the mdm2 oncoprotein promotes apoptosis in p53- deficient...

The MDM2 Oncoprotein Promotes Apoptosis in p53-Deficient Human Medullary Thyroid Carcinoma Cells*

TATIANA DILLA, JUAN A. VELASCO†, DIEGO L. MEDINA,J. FERNANDO GONZALEZ-PALACIOS, AND PILAR SANTISTEBAN

Instituto de Investigaciones Biomedicas Alberto Sols, Consejo Superior de Investigaciones Cientıficas,Universidad Autonoma de Madrid (T.D., J.A.V., D.L.M., P.S.), 28029 Madrid; and the Department ofPathology, Hospital Ramon y Cajal, Universidad de Alcala de Henares (J.F.G-P.), 28034Madrid, Spain

ABSTRACTThe MDM2 oncoprotein has been shown to inhibit p53-mediated

growth arrest and apoptosis. It also confers growth advantage todifferent cell lines in the absence of p53. Recently, the ability of MDM2to arrest the cell cycle of normal human fibroblasts has also beendescribed. We report a novel function for this protein, showing thatoverexpression of MDM2 promotes apoptosis in p53-deficient, humanmedullary thyroid carcinoma cells. These cells, devoid of endogenousMDM2 protein, exhibited a significant growth retardation after stabletransfection with mdm2. Cell cycle distribution of MDM2 transfec-tants [medullary thyroid tumor (MTT)-mdm2] revealed a fraction ofthe cell population in a hypodiploid status, suggesting that MDM2 issufficient to promote apoptosis. This circumstance is further demon-strated by annexin V labeling. MDM2-induced apoptosis is partially

reverted by transient transfection with p53 and p19ARF. Both MTTand MTT-mdm2 cells were tumorigenic when injected into nude mice.However, the percentage of apoptotic nuclei in tumor sections derivedfrom MDM2-expressing cells was significantly higher relative to thatin the parental cell line. MDM2-mediated programmed cell death isat least mediated by a down-regulation of the antiapoptotic proteinBcl-2. Protein levels of caspase-2, which are undetectable in the pa-rental cell line, appear clearly elevated in MTT-mdm2 cells.Caspase-3 activation does not participate in MDM2-induced apopto-sis, as determined by protein levels or poly(ADP-ribose) polymerasefragmentation. The results observed in this medullary carcinoma cellline show for the first time that the product of the mdm2 oncogenemediates cell death by apoptosis in p53-deficient tumor cells. (Endo-crinology 141: 420–429, 2000)

MEDULLARY THYROID carcinoma (MTC) is a neu-roendocrine tumor of the parafollicular C cells that

accounts for up to 10% of all thyroid tumors (1). One fourthof all MTC appear to be genetically determined and areassociated with inherited clinical syndromes (multiple en-docrine neoplasia 2A and 2B and familial MTC). The re-maining cases of MTC are sporadic and therefore occur as theconsequence of somatic alterations caused by both geneticand epigenetic factors (2). Established cell lines from humanand animal MTC tumors provide a valuable system to an-alyze genes involved in the development of this neoplasia.Human medullary thyroid tumor cells (MTT), recently char-acterized in our laboratory (3), show all of the major prop-erties described for MTC cells. They immunoreact with spe-cific calcitonin antibodies (our unpublished observations)and express somatostatin and somatostatin receptors 2, 3, 4,and 5 (4). The transformed phenotype of these cells is at leastdue to a loss of expression of the tumor suppressor gene p53and a genetic deletion involving exon 11 of the ret protoon-cogene (3).

