. Doxycycline addition caused a reproducible reduction incell number in NIH-MDM4 cells, whereas NIHpTRE cells werenot affected (Fig. 3, compare C and D). Analysis of cell cycleprofiles showed a significant increase in the sub-G1 area inNIH-MDM4 compared with NIHpTRE cells (Fig. 3E), confirm-ing that MDM4 overexpression decreases cell viability in theseconditions too. However, NIH-MDM4 as well as control cellsshowed a progressive and comparable accumulation of cells inthe G1 phase (Fig. 3F). Similarly to Adr treatment, p53 levelswere increased in NIH-MDM4-expressing cells relative toNIHpTRE cells following serum starvation (data not shown).

These data confirm that under stress conditions, MDM4overexpression decreases cell survival without interfering withgrowth arrest.

The COOH-terminal Domain of MDMX Is Necessary for p53Stabilization and Apoptosis—Previous reports have shownthat deletion mutants of MDM4, lacking the ring finger domainare unable to stabilize exogenously expressed p53 (15, 19). Toconfirm that stabilization of p53 by MDM4 is the main medi-ator of the apoptotic phenotype, we transiently transfectedNIH3T3 with a deleted MDM4 construct. We used a naturallyoccurring isoform named p54 that we had previously charac-terized (23). p54 derives from caspase cleavage of full-lengthMDM4 and is devoid of the carboxyl-terminal amino acids361–489 of the WT protein. The expression of p54 in compar-ison with control vector, �-galactosidase, did not alter p53 basallevels; nor did it induce further increase of p53 levels followingAdr treatment (Fig. 4A). Concomitantly, the analysis of cellviability did not evidence a significant alteration of the growthproperties of NIH3T3 cells (Fig. 4B). These data confirm thatthe stabilization of p53 induced by MDM4 overexpression is the

main factor responsible for the activation of the apoptotic re-sponse Moreover, the COOH terminus region is necessary forthis effect. The analysis of p21 protein levels showed a parallelincrease in both cell populations (Fig. 4A), indicating indirectlythat p53 is transcriptionally active in �-galactosidase as well asin p54-transfected cells. Since p54 contains the MDM4 p53binding domain and is able to repress p53 transcriptional ac-tivity under normal growth conditions (data not shown), thesedata would confirm that MDM4 is ineffective in repressing p53transcriptional function following p53 activation by Adr.

MDM4 Reduces the Association between MDM2 and p53following Adr Treatment—Stabilization of p53 by MDM4 hasbeen reported by other authors, and different hypotheses havebeen proposed to explain its mechanism. Squelching of MDM2binding to p53 has been observed when MDM4 largely exceedsMDM2 (13). Our previous data indicate that the presence ofMDM4 p53 binding domain is not sufficient to induce suchstabilization and that the COOH terminus, where the MDM2binding domain resides (44), plays a role. We therefore inves-tigated which mechanisms underlie the p53 enhancement ob-served in our system. Since different MDM4 levels could dif-ferently alter the composition of the immunocomplexes, all celllysates were collected at the same time after Dox induction byadding adriamycin according to the schedule in Fig. 5A.

p53 was immunoprecipitated by Ab 1, and the MDM2 proteinpresent in the immnunocomplexes was analyzed by Westernblot. In NIHpTRE control cells, following Adr treatment, theamount of MDM2 protein associated with p53 did not increase,despite the increases in p53 levels, as previously reported (40)(Fig. 5B). In MX-18 cells, the fraction of MDM2 associated withp53 was comparably lower if considering the higher levels ofp53 observed in these cells and further decreased during Adrtreatment (Fig. 5C). The fraction of MDM4 associated with p53in the same immunocomplexes did not vary. To confirm that ahigher fraction of p53 was free from MDM2 inhibition, thesame lysates were depleted of the MDM2-bound p53 fraction byimmunoprecipitation of the MDM2-associated complex with�-MDM2 antibody 2A10, and the levels of p53 in the superna-tant were analyzed. As expected, higher levels of p53 wereobserved in the MX-18 cell lysates in comparison with NIH3T3lysates (Fig. 5D). A further immunoprecipitation of the samesupernatants with a-MDM2 2A10 did not reveal detectablelevels of MDM2, indicating that the differences in p53 were notcaused by different amounts of MDM2 remaining from the firstimmunoprecipitation (data not shown).

