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• W2101003419 abstract "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. 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. 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 (1Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6759) Google Scholar). To ensure a proper cell growth under physiological conditions, p53 function is tightly controlled by maintaining the protein at low levels and partially inactive (1Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6759) Google Scholar, 2Ashcroft M. Vousden K.H. Oncogene. 1999; 18: 7637-7643Crossref PubMed Scopus (373) Google Scholar, 3Oren M. J. Biol. Chem. 1999; 274: 36031-36034Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar). A key molecule in the regulation of p53 basal levels and activity is the MDM2 protein (3Oren M. J. Biol. Chem. 1999; 274: 36031-36034Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar, 4Mendrysa S.M. McElwee M.K. Michalowski J. O'Leary K.A. Young K.M. Perry M.E. Mol. Cel. Biol. 2003; 23: 462-473Crossref PubMed Scopus (194) Google Scholar). MDM4 (MDMX) was identified in 1996 as a p53-binding protein, structurally related to the p53 negative regulator, MDM2 (5Shvarts A. Steegenga W.T. Riteco N. van Laar T. Dekker P. Bazuine M. van Ham R.C. van der Houven van Oordt W. Hateboer G. van der Eb A J. Jochemsen A.G. EMBO J. 1996; 15: 5349-5357Crossref PubMed Scopus (523) Google Scholar). 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 (6Parant J. Chavez-Reyes A. Little W Yan N.A. Reinke V. Jochemsen A.G. Lozano G. Nat. Genet. 2001; 29: 92-95Crossref PubMed Scopus (416) Google Scholar, 7Finch R.A. Donoviel D.B. Potter D. Shi M. Fan A. Freed D.D. Wang C. Zambrowicz B.P. Ramirez-Solis R. Sands A.T. Zhang N. Cancer Res. 2002; 62: 3221-3225PubMed Google Scholar, 8Migliorini D. Denchi E.L. Danovi D. Jochemsen A.G. Capillo M. Gobbi A. Helin K. Pelicci P.G. Marine J.C. Mol. Cel. Biol. 2002; 22: 5527-5538Crossref PubMed Scopus (263) Google Scholar). 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 (6Parant J. Chavez-Reyes A. Little W Yan N.A. Reinke V. Jochemsen A.G. Lozano G. Nat. Genet. 2001; 29: 92-95Crossref PubMed Scopus (416) Google Scholar, 9Jones S.N. Roe A.E. Donehower L.A. Bradley A. Nature. 1995; 378: 206-208Crossref PubMed Scopus (1071) Google Scholar, 10Montes de Oca-Luna R. Wagner D.S. Lozano G. Nature. 1995; 378: 203-206Crossref PubMed Scopus (1211) Google Scholar). 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 (6Parant J. Chavez-Reyes A. Little W Yan N.A. Reinke V. Jochemsen A.G. Lozano G. Nat. Genet. 2001; 29: 92-95Crossref PubMed Scopus (416) Google Scholar, 7Finch R.A. Donoviel D.B. Potter D. Shi M. Fan A. Freed D.D. Wang C. Zambrowicz B.P. Ramirez-Solis R. Sands A.T. Zhang N. Cancer Res. 2002; 62: 3221-3225PubMed Google Scholar, 8Migliorini D. Denchi E.L. Danovi D. Jochemsen A.G. Capillo M. Gobbi A. Helin K. Pelicci P.G. Marine J.C. Mol. Cel. Biol. 2002; 22: 5527-5538Crossref PubMed Scopus (263) Google Scholar) and express high levels of p53 and of its target gene p21, a well known negative regulator of cyclin/cyclin-dependent kinases (11el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7957) Google Scholar, 12Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5250) Google Scholar), 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 (13Gu J. Kawai H. Nie L. Kitao H. Wiederschain D. Yuan Z.M. J. Biol. Chem. 2002; 31: 19251-19254Abstract Full Text Full Text PDF Scopus (212) Google Scholar), 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 (5Shvarts A. Steegenga W.T. Riteco N. van Laar T. Dekker P. Bazuine M. van Ham R.C. van der Houven van Oordt W. Hateboer G. van der Eb A J. Jochemsen A.G. EMBO J. 1996; 15: 5349-5357Crossref PubMed Scopus (523) Google Scholar, 14Jackson M.W. Berberich S.J. Mol. Cell Biol. 2000; 20: 1001-1007Crossref PubMed Scopus (183) Google Scholar, 15Stad R. Ramos Y.F. Little N. Grivell S. Attema J. van der Eb A.J. Jochemsen A.G. J. Biol. Chem. 2000; 275: 28039-28044Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 16Wang X.Q. Arooz T. Siu W.Y. Chiu C.H. Lau A. Yamashita K. Poon R.Y. FEBS Lett. 2001; 16: 202-208Crossref Scopus (76) Google Scholar) and (ii) antagonism of MDM2-driven degradation of the p53 protein (14Jackson M.W. Berberich S.J. Mol. Cell Biol. 2000; 20: 1001-1007Crossref PubMed Scopus (183) Google Scholar, 15Stad R. Ramos Y.F. Little N. Grivell S. Attema J. van der Eb A.J. Jochemsen A.G. J. Biol. Chem. 2000; 275: 28039-28044Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 16Wang X.Q. Arooz T. Siu W.Y. Chiu C.H. Lau A. Yamashita K. Poon R.Y. FEBS Lett. 2001; 16: 202-208Crossref Scopus (76) Google Scholar, 17Sharp D.A. Kratowicz S.A. Sank M.J. George D.L. J. Biol. Chem. 1999; 274: 38189-38196Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 18Stad R. Little N. Xirodimas D.P. Frenk R. van der Eb A.J. Lane D.P. Saville M.K. Jochemsen A.G. EMBO Rep. 2001; 2: 1029-1034Crossref PubMed Scopus (188) Google Scholar, 19Migliorini D. Danovi D. Colombo E. Carbone R. Pelicci P.G. Marine J.C. J. Biol. Chem. 2002; 277: 7318-7323Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). 