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Originally published In Press as doi:10.1074/jbc.M311793200 on December 1, 2003

J. Biol. Chem., Vol. 279, Issue 9, 8169-8180, February 27, 2004
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MDM4 (MDMX) Overexpression Enhances Stabilization of Stress-induced p53 and Promotes Apoptosis*

Francesca Mancini{ddagger}, Francesca Gentiletti{ddagger}, Marco D'Angelo{ddagger}, Simona Giglio{ddagger}, Simona Nanni{ddagger}§, Carmen D'Angelo¶, Antonella Farsetti{ddagger}||, Gennaro Citro**, Ada Sacchi{ddagger}, Alfredo Pontecorvi{ddagger}{ddagger}{ddagger}, and Fabiola Moretti{ddagger}||§§

From the {ddagger}Laboratory of Molecular Oncogenesis, Laboratory of Experimental Chemotherapy, and **Animal House Service, Regina Elena Cancer Institute, Via delle Messi D'Oro 156, Rome 00158, Italy, {ddagger}{ddagger}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

Received for publication, October 28, 2003 , and in revised form, November 26, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rescue of embryonic lethality in MDM4-/- mice through concomitant loss of p53 has revealed a functional partnership between the two proteins. Biochemical studies have suggested that MDM4 may act as a negative regulator of p53 levels and activity. On the other hand, MDM4 overexpression has been reported to stabilize p53 levels and to counteract MDM2-degradative activity. We have investigated the functional role of MDM4 overexpression on cell behavior. In both established and primary cells cultured under stress conditions, overexpression of MDM4 significantly increased p53-dependent cell death, in correlation with enhanced induction of the endogenous p53 protein levels. This phenomenon was associated with induced p53 transcriptional activity and increased levels of the proapoptotic protein, Bax. Further, p53 stabilization was accompanied by decreased association of the protein to its negative regulator, MDM2. These findings reveal a novel role for MDM4 by demonstrating that in non-tumor cells under stress conditions it may act as a positive regulator of p53 activity, mainly by controlling p53 levels. They also indicate a major distinction between the biological consequences of MDM4 and MDM2 overexpression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
p53 is the most frequently inactivated tumor suppressor gene in human cancer. Following different stress conditions, the p53 protein is stabilized and functionally activated, resulting in two main outcomes: cell cycle arrest or apoptosis (1). To ensure a proper cell growth under physiological conditions, p53 function is tightly controlled by maintaining the protein at low levels and partially inactive (1–3). A key molecule in the regulation of p53 basal levels and activity is the MDM2 protein (3, 4).

MDM4 (MDMX) was identified in 1996 as a p53-binding protein, 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 the double knock-out p53-/- MDM4-/- mouse is alive and develops normally (6–8). The comparison of MDM4-/- and MDM2-/- mice, both characterized by embryonic lethality and rescued by simultaneous knock-out of the p53 gene, has revealed a main difference in the determinants of lethality; MDM4-/- embryos do not develop beyond 7–11 days due to loss of cell proliferation, whereas MDM2-/- embryos die by massive apoptosis at the blastula stage (6, 9, 10). These results, while confirming a role for 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-/- embryo fibroblasts undergo growth arrest in vivo and in vitro (6–8) and express high levels of p53 and of its target gene p21, a well known 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 demonstrated that in the absence of MDM4, MDM2 degrades p53 less efficiently (13), suggesting that the higher p53 levels observed in MDM4-/- mice could be due to impairment of this MDM2 function.

Biochemical studies based on transient overexpression of MDM4 in different cell types, have revealed two distinct activities of the protein toward p53: (i) inhibition of p53 transacting activity (5, 14–16) and (ii) antagonism of MDM2-driven degradation of the p53 protein (14–19). The apparent contradiction between the latter effect and the hypothesis that MDM4 is a negative regulator of p53 levels has been partially solved by Gu et al. (13), who have shown that the effects on p53 levels depend on the relative ratio of MDM4 and MDM2. Antagonism of p53 degradation prevails when MDM4 levels largely exceed those of MDM2, whereas in all of the other conditions, the two proteins cooperate in the degradation of p53 (13). Since MDM2 levels vary within the cell depending upon stress signals of different intensity (20–22), and MDM4 appears to be preferentially degraded under conditions that activate p53-induced growth arrest (23–25), it is reasonable to hypothesize that MDM4 may differentially affect p53 levels in different growth conditions.

The negative regulation of p53 transactivating properties appears to depend on the presence of MDM2 (13) and to be affected by MDM4 subcellular localization (13, 19). In turn, stress conditions as well as p53 activation induce nuclear translocation of overexpressed MDM4 (26). However, the effects of MDM4 on p53 transactivating function under these conditions have not been examined.

In order to investigate the biological consequences of MDM4 on p53 function, we overexpressed MDM4 cDNA in different cells expressing endogenous wild-type (WT)1 p53 and normal levels of MDM2. Our results show that in NIH3T3 cells, stable overexpression 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 that correlates with increased p53 protein levels and transcriptional activity and with increased dissociation of p53 from its negative regulator MDM2. Similarly, overexpression of human MDM4 in human primary cells (human fibroblasts (HF) and human embryo kidney (HEK) cells) causes significant decrease of cell viability correlated with enhanced p53 induction and activity following adriamycin treatment, whereas no effects were observed in MEF p53-/-.