The oncogenic potential of the murine double-minute-2(mdm2) gene was originally detected in spontaneously trans-formed murine fibroblasts (5). Thereafter, genetic amplifica-tion of the mdm2 gene was detected in different human tu-mors and cell lines (6, 7). More recently, the oncogenicfunction of the mdm2 gene product (MDM2) has also beendetermined in transgenic mice expressing MDM2 in themammary gland. These animals, which show major alter-ations of the cell cycle, have a high incidence of breast tumors(8). Coimmunoprecipitation experiments determined thatMDM2 physically interacts with the p53 tumor suppressorgene product (9), leading to the idea that, as described forproteins such as the simian virus 40 large T antigen or thepapillomavirus E6 protein, the oncogenic potential of MDM2is based on its ability to bind to and inactivate p53. Thus, theinactivation of p53 function by MDM2 results in the abro-gation of both p53-mediated cell cycle arrest and apoptosis.Recent findings indicate that inactivation of p53 by MDM2occurs by promoting the degradation of the tumor suppres-sor protein through the ubiquitin-proteasome pathway (10,11). In addition, the discovery that p53 is able to transcrip-tionally activate the expression of mdm2 (12) led to the hy-pothesis that a feedback autoregulatory loop provides a pre-cise time frame for p53 signaling to regulate the cell cycle.MDM2 also interacts with other proteins important in theregulation of cell cycle transition, such as the retinoblastomagene product, the TATA-binding protein, the transcriptionfactor E2F, and the INK4a-ARF tumor suppressor gene prod-uct p19ARF (13–15).

Recently, studies in NIH-3T3 fibroblasts revealed that

Received June 11, 1999.Address all correspondence and requests for reprints to: Dr. Pilar

Santisteban, Instituto de Investigaciones Biomedicas, Consejo Superiorde Investigaciones Cientıficas, Arturo Duperier 4, 28029 Madrid, Spain.E-mail: [email protected].

* This work was supported by Grants DGICYT (PM97–0065) andCAM (08.1/0025/1997), Fundacion Salud 2000 (Spain), and fellowshipsfrom the Fondo de Investigaciones Sanitarias (to T.D.) and the SpanishMinisterio de Educacion y Cultura (to D.L.M.).

† Current address: Centro Nacional de Investigaciones OncologicasCarlos III, 28220 Madrid, Spain.

0013-7227/00/$03.00/0 Vol. 141, No. 1Endocrinology Printed in U.S.A.Copyright © 2000 by The Endocrine Society

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MDM2 arrests the cell cycle, causing a specific inhibition ofG0/G1-S transition (16). In the present report we demonstratethat MDM2 is sufficient to promote apoptosis in the MTT cellline. Transfection of these cells with mdm2 resulted in theisolation of clones that constitutively express MDM2. Theseclones exhibit a growth retardation compared with the pa-rental cell line. Cell cycle analysis and annexin V labelingshow a significant fraction of these MDM2 transfectants un-dergoing apoptosis, thus providing a direct link betweenMDM2 expression and programmed cell death.

Materials and MethodsCell culture

The human MTC cell line MTT (3) was maintained in RPMI 1640medium supplemented with 10% FBS, 2 mm glutamine, 100 mg/mlsodium pyruvate, 100 U/ml penicillin, and 100 mg/ml streptomycin.The human follicular thyroid carcinoma cell lines FRO, ARO, and NPAwere provided by Dr. J. A. Fagin (University of Cincinnati, Cincinnati,OH) and Dr. Juillard (University of California, Los Angeles, CA). Theywere maintained in the same conditions as those used for the MTT cells.Human breast cancer MCF-7 cells were grown in DMEM supplementedwith 10% FBS, 2 mm glutamine, 100 U/ml penicillin, and 100 mg/mlstreptomycin.