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. (13Gu J. Kawai H. Nie L. Kitao H. Wiederschain D. Yuan Z.M. J. Biol. Chem. 2002; 31: 19251-19254Abstract Full Text Full Text PDF Scopus (212) Google Scholar), 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 (13Gu J. Kawai H. Nie L. Kitao H. Wiederschain D. Yuan Z.M. J. Biol. Chem. 2002; 31: 19251-19254Abstract Full Text Full Text PDF Scopus (212) Google Scholar). Since MDM2 levels vary within the cell depending upon stress signals of different intensity (20Latonen L. Taya Y. Laiho M. Oncogene. 2001; 20: 6784-6793Crossref PubMed Scopus (111) Google Scholar, 21Saucedo L.J. Carstens B.P. Seavey S.E. Albee II, L.D. Perry M.E. Cell Growth Differ. 1998; 9: 119-130PubMed Google Scholar, 22Wu L. Levine A.J. Mol. Med. 1997; 3: 441-451Crossref PubMed Google Scholar), and MDM4 appears to be preferentially degraded under conditions that activate p53-induced growth arrest (23Gentiletti F. Mancini F. D'Angelo M. Sacchi A. Pontecorvi A. Jochemsen A.G. Moretti F. Oncogene. 2002; 31: 867-877Crossref Scopus (35) Google Scholar, 24Pan Y. Chen J. Mol. Cell Biol. 2003; 23: 5113-5121Crossref PubMed Scopus (198) Google Scholar, 25Kawai H. Wiederschain D. Kitao H. Stuart J. Tsai K.K. Yuan Z.M. J. Biol. Chem. 2003; 278: 45946-45953Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), 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 (13Gu J. Kawai H. Nie L. Kitao H. Wiederschain D. Yuan Z.M. J. Biol. Chem. 2002; 31: 19251-19254Abstract Full Text Full Text PDF Scopus (212) Google Scholar) and to be affected by MDM4 subcellular localization (13Gu J. Kawai H. Nie L. Kitao H. Wiederschain D. Yuan Z.M. J. Biol. Chem. 2002; 31: 19251-19254Abstract Full Text Full Text PDF Scopus (212) Google Scholar, 19Migliorini D. Danovi D. Colombo E. Carbone R. Pelicci P.G. Marine J.C. J. Biol. Chem. 2002; 277: 7318-7323Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In turn, stress conditions as well as p53 activation induce nuclear translocation of overexpressed MDM4 (26Li C. Chen L. Chen J. Mol. Cell Biol. 2002; 22: 7562-7571Crossref PubMed Scopus (68) Google Scholar). 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) 1The 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. 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. 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 (50×; Invitrogen) nonessential amino acids (100×; Invitrogen), BME vitamin solution (100×; 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 (27Stewart N. Bacchetti S. Virology. 1991; 180: 49-57Crossref PubMed Scopus (146) Google Scholar) were cultured in α-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 (23Gentiletti F. Mancini F. D'Angelo M. Sacchi A. Pontecorvi A. Jochemsen A.G. Moretti F. Oncogene. 2002; 31: 867-877Crossref Scopus (35) Google Scholar). 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 pCMVβgal 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 (28Miyashita T. Reed J.C. Cell. 1995; 80: 293-299Abstract Full Text PDF PubMed Scopus (305) Google Scholar) or 800 ng of p21-Luc plasmids plus 0.25 μg of cytomegalovirus β-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 × 102, and 5 × 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 × 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 μlof2× 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. (29Candi E. Oddi S. Paradisi A. Terrinoni A. Ranalli M. Teofoli P. Citro G. Scarpato S. Puddu P. Melino G. J. Invest. Dermatol. 2002; 119: 670-677Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar)), 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-α-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. (30Boyd K.E. Wells J. Gutman J. Bartely S.M. Farnham P.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13887-13892Crossref PubMed Scopus (246) Google Scholar). The chromatin solution was precleared by the addition of Protein G (Pierce) for1hat4 °C and incubated with 2 μgof α-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 (30Boyd K.E. Wells J. Gutman J. Bartely S.M. Farnham P.