These results provide evidence for a potential new role for MDM4 as a positive regulator of p53 function under stress conditions and indicate a major distinction between MDM4 and MDM2 activities.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture, Plasmids, and Transfections—Mouse NIH3T3 fibroblasts were cultured at 37 °C in F15 medium (minimum essential medium with 26 mM NaHCO3, 2 mg/liter biotin, 10 mM glucose, 4 mM glutamine, essential amino acids (50x; Invitrogen) nonessential amino acids (100x; Invitrogen), BME vitamin solution (100x; Invitrogen)) supplemented with 8% TET system-approved fetal bovine serum (Clontech).

MEF p53-/- (Dr. S. Soddu (CRS-IRE, Rome)) and HF, derived from a foreskin human explant, were cultured in Dulbecco's modified Eagle's medium high glucose supplemented with 10% fetal bovine serum (Hyclone). All experiments were done between passages 8 and 10. HEK cells (27) were cultured in {alpha}-minimum essential medium supplemented with 10% fetal bovine serum (Invitrogen) and used between passages 3 and 5.

NIH3T3 cells stably transfected with MDM4, MDM2, or pTRE were maintained in medium containing 400 µg/ml G418 (23).

Transient transfections were performed by the calcium phosphate precipitation technique or LipofectAMINE for full-length human MDM4 (hMDM4) and p54. Briefly, in 60-mm plates, a fixed number of cells were transfected with 8 µg of hMDM4 or p54 or pCMV{beta}gal or pcDNA3.1 plus 0.8 µg of pEGFP plasmid (Invitrogen), as internal control of transfection efficiency. For transcriptional assays, cells were transfected with 0.5 µg of Bax-Luc (28) or 800 ng of p21-Luc plasmids plus 0.25 µg of cytomegalovirus {beta}-galactosidase plasmid, as internal control of transfection efficiency. Cells were harvested 48 h after transfection, and Luc activity was assayed on whole cell extracts.

Clonogenicity Assay—Different cell numbers (102, 2 x 102, and 5 x 102) were plated in quadruplicate in 6-cm dishes in the presence or absence of 10 µM doxycycline (Dox). Every 3 days, medium was changed, and the Dox dose was renewed. 10 days after plating, dishes were stained by crystal violet (0.25% in methanol) for 10 min and air-dried, and colonies (>=1-mm diameter) were counted.

Proliferation Rate and Cell Cycle Analysis—Cell proliferation rate was assessed by determining cell number in a Thomas's hemocytometer, using trypan blue exclusion as a cell viability test.

Cell proliferation under growth factor deprivation was determined by plating 105 cells in 6-cm dishes; after cell adhesion, culture medium was replaced with medium containing 0.25% fetal calf serum. After 48 h, 10 µM Dox was added. As control, the same cells were grown in the absence of Dox.

Cell cycle profiles were evaluated by fixing 2 x 105 cells in cold ethanol (70%) overnight and staining DNA for 30 min at room temperature with 50 µg/ml propidium iodide in phosphate-buffered saline containing 1 mg/ml RNase A. Percentages of cells in the different phases of the cycle were measured by flow cytometric analysis of propidium iodide-stained nuclei 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 temperature and permeabilized in 0.1% Triton X-100, 0.1% sodium citrate for 2 min on ice. Apoptotic nuclei were detected using a TUNEL labeling reaction according to the manufacturer's instructions (Roche Applied Science). TUNEL labeling and phase contrast images were analyzed by the AXIO VISION version 3.0 program.

Immunoprecipitation and Western Blot Analysis—For immunoprecipitation 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) containing a mixture of protease inhibitors (Roche Applied Science), and whole cell extracts were centrifuged at 14,000 rpm for 30 min to remove cell debris. Protein concentration was determined by a colorimetric assay (Bio-Rad). Immunoprecipitations were performed by incubating whole cell extracts with the indicated antibody, preincubated with protein G-Sepharose (Pierce), under gentle rocking at 4 °C overnight. Immunoprecipitates were washed three times with Giordano's buffer supplemented with protease inhibitors, resuspended in 40 µlof2x SDS Laemmli sample buffer, and then resolved by SDS-PAGE. For Western blot analysis, cells were lysed in radioimmune precipitation buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA) supplemented with a mixture of protease inhibitors (Roche Applied Science). Whole lysates were resolved by SDS-PAGE and subsequently transferred onto polyvinylidene difluoride membranes (Millipore). Before immunoblotting, membranes were stained with Ponceau Red to ensure equal protein loading. After blocking, membranes were incubated for 2 h using the following primary antibody: anti-MDM4 monoclonal antibody (6B1A, 114FD, and 12G11G), anti-p54 rat polyclonal antibody (raised against full-length p54, according to Candi et al. (29)), anti-p21 polyclonal antibody specific for the mouse protein (kindly provided from Dr. C. Schneider), anti-p21 F5 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 polyclonal antibody, N-20 (Santa Cruz Biotechnology), anti-MDM2 monoclonal antibody 2A10, anti-{alpha}-tubulin monoclonal antibody, DM1A (Sigma), and anti-Hsp70 mouse monoclonal antibody SPA-820. Membranes were developed using ECL (Amersham Biosciences).