Thus, under conditions that activate p53 function, overex-pression of MDM4 causes a decreased association of MDM2with p53, resulting in an increased fraction of p53 not subjectedto MDM2-mediated inhibition. However, the decreased associ-ation of MDM2 with p53 is not accompanied by a proportionalincrease in MDM4 association with p53, indicating that amechanism other than physical squelching is functioning andconfirming previous data from p53 transfection (see Fig. 4A).

Western blot analysis of MDM2 in the same lysates assayedby immunoprecipitation demonstrated that MDM2 protein lev-els were similarly increased in both NIHpTRE and MX-18 cellsfollowing adriamycin treatment (Fig. 5E), thus excluding thepossibility that the minor fraction of MDM2 associated withp53 in MX-18 cells is mainly imputable to lower levels of totalMDM2. Under normal growth conditions, the fraction of theMDM2 molecules bound to p53 was not decreased by MDM4overexpression (Fig. 5F), indicating that under these condi-tions, MDM4 is ineffective in counteracting p53 degradationand confirming that the positive role played by MDM4 towardp53 takes place only under stress conditions.

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FIG. 3. Effects of MDM4 overexpression on cell growth under stress conditions. A, equal number of cells were plated and cultured in thepresence of 10 �M Dox. 16 h later, Adr (0.9 �M) was added, and at the indicated time points, viable cells were determined by trypan blue exclusion.The number of starting viable cells was set to 100, and the following determinations were calculated as percentages of this number. The barsrepresent the mean of three different experiments in duplicate, and lines indicate S.D. values. B, merge of TUNEL immunofluorescence andphase-contrast microscopy of NIHpTRE, MX-18, and MX-24 cells grown in the presence of 10 �M Dox and 0.9 �M Adr for 4 h. TUNEL-positive cellsshow green nuclei. C and D, 105 cells were plated in 6-cm dishes; after cell adhesion, culture medium was replaced with medium containing 0.25%fetal calf serum. 48 h later, 10 �M Dox (C) or vehicle (D) was added to the medium. Every 3 days, medium was changed, and the Dox dose was renewed.Viable cells were determined by trypan blue exclusion. E, cells were grown in medium containing 0.25% fetal calf serum. 24 h later, Dox 10 �M wasadded, and at the indicated times cells were collected for fluorescence-activated cell sorting analysis. The histogram represents the fraction of cells inthe sub-G1 area calculated as a percentage of G1 � S � G2. F, fluorescence-activated cell sorting analysis of cells grown as described for E.

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MDM4 Overexpression Causes p53-mediated Apoptosis inDifferent Nontransformed Cell Contexts—The above resultswere obtained in the NIH3T3 cell line. We therefore askedwhether the phenotype observed following MDM4 overexpres-sion is limited to this cell context or is common to other non-transformed cells, particularly those of human origin. To thisend, we transiently overexpressed hMDM4 in two human pri-mary cell strains: HF and HEK. 16 h after transfection, adria-mycin was added, and at different times cell viability wasevaluated. In both cell strains, overexpression of hMDM4 sig-nificantly reduced cell viability in comparison with controlcells, either transfected with the empty vector (Fig. 6, A and C,CTRL) or a �-galactosidase coding plasmid (data not shown),confirming the results obtained in NIH3T3 cells. Western blotanalysis of cell lysates collected at the same time pointsshowed, as expected, the induction of p53 levels by adriamycintreatment in control cells. Notably, hMDM4 overexpressioncaused a further enhancement of p53 levels (Fig. 6, B and D).This effect was more pronounced in the HEK cells, whichshowed concomitantly a stronger decrease in cell viability. Con-

versely, transient overexpression of MDM4 in mouse embry-onic fibroblast knock-out for the p53 gene (MEF p53�/�) did notalter cell viability following adriamycin treatment (Fig. 6E),despite the strong and persistent expression of MDM4 obtainedin this cell strain (Fig. 6, compare F with B and D). Thus, theconsequences of MDM4 overexpression represent a commonfinding also in human cells and are mainly imputable to theeffects on p53, as indicated by the data from MEF p53�/�.