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13887-13892Crossref PubMed Scopus (246) Google Scholar). 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, 1× 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 (31Bouvard V. Zaitchouk T. Vacher M. Duthu A. Canivet M. Choisy-Rossi C. Nieruchalski M. May E. Oncogene. 2000; 19: 649-660Crossref PubMed Scopus (168) Google Scholar) 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. (32He T.C. Zhou S. da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3256) Google Scholar). 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 (32He T.C. Zhou S. da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3256) Google Scholar). 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 (33Graham F. Prevec L. Methods Mol. Biol. 1991; 7: 109-128PubMed Google Scholar). 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. 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 (23Gentiletti F. Mancini F. D'Angelo M. Sacchi A. Pontecorvi A. Jochemsen A.G. Moretti F. Oncogene. 2002; 31: 867-877Crossref Scopus (35) Google Scholar). 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) (23Gentiletti F. Mancini F. D'Angelo M. Sacchi A. Pontecorvi A. Jochemsen A.G. Moretti F. Oncogene. 2002; 31: 867-877Crossref Scopus (35) Google Scholar). 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. We had previously shown that under normal growth conditions, MDM4 inhibits p53 transcriptional activity in this system (23Gentiletti F. Mancini F. D'Angelo M. Sacchi A. Pontecorvi A. Jochemsen A.G. Moretti F. Oncogene. 2002; 31: 867-877Crossref Scopus (35) Google Scholar). Accordingly, it has been proposed that MDM4 could be a negative regulator of p53 function (7Finch R.A. Donoviel D.B. Potter D. Shi M. Fan A. Freed D.D. Wang C. Zambrowicz B.P. Ramirez-Solis R. Sands A.T. Zhang N. Cancer Res. 2002; 62: 3221-3225PubMed Google Scholar, 8Migliorini D. Denchi E.L. Danovi D. Jochemsen A.G. Capillo M. Gobbi A. Helin K. Pelicci P.G. Marine J.C. Mol. Cel. Biol. 2002; 22: 5527-5538Crossref PubMed Scopus (263) Google Scholar, 19Migliorini D. Danovi D. Colombo E. Carbone R. Pelicci P.G. Marine J.C. J. Biol. Chem. 2002; 277: 7318-7323Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 34Ramos Y.F. Stad R. Attema J. Peltenburg L.T.C. van der Eb A. Jochemsen A.G. Cancer Res. 2001; 61: 1839-1842PubMed Google Scholar, 35Riemenschneider M.J. Buschges R. Wolter M. Reifenberger J. Bostrom J. Kraus J.A. Schlegel U. Reifenberger G. Cancer Res. 1999; 59: 6091-6096PubMed Google Scholar). Since cultured fibroblasts deficient for p53 function have an increased proliferation rate (36Garcia-Cao I. Garcia-Cao M. Martin-Caballero J. Criado L.M. Klatt P. Flores J.M. Weill J.C. Blasco M.A. Serrano M. EMBO J. 2002; 21: 6225-6235Crossref PubMed Scopus (411) Google Scholar, 37Harvey M. Sands A.T. Weiss R.S. Hegi M.E. Wiseman R.W. Pantazis P. Giovanella B.G. Tainsky M.A. Bradley A. Donehower L.A. Oncogene. 1993; 8: 2457-2467PubMed Google Scholar, 38Jones S.N. Sands A.T. Hancock A.R. Vogel H. Donehower L.A. Linke S.P. Wahl G.M. Bradley A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14106-14111Crossref PubMed Scopus (78) Google Scholar), 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 (37Harvey M. Sands A.T. Weiss R.S. Hegi M.E. Wiseman R.W. Pantazis P. Giovanella B.G. Tainsky M.A. Bradley A. Donehower L.A. Oncogene. 1993; 8: 2457-2467PubMed Google Scholar, 38Jones S.N. Sands A.T. Hancock A.R. Vogel H. Donehower L.A. Linke S.P. Wahl G.M. Bradley A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14106-14111Crossref PubMed Scopus (78) Google Scholar). 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 (39Kastan M.B. Onyekwere O. Sindransky D. Vogelstein B. Craig R.W. Cancer Res. 1991; 51: 6304-6311PubMed Google Scholar) and arrests NIH3T3 cells in the G1/G2 phase of cell cycle (23Gentiletti F. Mancini F. D'Angelo M. Sacchi A. Pontecorvi A. Jochemsen A.G. Moretti F. Oncogene. 2002; 31: 867-877Crossref Scopus (35) Google Scholar). Since enhancement of p53 levels is a common finding following Adr stimulus (39Kastan M.B. Onyekwere O. Sindransky D. Vogelstein B. Craig R.W. Cancer Res. 1991; 51: 6304-6311PubMed Google Scholar), 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 sy" @default.
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• W2101003419 title "MDM4 (MDMX) Overexpression Enhances Stabilization of Stress-induced p53 and Promotes Apoptosis" @default.
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