Formaldehyde Cross-linking and Immunoprecipitation of Chromatin—Cells grown as previously described were washed twice with phosphate-buffered saline and cross-linked as described in Boyd et al. (30). The chromatin solution was precleared by the addition of Protein G (Pierce) for1hat4 °C and incubated with 2 µgof {alpha}-p53 (Ab 7), anti-IgG, or no antibody overnight at 4 °C with mild shaking. Before use, Protein G was blocked with 1 µg/µl sonicated salmon sperm DNA and 1 µg/µl bovine serum albumin for 2 h at 4 °C. Chromatin immunoprecipitation, washing, and elution of immune complexes were carried out as previously described (30). DNA fragments were recovered by centrifugation and resuspended in 30 µl of double-distilled H2O and analyzed by PCR. Total input sample was resuspended in 100 µl of double-distilled H2O and then diluted 1:100 before PCR. Each reaction mixture contained 0.5–1 µl of immunoprecipitated chromatin, 70 ng of each primer, 250 µM deoxynucleoside triphosphates (Roche Applied Science), 2 mM MgCl2, 1x Taq reaction buffer, and 1.25 units of Taq polymerase (ABgene House, Epsom, UK) in a final volume of 30 µl. After 32–35 cycles of amplification, PCR products were run on a 2% agarose gel and analyzed by ethidium bromide staining. For PCR analysis of the p21 and bax promoters, the following oligonucleotides spanning the p53 binding elements (31) were used: p21-up, 5'-GAG TTT GTG TGG AGG TGA CTT CTT C-3'; p21-down, 5'-CTG GTA GTT GGG TAT CAT CAG GTC 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 PCR analysis of the tubulin gene, the following oligonucleotides were used: tub-up, 5'-GCA CTC TGA TTG TGC CTT CA-3'; tub-down, 5'-AGC AGG CAT TGG TGA TCT CT-3'. The linearity of the PCRs was verified by analyzing (i) a 10-fold dilution of the DNA samples and (ii) PCR products obtained from increasing amplification cycles.

Adenovirus Generation and Infection—The strategy to create recombinant adenovirus was as previously described by He et al. (32). The BamHI fragment of cDNA coding for mouse MDM4 (nucleotides 171–1645) was cloned into the pAdShuttle-CMV (Stratagene). The resulting construct and the control construct (pAdTrack-CMV-ATCC, carrying the cDNA sequence of the green fluorescent protein gene) were linearized with PmeI and transfected by electroporation, together with pAdEasy1 vector (Stratagene) in electrocompetent E. coli BJ5183 cells (Stratagene). Recombinant colonies were selected with kanamycin and screened by restriction endonuclease digestion. The resulting recombinant adenoviral constructs were digested by PacI and transfected into the packaging cell line 293A (Invitrogen) using the LipofectAMINE Plus protocol (Invitrogen). Transfected cells were collected 10–12 days after transfection, and the viral lysates were obtained as previously described (32).

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

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



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

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Stable MDM4 Overexpression Does Not Affect p53 Levels or Change the Proliferative Capability of NIH3T3 Cells—In order to analyze the functional effects of MDM4 overexpression, we used immortalized NIH3T3 mouse fibroblasts, expressing WT p53, stably transfected with MDM4 cDNA under control of a tetracycline-inducible promoter (Tet-ON). Following 24-h treatment with 10 µM Dox, several clones were isolated and screened for MDM4 expression (NIH-MDM4), as previously reported (23). As a control, a mixed population of NIH3T3 cells stably transfected with the pTet-ON coding plasmid and the pTRE empty vector, was used (NIHpTRE) (23).

First, we investigated the effects of stable MDM4 overexpression on the endogenous p53 levels. Analysis of p53 protein in NIH-MDM4 clones (MX-18 and MX-24) at different time points following Dox addition did not show changes in p53 levels during cell growth or differences in comparison with NIHpTRE cells (Fig. 1A), indicating that MDM4 overexpression does not alter p53 basal levels in NIH3T3.



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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 indicated time. Cell lysates were then collected and analyzed by Western blot using the anti-MDM4 monoclonal antibody 6B1A. p53 levels were detected by immunoprecipitating (Ip) 200 µg of total cell lysate with anti-p53 monoclonal antibody 421 and further Western blot analysis (WS) with anti-p53 polyclonal antibody 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 the presence (B) or in the absence (C)of10 µM Dox. Every 3 days, medium was changed, and the Dox dose was renewed. Viable cells were determined by trypan blue 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 µM. 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 (>=1 mm 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 was statistically evaluated by a paired one-sided t test.