Plasmids and transfections

pCMDM2 was constructed by ligation of the human mdm2 comple-mentary DNA (cDNA) (6) containing the complete open reading frameinto the BamHI site of the pCDNA3 eukaryotic expression vector (In-vitrogen BV, Leek, The Netherlands). DNAs (pCMDM2 and pCDNA3)were transferred into MTT cultures (106 cells/plate) using lipofectin,following the manufacturer’s directions. For all experiments reported,early passage cells (,10) were used. Optimum conditions for DNAtransfer were found by mixing equal amounts of lipofectin reagent andDNA (2–10 mg) and maintaining the lipid-DNA complex in serum-freemedium cultures for 8–12 h. G418 (200 mg/ml) was added to the culturesfor selection. Nuclear extracts from transient experiments were collected72 h after transfection. For the scoring colony formation assay, 4 weeksafter transfection, colonies were fixed in 70% methanol and stained with0.5% crystal violet. In these experiments, a retroviral construct express-ing wild-type human p53 (17), and an expression vector carrying thep19ARF cDNA (18) were also used. For establishment of constitutivetransfectants, resistant colonies were either isolated from the platesindividually or pooled and expanded to generate cell lines. Unlessotherwise indicated, reagents were purchased from Life Technologies,Inc. (Gaithersburg, MD).

Cell growth and tumorigenicity assays

Cells (2 3 104) were seeded in 6-cm plates, and the number of viablecells was determined every 24 h for 4 consecutive days by the trypan bluedye exclusion test. Experiments were performed in triplicate. For tu-morigenicity assays, 5 3 106 cells from each cell line were trypsinized,collected in 100 ml PBS, and injected sc into nude mice. Tumor formationwas monitored weekly, and tumorigenicity was scored as the numberof tumors per site after 4 weeks.

Detection of apoptosis

To determine cell cycle distribution, asynchronous cultures weretrypsinized and fixed in 70% ethanol. Cells were pelleted, resuspendedin PBS, and stained with propidium iodide. Stained samples were an-alyzed in a FACScan flow cytometer (Becton Dickinson, San Jose, CA).Histograms containing at least 10,000 events were generated using LysisII software (Becton Dickinson and Co.).

Apoptosis was also monitored by annexin V labeling and fluores-cence microscopy (19). Cells were washed with PBS and then treatedwith annexin V-fluorescein (Roche Molecular Biochemicals, Mannheim,Germany) for 15 min. After a 488-nm excitation, green fluorescence was

visualized and recorded at 515 nm. Phase contrast microscopic imagesfrom the same preparations were also obtained.

Apoptotic cells from tumor sections were identified by TUNEL (ter-minal deoxynucleotidyltransferatse-mediated deoxy-UTP-biotin nickend labeling) staining (20), with minor modifications, as previouslydescribed (21).

Northern analysis

Total RNA was extracted from guanidinium isothyocianate cell ly-sates (22) with phenol-chloroform and isopropanol precipitation. RNAsamples (20 mg) were separated by 1% agarose electrophoresis underdenaturing conditions (1.1 m formaldehyde and 50% formamide) andtransferred to Nytran filters (Schleicher & Schuell, Inc., Keene, NH).Prehybridization and hybridization were performed at 42 C for 6 and24 h, respectively, in a buffer containing 50 mm Na2HPO4 (pH 6.5), 5 3SSC (standard saline citrate), 0.2% SDS, 5 3 Denhardt’s solution, and50% formamide. Blots were washed three times at room temperature in2 3 SSC-0.1% SDS and twice at 42 C in 0.1% SSC-0.1% SDS. A 1.6-kbhuman mdm2 probe, obtained after SalI/BamHI digestion of pcMDM2,was used for hybridization. DNA fragments were purified using Ge-neclean (BIO 101, La Jolla, CA) and labeled with [a-32P]deoxy-CTP byrandom priming. Specific activity was usually about 5 3 108 cpm/mg.To assess equal loading of the samples, the same blots were hybridizedwith a b-actin probe.