We then asked whether the enhancement of p53 levels iscaused by the overexpression of MDM4 per se or whether levelsof MDM4 are relevant for this phenomenon. To this end, wegenerated recombinant adenovirus carrying MDM4 cDNA andinfected human HEK and HF cells with increasing doses (50–1000 plaque-forming units/cell) of this adenovirus. Cells in-fected with an adenovirus carrying enhanced green fluorescentprotein (AdCTRL) were used as a control. 24 h after infection,cells were treated with Adr, and after a further 24 h, viablecells were counted. Under normal conditions of growth, infec-tion of HEK cells with AdMDM4 did not significantly changecell viability in comparison with cells infected with AdCTRL

FIG. 4. Effects of overexpression ofp54 on growth properties of NIH3T3.A, the same number of NIH3T3 cells wereplated, and the expression vectors for p54or �-galactosidase (�-gal) as control weretransfected by LipofectAMINE. 24 hlater, Adr (0.9 �M) was added, and at dif-ferent time points as indicated, cell ly-sates were collected and analyzed byWestern blot using anti-p54 polyclonalantibody, anti-�-galactosidase monoclonalantibody, anti-p53 Ab 7 polyclonal anti-body, anti-p21 polyclonal antibody, anti-Bax N-20 polyclonal antibody, and anti-Hsp70 SPA-820 as loading control (LC).B, cells were treated as in A, and at theindicated times, cell viability was evalu-ated by trypan blue exclusion. The num-ber of starting viable cells was set to 100,and the following determinations werecalculated as a percentage of this number.The bars represent the means of two dif-ferent experiments in duplicate, and linesindicate S.D. values.

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(Fig. 7A). Conversely, in the presence of Adr, a progressivedecrease of cell viability more pronounced than with AdCTRLwas observed with increasing doses of AdMDM4 (Fig. 7B). Thisdecrease was significant only at the higher doses of infection(see asterisks in Fig. 7B) and correlated with increasingamounts of MDM4 expressed in the cells (the higher exposureof the film showed detectable levels of MDM4 also at 50 plaque-forming units/cell; data not shown) and with concomitant in-crease of p53 (Fig. 7C). In comparison, p53 levels in HEK cellsinfected with AdCTRL were increased by Adr treatment but nofurther by increasing the multiplicity of infection (Fig. 7D). Theanalysis of p21 protein showed an increase in both cell typesfollowing Adr treatment. On the contrary, Bax levels wereincreased only in AdMDM4 cells and at the higher doses ofinfection in association with the higher levels of p53.

These data demonstrate that p53 levels, cell death, andMDM4 expression in HEK cells are all strictly dose-dependent,supporting the hypothesis that the MDM4-associated celldeath is dependent on the enhancement of induced p53 proteinand not simply on MDM4 expression.

Moreover, these data confirm that following Adr treatment,overexpressed MDM4 allows the induction of the proapoptoticBax and does not repress the induction of the p53 target p21.However, in the absence of Adr, p21, and to a lesser extent Bax,basal levels appeared lower in AdMDM4 in comparison withAdCTRL cells. Comparison of p21 and Bax levels in theselysates on a same gel confirmed these data (data not shown).These results are in accord with data from NIH3T3 cells (seeFig. 2E) and confirm that in the absence of stress, MDM4contributes to inactivate p53 transcriptional function. Compa-rable data were obtained in HF cells (Fig. 7, E and F).

DISCUSSION

Animal models have provided evidence for a functional linkbetween MDM4 and p53 (6–8). Particularly, the rescue ofMDM4�/� embryo lethality by simultaneous knock-out of thep53 gene has suggested a role for MDM4 as negative regulatorof p53 function under physiological conditions.

In this study, we investigated the biological effects of overex-pressed MDM4 in different non-tumor cell systems, character-ized by wild-type p53 status and function. We show that stableMDM4 overexpression does not alter p53 levels; nor does it confera proliferative advantage to exponentially growing NIH3T3 cells.These results indicate that under normal growth conditions,MDM4 overexpression per se does not affect cell growth andindirectly support the hypothesis that MDM4 is mainly devotedto maintaining a pool of inactive p53 in undamaged cells (14).

On the other hand, our results demonstrate that under dif-ferent stress conditions such as Adr treatment and growthfactor deprivation, MDM4 overexpression causes cell death.This event correlates with higher and prolonged enhancementof the p53 protein levels, concomitant increase in its transcrip-tional function, and increased levels of the proapoptotic proteinBax. This phenotype was confirmed by transient overexpres-sion of MDM4 in other cell systems, suggesting that cell deathassociated with MDM4 overexpression is a common finding ina nontransformed cell context. These effects are strictly de-pendent on the p53 protein, as demonstrated by the lack ofresponse in MEF p53�/� cells.