 
We had previously shown that under normal growth conditions, MDM4 inhibits p53 transcriptional activity in this system (23). Accordingly, it has been proposed that MDM4 could be a negative regulator of p53 function (7, 8, 19, 34, 35). Since cultured fibroblasts deficient for p53 function have an increased proliferation rate (36–38), the growth of MDM4-expressing clones was compared with that of NIHpTRE control cells. In the presence of Dox, all cells had a similar proliferation rate (Fig. 1B) and fraction of viable cells (data not shown), indicating that overexpression of MDM4 does not alter these parameters. At confluence, whereas NIHpTRE cells remained viable, the NIH-MDM4 clones (MX-18 and MX-24) exhibited a reproducible reduction of viability, a phenomenon not observed in the absence of Dox (Fig. 1, compare B with C). It has also been reported that p53 inactivation correlates with increased ability of cells to survive and proliferate when plated at low densities (37, 38). Therefore, a colony-forming assay with different NIH-MDM4 clones was performed. Cells at different densities were plated and, every 3 days, refed with fresh medium with Dox. Surprisingly, at the lowest density (100 cells/6-cm dish), MDM4 overexpression significantly reduced colony formation in comparison with NIHpTRE control cells (Fig. 1D).

All of these data show that MDM4 overexpression per se does not alter the growth properties of NIH3T3 cells or affect endogenous p53 protein basal levels and rather suggest that it might increase cell susceptibility to stress conditions.

MDM4 Overexpression Stabilizes Transcriptionally Active p53—To ascertain whether MDM4 does indeed affect cell viability under stress conditions, we analyzed the cell response following treatment with Adriamycin (Adr), a DNA damage-inducing drug that activates the tumor suppressor p53 (39) and arrests NIH3T3 cells in the G1/G2 phase of cell cycle (23). Since enhancement of p53 levels is a common finding following Adr stimulus (39), we first tested the effects of MDM4 overexpression on p53 levels.

Adr (0.9 µM) was added 16 h after induction of MDM4 by Dox, and p53 protein levels were evaluated. Indeed, Adr treatment increased p53 levels in both NIHpTRE and MX-18 cells (Fig. 2A), but more substantially and for a longer period of time in the latter, indicating that MDM4 overexpression enhances p53 levels under these conditions. We then tested whether the induced p53 protein retained transcription activating competence. We performed transient transfection assays using the p53-responsive promoters of the bax and p21 genes. MDM4 expression was induced 8 h before transfection, cells were then transfected and treated with adriamycin for 24 h before lysis. In all NIH-MDM4 clones, the addition of doxycycline caused a reproducible increase of p21 and more strongly of bax promoter activity (Fig. 2B), whereas in NIHpTRE control cells, it did not cause further induction of either promoter. Overexpression of MDM2, achieved through the same inducible system (M2–23 clone) (23), caused inhibition of both promoters, as expected (40), indicating that the induction observed in NIH-MDM4 clones is specifically related to MDM4 overexpression.



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  		FIG. 2.
Effects of MDM4 on p53 levels and activity. A, 200 µg of whole cell extract, from NIHpTRE and MX-18 cells, treated with Adr as described under "Results," were immunoprecipitated (Ip) with anti-p53 monoclonal antibody 421 and analyzed by Western blot (WS) with the anti-p53 polyclonal antibody, Ab 7. B, NIHpTRE, MX-18, MX-24, and M2–23 cells were plated in quadruplicate and cultured in the absence or presence of Dox, and 8 h later, they were transiently transfected by the calcium phosphate method. After removal of calcium phosphate precipitates, cells were cultured for 48 h. Adr was added 24 h before lysis. The histogram shows the ratio offold induction of luciferase activity(corrected for {beta}-galactosidase) from cells cultured in the presence or absence of Dox. The values represent the mean of two different experiments in 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 indicated and formaldehyde-cross-linked, and the chromatin was immunoprecipitated with anti-p53 Ab 7 antibody or an anti-IgG unspecific antibody. The histograms represent the relative signal intensities of the PCR products using specific primers for p21 (C) or bax (D) promoters. PCR products resolved by electrophoresis were stained with ethidium bromide and quantified by densitometry. All signal intensities were corrected for the corresponding signal obtained from PCR of the tubulin gene. The signal intensity obtained from PCR of chromatin from cells grown under normal conditions was set to 1 (white bars). The graphs are representative of at least three independent PCR amplifications of two independent sets of DNA preparations. 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 analyzed by Western blot using the anti-Bax polyclonal antibody N-20, anti-p21 polyclonal antibody, anti-MDM4 monoclonal antibody 6B1A, or anti-tubulin DM1A as loading control (LC).

 
These observations lead us to conclude that stable MDM4 overexpression differentially affects p53 levels under normal or stress conditions of growth in NIH3T3 cells, by increasing the levels of activated p53. Further, the induced p53 appears to retain its transactivating capability and particularly is functional in the activation of its proapoptotic target, Bax.