RT-PCR amplification

MTC tumor samples were provided by Drs. E. Mato and X. Matias-Guiu (Hospital de la Santa Creu i Sant Pau, Barcelona, Spain). Total RNAfrom the tumor samples was extracted as described above (22). RNApreparations (1 mg) were reverse transcribed using Moloney murineleukemia virus reverse transcriptase (Pharmacia Biotech, Piscataway,NJ) for first strand synthesis. Aliquots of the reactions were then usedfor PCR amplification using Taq polymerase (Perkin-Elmer Corp.,Norwalk, CT). Forward and reverse primers for mdm2 amplificationwere 59-GCTGAAGAGGGCTTTGAT-39 and 59-TGGTGTAAAGGAT-GAGCT-39. Amplification was carried out for 40 cycles, and PCR cycleparameters were: denaturation at 94 C for 1 min, annealing at 55 C for1 min, and extension at 72 C for 1 min. Control amplification of glyc-eraldehyde-3-phosphate dehydrogenase (GAPDH) was performed withthe following forward and reverse primers: 59-GACCCACATCGCTCA-GAC-39 and 59-TTCTCCATGGTGGTGAAG-39. Amplification was per-formed in 40 cycles with these PCR cycle parameters: denaturation at 94C for 1 min, annealing at 62 C for 30 sec, and extension at 72 C for 90sec. PCR products were separated and visualized in ethidium bromide-stained 2% agarose gels.

Western analysis

Nuclear extracts were obtained as previously described (23). Equalamounts of nuclear proteins (20 mg) were subjected to SDS-PAGE andtransferred to nitrocellulose membranes (Schleicher & Schuell, Inc.,Keene, NH). After blocking membranes with 5% low fat dry milk inTris-buffered saline-0.05% Tween-20, immunodetection of MDM2 wasperformed using a commercial monoclonal antibody (Santa Cruz Bio-technology, Inc., Santa Cruz, CA). After incubation with a horseradishperoxidase-conjugated secondary antibody, immunoreactive proteinswere visualized by Western blotting luminol reagent (Santa Cruz Bio-technology, Inc.). Starting from total protein extracts, similar protocolswere used to detect the apoptosis-related proteins Bcl-2, Bcl-x, caspase-2,caspase-3, and receptor interactin protein (RIP), using antibodies ob-tained from Transduction Laboratories (Lexington, KY). Poly(ADP-ri-bose) polymerase (PARP) and actin antibody were purchased fromSanta Cruz Biotechnology, Inc.

Statistical analysis

Statistical significance among experimental groups was determined us-ing Student’s t test. Differences were considered significant at P , 0.05.

INDUCTION OF APOPTOSIS BY MDM2 IN MTT CELLS 421



ResultsThe mdm2 protooncogene is not expressed in the MTTcell line

We have previously reported that overexpression of p53 inMTT cells causes a partial G1-specific arrest, as p53 clones areable to partially overcome the G1 block and progress throughthe cell cycle (3). In this study we have searched for cell cycleregulatory pathways operating in this p53-null cell line andanalyzed the participation of the MDM2 oncoprotein.

We initially characterized the expression levels of mdm2 byNorthern analysis. To our knowledge, expression of mdm2had never been tested in any thyroid-derived tumor cell line,so we included a panel with three follicular tumor cell lines(FRO, ARO, and NPA). Total RNA was extracted and hy-bridized with a mdm2 cDNA probe. As shown in Fig. 1A, a5.5-kb mdm2 transcript was detected in the three follicularthyroid carcinoma cell lines. The mol wt for mdm2 messengerRNA (mRNA) was as previously described (6). Expressionwas maximum in FRO cells and was also detected in AROand NPA. However, expression of mdm2 was absent in MTTcells.

The absence of mdm2 transcripts in the MTT cell line

prompted us to analyze whether this observation was re-stricted to this particular cell line or could be extended toother MTC samples. To address this question, RNA fromfour MTC tumors was analyzed for the presence of mdm2 byRT-PCR. Positive and negative control experiments includedNPA and MTT samples, respectively. Results show that noneof the tumors analyzed expressed mdm2, whereas a band ofthe expected size was amplified from NPA cells. In all cases,the integrity of the RNA samples was confirmed using prim-ers for GAPDH (Fig. 1B).