The mechanisms by which MDM4 activates p53 apoptoticfunction seem to rely mainly on the stabilization of p53 levels (45,46). Particularly, the experiments we performed in NIH3T3 cellsshow that MDM4 overexpression counteracts MDM2 binding top53. Since this binding is necessary for the degradation of the p53protein (47, 48), the interference by MDM4 on p53-MDM2 asso-ciation appears to be a reasonable cause for p53 stabilization.However, our results do not support the model of physicalsquelching (13); (i) MDM4 binding to p53 does not increase cor-respondingly with the decrease of MDM2, and (ii) the p54 formthat lacks the ring finger domain is unable to cause p53 stabili-zation. A role of MDM4 in the recruitment of other factors caus-ing MDM2 dissociation and stabilization of p53 is under study.

In agreement with our results, it has been demonstrated thatthe inhibition of MDM2-driven degradation of p53 preferen-tially produces activation of the p53 apoptotic function (49).Moreover, we observed that the levels of p21/Waf1/Cip1, themajor determinant of growth arrest, were increased to a simi-lar extent in MDM4-overexpressing and control cells, indicat-ing that MDM4-induced apoptosis is not caused by inhibition ofcell cycle arrest (50, 51). On the other hand, the molecularmechanisms that underlie the activation of the apoptotic phe-notype and particularly the Bax protein in our system remainto be elucidated. It has been reported that the p53 familymember, p73, actively cooperates with p53 in the induction ofapoptosis (52, 53). Since MDM4 stabilizes p73 (54), a modifica-tion of the p73 levels by MDM4 could contribute to this induc-

FIG. 5. Effects of stable MDM4 overexpression on MDM2/p53association in NIH3T3 under different conditions. A, schematicdiagram of cell lysate collection following different Adr exposure times.B and C, 400 �g of whole cell extract, collected from NIHpTRE (B) andMX-18 cells (C), as schematized in A, were immunoprecipitated (Ip)with anti-p53 monoclonal antibody 421 and analyzed by Western blotwith the anti�p53 polyclonal antibody Ab 7, anti-MDM4 monoclonalantibody 6B1A, and anti-MDM2 monoclonal antibody 2A10. D, lysatescollected from NIHpTRE and MX-18 cells as schematized in Fig. 5Awere immnunoprecipitated with anti-MDM2 monoclonal antibody2A10. The relative supernatants were analyzed by Western blot withanti-p53 polyclonal antibody Ab 7 or anti-tubulin DM1A as loadingcontrol (LC). E, lysates collected from NIHpTRE and MX-18 cells asschematized in A were analyzed by Western blot using the anti-MDM2monoclonal antibody 2A10 and anti-tubulin DM1A as loading control(LC). F, 400 �g of whole cell extract, collected from MX-18 cells grownunder subconfluent conditions, were immunoprecipitated with anti-p53monoclonal antibody 421 and analyzed by Western blot with the anti-p53 polyclonal antibody Ab 7, anti-MDM4 monoclonal antibody 6B1A,and anti-MDM2 monoclonal antibody 2A10.

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tion. To date, however, we have been unable to detect endoge-nous p73 in our system.

Previous studies have shown an inhibitory activity of MDM4toward p53 transcriptional function. So far, evidence of MDM4activity on p53 transactivating function under stress conditionshas not been reported. Recently, it has been shown in p53�/

�MDM2�/� MEF that inhibition of the p53 transcriptionalactivity by MDM4 requires cooperation of MDM2, an observa-tion that indicates that MDM4 per se is not able to exert suchinhibition and indirectly supports our data (13). Moreover,since conditions that activate p53 function alleviate MDM2

binding and inhibition of p53 (3, 55–64), they may also allevi-ate MDM4 inhibitory activity. Indeed, following p53 inductionby Adr, we do not observe a concomitant increase of MDM4binding to p53, in analogy to what was reported for MDM2 (40).The lack of inhibitory activity toward p53 by p54, under thesame conditions, also supports this hypothesis.

We propose that, in normal cells, stress may abrogate thecooperation between MDM2 and MDM4 in inhibiting p53 tran-scriptional activity. Under these conditions, in the presence ofhigher levels of MDM4 with respect to MDM2, the ability ofMDM4 to antagonize the MDM2-dependent degradation of p53

FIG. 6. Effects of transient MDM4 overexpression in nontransformed cells. A, C, and E, the same number of HF (A), HEK (C), andp53�/� MEF cells (E) were plated, and MDM4 or control (CTRL) expression vector was transfected by LipofectAMINE 2000. 16 h later, Adr (0.9�M) was added, and at different time points as indicated, viable cells were determined by trypan blue exclusion. The number of starting viable cellswas set to 100, and the following determinations were calculated as percentages of this number. The bars represent the mean of at least twodifferent experiments in duplicate, and lines indicate S.D. values. The asterisks indicate significantly different values. The significance of the datawas statistically evaluated by paired one-sided t test. B, D, and F, lysates collected from HF (B), HEK (D), and p53�/�MEF cells (F) treated asdescribed for A were analyzed by Western blot using anti-p53 FL393 polyclonal antibody, anti-MDM4 mix 6B1A/114FD/12G11G, anti-greenfluorescent protein (GFP) polyclonal antibody as transfection efficiency control, or anti-tubulin DM1A as loading control (LC).