To confirm this, the binding of p53 to endogenous p21 and bax promoters was tested by chromatin immunoprecipitation assay. Under normal growth conditions and at different time points after adriamycin treatment, cells were formaldehyde-cross-linked, and chromatin was immunoprecipitated by p53 antibody Ab 7 or an unrelated antibody as control. PCR amplification of the immunoprecipitated DNAs with specific primers for the p53-binding regions, revealed an increasing signal of the p21 promoter in both NIH-MDM4 clones and in control cells although delayed in the first (Fig. 2C). On the contrary, analysis of the bax promoter fragment showed increased signal only in the MDM4-expressing cells (Fig. 2D). Thus, following Adr treatment, p53 was indeed recruited onto the endogenous p21 promoter in both NIH-MDM4 clones and in control cells, whereas it was recruited onto the bax promoter exclusively in NIH-MDM4-expressing clones, in agreement with results on p53 transcriptional activity (Fig. 2B).

To definitely confirm these data, the protein levels of p21 and Bax were analyzed by Western blot. Adriamycin treatment caused a progressive increase in Bax levels in NIH-MDM4 clones over 24 h, whereas in control cells, Bax levels were stable (Fig. 2E), in good agreement with previous results (see Fig. 2D). Levels of p21 analyzed in the same cell lysates were increased in all populations (Fig. 2E) and were not increased further by p53 stabilization. Analysis of MDM4 levels under the same conditions did not show evident modifications (Fig. 2E).

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

MDM4 Overexpression Causes Cell Apoptosis under Stress Conditions—Since following Adr treatment, MDM4 stabilizes functional p53 and causes Bax induction, we tested whether cell growth of NIH-MDM4 was affected under these conditions. A decrease in cell viability was observed in MDM4-overexpressing clones after as little as 4 h of treatment and became more pronounced at later times (Fig. 3A). In comparison, NIHpTRE cells did not show a significant decrease in viability. A TUNEL assay further showed the presence of apoptotic cells only in MDM4-expressing clones (Fig. 3B). These data clearly indicate that MDM4 overexpression causes an apoptotic response, following Adr treatment.



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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 the presence of 10 µM Dox. 16 h later, Adr (0.9 µM) 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 bars represent the mean of three different experiments in duplicate, and lines indicate S.D. values. B, merge of TUNEL immunofluorescence and phase-contrast microscopy of NIHpTRE, MX-18, and MX-24 cells grown in the presence of 10 µM Dox and 0.9 µM Adr for 4 h. TUNEL-positive cells show 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 µM 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 µM was added, and at the indicated times cells were collected for fluorescence-activated cell sorting analysis. The histogram represents the fraction of cells in the sub-G1 area calculated as a percentage of G1 + S + G2. F, fluorescence-activated cell sorting analysis of cells grown as described for E.

 
MDM4-/- fibroblasts are blocked at the G0/G1 phase of cell cycle and express high levels of p21 (6–8). This phenotype is rescued by concomitant knock-out of the p53 gene, leading to the hypothesis that MDM4 interferes with p53-mediated growth arrest (8). We had previously shown that MDM4 overexpression down-regulates basal levels of p21 (see Fig. 2E). To ascertain whether the apoptosis associated with MDM4 overexpression is not related to inhibition of growth arrest, we analyzed 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 medium containing 0.25% fetal calf serum had a reduced proliferation rate. Doxycycline addition caused a reproducible reduction in cell number in NIH-MDM4 cells, whereas NIHpTRE cells were not affected (Fig. 3, compare C and D). Analysis of cell cycle profiles showed a significant increase in the sub-G1 area in NIH-MDM4 compared with NIHpTRE cells (Fig. 3E), confirming that MDM4 overexpression decreases cell viability in these conditions too. However, NIH-MDM4 as well as control cells showed a progressive and comparable accumulation of cells in the G1 phase (Fig. 3F). Similarly to Adr treatment, p53 levels were increased in NIH-MDM4-expressing cells relative to NIHpTRE cells following serum starvation (data not shown).

These data confirm that under stress conditions, MDM4 overexpression decreases cell survival without interfering with growth arrest.

The COOH-terminal Domain of MDMX Is Necessary for p53 Stabilization and Apoptosis—Previous reports have shown that deletion mutants of MDM4, lacking the ring finger domain are unable to stabilize exogenously expressed p53 (15, 19). To confirm that stabilization of p53 by MDM4 is the main mediator of the apoptotic phenotype, we transiently transfected NIH3T3 with a deleted MDM4 construct. We used a naturally occurring isoform named p54 that we had previously characterized (23). p54 derives from caspase cleavage of full-length MDM4 and is devoid of the carboxyl-terminal amino acids 361–489 of the WT protein. The expression of p54 in comparison with control vector, {beta}-galactosidase, did not alter p53 basal levels; nor did it induce further increase of p53 levels following Adr treatment (Fig. 4A). Concomitantly, the analysis of cell viability did not evidence a significant alteration of the growth properties of NIH3T3 cells (Fig. 4B). These data confirm that the stabilization of p53 induced by MDM4 overexpression is the main factor responsible for the activation of the apoptotic response Moreover, the COOH terminus region is necessary for this effect. The analysis of p21 protein levels showed a parallel increase in both cell populations (Fig. 4A), indicating indirectly that p53 is transcriptionally active in {beta}-galactosidase as well as in p54-transfected cells. Since p54 contains the MDM4 p53 binding domain and is able to repress p53 transcriptional activity under normal growth conditions (data not shown), these data would confirm that MDM4 is ineffective in repressing p53 transcriptional function following p53 activation by Adr.



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  		FIG. 4.
Effects of overexpression of p54 on growth properties of NIH3T3. A, the same number of NIH3T3 cells were plated, and the expression vectors for p54 or {beta}-galactosidase ({beta}-gal) as control were transfected by LipofectAMINE. 24 h later, Adr (0.9 µM) was added, and at different time points as indicated, cell lysates were collected and analyzed by Western blot using anti-p54 polyclonal antibody, anti-{beta}-galactosidase monoclonal antibody, anti-p53 Ab 7 polyclonal antibody, 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 the indicated times, cell viability was evaluated by trypan blue exclusion. The number of starting viable cells was set to 100, and the following determinations were calculated as a percentage of this number. The bars represent the means of two different experiments in duplicate, and lines indicate S.D. values.

 
MDM4 Reduces the Association between MDM2 and p53 following Adr Treatment—Stabilization of p53 by MDM4 has been reported by other authors, and different hypotheses have been proposed to explain its mechanism. Squelching of MDM2 binding to p53 has been observed when MDM4 largely exceeds MDM2 (13). Our previous data indicate that the presence of MDM4 p53 binding domain is not sufficient to induce such stabilization and that the COOH terminus, where the MDM2 binding domain resides (44), plays a role. We therefore investigated which mechanisms underlie the p53 enhancement observed in our system. Since different MDM4 levels could differently alter the composition of the immunocomplexes, all cell lysates were collected at the same time after Dox induction by adding adriamycin according to the schedule in Fig. 5A.



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  		FIG. 5.
Effects of stable MDM4 overexpression on MDM2/p53 association in NIH3T3 under different conditions. A, schematic diagram of cell lysate collection following different Adr exposure times. B and C, 400 µg of whole cell extract, collected from NIHpTRE (B) and MX-18 cells (C), as schematized in A, were immunoprecipitated (Ip) with anti-p53 monoclonal 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. D, lysates collected from NIHpTRE and MX-18 cells as schematized in Fig. 5A were immnunoprecipitated with anti-MDM2 monoclonal antibody 2A10. The relative supernatants were analyzed by Western blot with anti-p53 polyclonal antibody Ab 7 or anti-tubulin DM1A as loading control (LC). E, lysates collected from NIHpTRE and MX-18 cells as schematized in A were analyzed by Western blot using the anti-MDM2 monoclonal antibody 2A10 and anti-tubulin DM1A as loading control (LC). F, 400 µg of whole cell extract, collected from MX-18 cells grown under subconfluent conditions, were immunoprecipitated with anti-p53 monoclonal antibody 421 and analyzed by Western blot with the antip53 polyclonal antibody Ab 7, anti-MDM4 monoclonal antibody 6B1A, and anti-MDM2 monoclonal antibody 2A10.

 
p53 was immunoprecipitated by Ab 1, and the MDM2 protein present in the immnunocomplexes was analyzed by Western blot. In NIHpTRE control cells, following Adr treatment, the amount 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 with p53 was comparably lower if considering the higher levels of p53 observed in these cells and further decreased during Adr treatment (Fig. 5C). The fraction of MDM4 associated with p53 in the same immunocomplexes did not vary. To confirm that a higher fraction of p53 was free from MDM2 inhibition, the same lysates were depleted of the MDM2-bound p53 fraction by immunoprecipitation of the MDM2-associated complex with {alpha}-MDM2 antibody 2A10, and the levels of p53 in the supernatant were analyzed. As expected, higher levels of p53 were observed in the MX-18 cell lysates in comparison with NIH3T3 lysates (Fig. 5D). A further immunoprecipitation of the same supernatants with a-MDM2 2A10 did not reveal detectable levels of MDM2, indicating that the differences in p53 were not caused by different amounts of MDM2 remaining from the first immunoprecipitation (data not shown).

Thus, under conditions that activate p53 function, overexpression of MDM4 causes a decreased association of MDM2 with p53, resulting in an increased fraction of p53 not subjected to MDM2-mediated inhibition. However, the decreased association of MDM2 with p53 is not accompanied by a proportional increase in MDM4 association with p53, indicating that a mechanism other than physical squelching is functioning and confirming previous data from p53 transfection (see Fig. 4A).

Western blot analysis of MDM2 in the same lysates assayed by immunoprecipitation demonstrated that MDM2 protein levels were similarly increased in both NIHpTRE and MX-18 cells following adriamycin treatment (Fig. 5E), thus excluding the possibility that the minor fraction of MDM2 associated with p53 in MX-18 cells is mainly imputable to lower levels of total MDM2. Under normal growth conditions, the fraction of the MDM2 molecules bound to p53 was not decreased by MDM4 overexpression (Fig. 5F), indicating that under these conditions, MDM4 is ineffective in counteracting p53 degradation and confirming that the positive role played by MDM4 toward p53 takes place only under stress conditions.