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FIG. 7. Overexpression of MDM4 by adenovirus in HEK and HF cells. A and B, 2 � 105 HEK cells plated in a 6-cm dish were infected withincreasing doses of AdMDM4 or AdCTRL (50–1000 plaque-forming units (pfu)/cell) for 1 h. 24 h later, 0.9 �M Adr (B) or vehicle (A) was added tothe culture medium, and after 24 h, cells were collected, and viable cells were counted by trypan blue exclusion. The bars represent the mean oftwo different experiments in duplicate, and lines indicate S.D. values. The asterisks indicate significantly different values. C and D, cell lysatescollected from HEK cultured as described for A and B were analyzed by Western blot using anti-p53 FL393 polyclonal antibody, anti-MDM4 mix6B1A/114FD/12G11G, anti-p21 F5 monoclonal antibody, anti-Bax N20 polyclonal antibody, or anti-tubulin DM1A as loading control (LC). E, 2 �105 HF cells plated in a 6-cm dish were infected and treated as in A and B. F, cell lysates collected from HF cultured as described for E wereanalyzed by Western blot using anti-p53 FL393 polyclonal antibody, anti-MDM4 mix 6B1A/114FD/12G11G, anti-green fluorescent protein (GFP)polyclonal antibody as transfection efficiency control, or anti-tubulin DM1A as loading control (LC).

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would prevail determining a net increase in p53 induction asobserved in NIH-MDM4 cells. Recently, Li et al. (26) reportedin U2OS cells the lack of changes in cell cycle distribution byoverexpressed MDM4 after stress treatment (26). Althoughthese data are not entirely comparable with ours, since theyused a transformed cell line, it is interesting to note that U2OSexpress high levels of MDM2 (65), thus indirectly supportingthe hypothesis that the balance between MDM2 and MDM4plays an important role in the regulation of p53 function. Inagreement with this model, it has been recently reported thatfollowing the activation of growth arrest pathways, MDM4 ispreferentially degraded by MDM2 (25, 26).

The lack in NIH-MDM4 cells of an influence of MDM4 on theinduction of MDM2, a transcriptional target of p53 (66), isapparently in contrast with the reported increase in p53 trans-activation. However, MDM2 expression is reduced or delayedduring p53-induced apoptosis in comparison with a situation ofgrowth arrest (20–22). Moreover, MDM2 induction by p53seems to be particularly sensitive to the function of the p300acetyltransferase transcriptional coactivator (67), and a recentreport showed that MDM4 decreases p300/CREB-binding pro-tein-mediated acetylation of p53 (68).

Ramos et al. (34) observed the overexpression of the MDM4protein in human tumor cell lines expressing WT p53 and sug-gested a possible oncogenic role for such deregulation (34), ahypothesis in apparent contrast with our model. However, it isinteresting to note in that study that the number of samples withconcomitant MDM4 and MDM2 overexpression is notably higherthan that with concomitant overexpression of sole MDM4 andWT p53 (nine versus two samples). These data again support thehypothesis that the balance between MDM4 and MDM2 mayplay a more relevant role than the sole overexpression of MDM4in the regulation of p53. Finally, an oncogenic role of the aberrantisoforms associated with MDM4 overexpression in many of thosetumor samples cannot be ruled out.

In summary, our data provide the first evidence of a positiveregulation of p53 activity by MDM4. In particular, we haveshown that this molecule may play a key role in the activationof the p53-mediated apoptotic function by controlling the in-duction of p53 levels. The absence of apoptosis in MDM4�/�

fibroblasts may be considered in agreement with our findings.Last, our data reveal an additional functional divergence be-tween MDM4 and the related MDM2, thus contributing to theunderstanding of the nonoverlapping regulatory activities ofthese proteins toward p53.

Acknowledgments—We thank Dr. A. J. Levine for anti-MDM2 anti-bodies and C. Schneider for anti-p21 antibody. We thank Dr. A. G.Jochemsen for the anti-MDM4 antibodies and for discussion. We areespecially grateful to Dr. Silvia Bacchetti and to Dr. Silvia Soddu forinvaluable help and discussion.

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