MDM4 Overexpression Causes p53-mediated Apoptosis in Different Nontransformed Cell Contexts—The above results were obtained in the NIH3T3 cell line. We therefore asked whether the phenotype observed following MDM4 overexpression is limited to this cell context or is common to other non-transformed cells, particularly those of human origin. To this end, we transiently overexpressed hMDM4 in two human primary cell strains: HF and HEK. 16 h after transfection, adriamycin was added, and at different times cell viability was evaluated. In both cell strains, overexpression of hMDM4 significantly reduced cell viability in comparison with control cells, either transfected with the empty vector (Fig. 6, A and C, CTRL) or a {beta}-galactosidase coding plasmid (data not shown), confirming the results obtained in NIH3T3 cells. Western blot analysis of cell lysates collected at the same time points showed, as expected, the induction of p53 levels by adriamycin treatment in control cells. Notably, hMDM4 overexpression caused a further enhancement of p53 levels (Fig. 6, B and D). This effect was more pronounced in the HEK cells, which showed concomitantly a stronger decrease in cell viability. Conversely, transient overexpression of MDM4 in mouse embryonic fibroblast knock-out for the p53 gene (MEF p53-/-) did not alter cell viability following adriamycin treatment (Fig. 6E), despite the strong and persistent expression of MDM4 obtained in this cell strain (Fig. 6, compare F with B and D). Thus, the consequences of MDM4 overexpression represent a common finding also in human cells and are mainly imputable to the effects on p53, as indicated by the data from MEF p53-/-.



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  		FIG. 6.
Effects of transient MDM4 overexpression in nontransformed cells. A, C, and E, the same number of HF (A), HEK (C), and p53-/- MEF cells (E) were plated, and MDM4 or control (CTRL) expression vector was transfected by LipofectAMINE 2000. 16 h later, Adr (0.9 µM) was added, and at different time points as indicated, 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 bars represent the mean of at least two different experiments in duplicate, and lines indicate S.D. values. The asterisks indicate significantly different values. The significance of the data was 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 as described for A were analyzed 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).

 
We then asked whether the enhancement of p53 levels is caused by the overexpression of MDM4 per se or whether levels of MDM4 are relevant for this phenomenon. To this end, we generated recombinant adenovirus carrying MDM4 cDNA and infected human HEK and HF cells with increasing doses (50–1000 plaque-forming units/cell) of this adenovirus. Cells infected with an adenovirus carrying enhanced green fluorescent protein (AdCTRL) were used as a control. 24 h after infection, cells were treated with Adr, and after a further 24 h, viable cells were counted. Under normal conditions of growth, infection of HEK cells with AdMDM4 did not significantly change cell viability in comparison with cells infected with AdCTRL (Fig. 7A). Conversely, in the presence of Adr, a progressive decrease of cell viability more pronounced than with AdCTRL was observed with increasing doses of AdMDM4 (Fig. 7B). This decrease was significant only at the higher doses of infection (see asterisks in Fig. 7B) and correlated with increasing amounts of MDM4 expressed in the cells (the higher exposure of the film showed detectable levels of MDM4 also at 50 plaque-forming units/cell; data not shown) and with concomitant increase of p53 (Fig. 7C). In comparison, p53 levels in HEK cells infected with AdCTRL were increased by Adr treatment but no further by increasing the multiplicity of infection (Fig. 7D). The analysis of p21 protein showed an increase in both cell types following Adr treatment. On the contrary, Bax levels were increased only in AdMDM4 cells and at the higher doses of infection in association with the higher levels of p53.

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

Moreover, these data confirm that following Adr treatment, overexpressed MDM4 allows the induction of the proapoptotic Bax 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 with AdCTRL cells. Comparison of p21 and Bax levels in these lysates on a same gel confirmed these data (data not shown). These results are in accord with data from NIH3T3 cells (see Fig. 2E) and confirm that in the absence of stress, MDM4 contributes to inactivate p53 transcriptional function. Comparable data were obtained in HF cells (Fig. 7, E and F).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Animal models have provided evidence for a functional link between MDM4 and p53 (6–8). Particularly, the rescue of MDM4-/- embryo lethality by simultaneous knock-out of the p53 gene has suggested a role for MDM4 as negative regulator of p53 function under physiological conditions.

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

On the other hand, our results demonstrate that under different stress conditions such as Adr treatment and growth factor deprivation, MDM4 overexpression causes cell death. This event correlates with higher and prolonged enhancement of the p53 protein levels, concomitant increase in its transcriptional function, and increased levels of the proapoptotic protein Bax. This phenotype was confirmed by transient overexpression of MDM4 in other cell systems, suggesting that cell death associated with MDM4 overexpression is a common finding in a nontransformed cell context. These effects are strictly dependent on the p53 protein, as demonstrated by the lack of response in MEF p53-/- cells.

The mechanisms by which MDM4 activates p53 apoptotic function seem to rely mainly on the stabilization of p53 levels (45, 46). Particularly, the experiments we performed in NIH3T3 cells show that MDM4 overexpression counteracts MDM2 binding to p53. Since this binding is necessary for the degradation of the p53 protein (47, 48), the interference by MDM4 on p53-MDM2 association appears to be a reasonable cause for p53 stabilization. However, our results do not support the model of physical squelching (13); (i) MDM4 binding to p53 does not increase correspondingly with the decrease of MDM2, and (ii) the p54 form that lacks the ring finger domain is unable to cause p53 stabilization. A role of MDM4 in the recruitment of other factors causing MDM2 dissociation and stabilization of p53 is under study.

In agreement with our results, it has been demonstrated that the inhibition of MDM2-driven degradation of p53 preferentially produces activation of the p53 apoptotic function (49). Moreover, we observed that the levels of p21/Waf1/Cip1, the major determinant of growth arrest, were increased to a similar extent in MDM4-overexpressing and control cells, indicating that MDM4-induced apoptosis is not caused by inhibition of cell cycle arrest (50, 51). On the other hand, the molecular mechanisms that underlie the activation of the apoptotic phenotype and particularly the Bax protein in our system remain to be elucidated. It has been reported that the p53 family member, p73, actively cooperates with p53 in the induction of apoptosis (52, 53). Since MDM4 stabilizes p73 (54), a modification of the p73 levels by MDM4 could contribute to this induction. To date, however, we have been unable to detect endogenous p73 in our system.

Previous studies have shown an inhibitory activity of MDM4 toward p53 transcriptional function. So far, evidence of MDM4 activity on p53 transactivating function under stress conditions has not been reported. Recently, it has been shown in p53-/-MDM2-/- MEF that inhibition of the p53 transcriptional activity by MDM4 requires cooperation of MDM2, an observation that indicates that MDM4 per se is not able to exert such inhibition 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 alleviate MDM4 inhibitory activity. Indeed, following p53 induction by Adr, we do not observe a concomitant increase of MDM4 binding to p53, in analogy to what was reported for MDM2 (40). The lack of inhibitory activity toward p53 by p54, under the same conditions, also supports this hypothesis.

We propose that, in normal cells, stress may abrogate the cooperation between MDM2 and MDM4 in inhibiting p53 transcriptional activity. Under these conditions, in the presence of higher levels of MDM4 with respect to MDM2, the ability of MDM4 to antagonize the MDM2-dependent degradation of p53 would prevail determining a net increase in p53 induction as observed in NIH-MDM4 cells. Recently, Li et al. (26) reported in U2OS cells the lack of changes in cell cycle distribution by overexpressed MDM4 after stress treatment (26). Although these data are not entirely comparable with ours, since they used a transformed cell line, it is interesting to note that U2OS express high levels of MDM2 (65), thus indirectly supporting the hypothesis that the balance between MDM2 and MDM4 plays an important role in the regulation of p53 function. In agreement with this model, it has been recently reported that following the activation of growth arrest pathways, MDM4 is preferentially degraded by MDM2 (25, 26).

The lack in NIH-MDM4 cells of an influence of MDM4 on the induction of MDM2, a transcriptional target of p53 (66), is apparently in contrast with the reported increase in p53 transactivation. However, MDM2 expression is reduced or delayed during p53-induced apoptosis in comparison with a situation of growth arrest (20–22). Moreover, MDM2 induction by p53 seems to be particularly sensitive to the function of the p300 acetyltransferase transcriptional coactivator (67), and a recent report showed that MDM4 decreases p300/CREB-binding protein-mediated acetylation of p53 (68).

Ramos et al. (34) observed the overexpression of the MDM4 protein in human tumor cell lines expressing WT p53 and suggested a possible oncogenic role for such deregulation (34), a hypothesis in apparent contrast with our model. However, it is interesting to note in that study that the number of samples with concomitant MDM4 and MDM2 overexpression is notably higher than that with concomitant overexpression of sole MDM4 and WT p53 (nine versus two samples). These data again support the hypothesis that the balance between MDM4 and MDM2 may play a more relevant role than the sole overexpression of MDM4 in the regulation of p53. Finally, an oncogenic role of the aberrant isoforms associated with MDM4 overexpression in many of those tumor samples cannot be ruled out.

In summary, our data provide the first evidence of a positive regulation of p53 activity by MDM4. In particular, we have shown that this molecule may play a key role in the activation of the p53-mediated apoptotic function by controlling the induction 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 between MDM4 and the related MDM2, thus contributing to the understanding of the nonoverlapping regulatory activities of these proteins toward p53.


   FOOTNOTES
 
* This work was supported by research grants from Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR), Ministero della Salute, Progetto "Oncologia" MIUR-CNR, and Associazione Italiana Ricerca sul Cancro. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported by a Fondazione Italiana Ricerca Cancro fellowship. Back

§§ To whom correspondence should be addressed: Laboratory of Molecular Oncogenesis, Regina Elena Cancer Institute, Via delle Messi D'Oro 156, Rome 00158, Italy. Tel.: 39-06-52662531; Fax: 39-06-4180526; E-mail: moretti{at}ifo.it.

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


   ACKNOWLEDGMENTS
 
We thank Dr. A. J. Levine for anti-MDM2 antibodies and C. Schneider for anti-p21 antibody. We thank Dr. A. G. Jochemsen for the anti-MDM4 antibodies and for discussion. We are especially grateful to Dr. Silvia Bacchetti and to Dr. Silvia Soddu for invaluable help and discussion.



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