Expression of mdm2 interferes with MTT cell growth

To analyze the participation of the mdm2 protooncogene inthe transformed phenotype of the MTT cell line, we intro-duced an exogenous mdm2 gene to study the effect on cellproliferation. A mammalian expression vector carrying thehuman mdm2 cDNA in sense orientation was transfected bylipofection into MTT cell line. A control experiment wasperformed using empty vector (pCDNA3). G418 was addedto the cultures 48 h after transfection, and clonal selectionwas maintained for 3 weeks. After that period, we observedthat the ability of individual colonies to progress was clearlyreduced in those cells receiving the mdm2 expression vector.Moreover, outgrowing colonies from mdm2 transfectionswere clearly smaller than those obtained with the emptyvector (Fig. 2, A and B). To quantify this observation, plateswere fixed with methanol and stained with crystal violet. Asa control for these experiments, two genes previously de-scribed to act as negative regulators of cell growth were used:p53, which has been shown to inhibit cell growth in thisparticular cell line (3), and the INK4a tumor suppressor genep19ARF, which interferes with cell proliferation in many celllines (18). Results are summarized in Table 1. Compared withthe control transfections, expression of mdm2 decreased col-ony formation about 3-fold. This reduction was similar tothat obtained with the tumor suppressor gene p19ARF. Amuch greater effect was observed with a p53 expressionvector. These results indicate that overexpression of mdm2has a negative effect on cell growth.

We next attempted to generate stable transfectants ex-pressing mdm2. For this purpose, G418 resistant colonieswere isolated and expanded as clonal cell lines, designatedMTT-mdm2 clones (c1 to c5). To avoid clonal heterogeneity,pools (-p) from the same transfection assays were also iso-lated and analyzed in parallel. A control cell line (pC-MTT)was originated by transfection of MTT cells with the emptyvector. MTT-mdm2 clones are viable and show major alter-ations on cell morphology with respect to the parental cellline (Fig. 2, C and D). Whereas asynchronous cultures of MTTcells have a typical criss-cross pattern and fibroblast-likemorphology, MDM2 transfectants exhibit lower saturationdensity values and appear more refringent under phase con-trast microscopy. This morphology was different from thatproduced by other genes tested in the assay that have anegative effect on cell growth. They did not show the cell tocell extensions found in p53-transfected cells (Fig. 2E), or thespindle-shaped morphology of MTT cells transfected withp19ARF (Fig. 2F).

Before further characterization, the expression levels of

FIG. 1. A, Expression of mdm2 in different follicular tumor thyroidcell line (ARO, FRO, and NPA) and in the MTT human medullarycarcinoma cell line. Total RNA from the different cell lines was ex-tracted, electrophoresed, and hybridized with a specific mdm2 cDNAprobe. Migration of the 28S ribosomal RNA is indicated. After expo-sure, the same blot was stripped and hybridized with a b-actin probeto assess equal loading of the RNA preparations. B, Detection ofmdm2 transcripts in human MTC tumors by RT-PCR. Total RNAfrom MTT, NPA cells, and four human tumor samples was reversetranscribed and amplified with specific primers. The sizes of the PCRfragments are indicated. GAPDH was used from the same RT reac-tions to assess the integrity of the preparations.

422 INDUCTION OF APOPTOSIS BY MDM2 IN MTT CELLS Endo • 2000Vol 141 • No 1



mdm2 mRNA in MTT-mdm2 clones were analyzed by North-ern blot (Fig. 3A). Specific transcripts corresponding to theexogenous mdm2 were detected in MTT-mdm2 cells, whereashybridization was absent in those cells transfected with thecontrol vector. To confirm that detected transcripts encodedfor a MDM2 protein, nuclear extracts from MTT-mdm2-c1,-c4, and -p1, which showed higher expression levels ofmRNA, were isolated, resolved by electrophoresis, and im-munoblotted with a specific human MDM2 monoclonal an-tibody. A polypeptide migrating at 90 kDa was observed inall MTT-mdm2 transfectants (Fig. 3B). Protein accumulationwas maximum in MTT-mdm2-c1. These results confirmedthe presence of MDM2 and indicated that the exogenousprotein is efficiently translocated to the nucleus. To obtain anestimation of the levels of protein achieved in MTT-mdm2clones, nuclear extracts from MCF-7 were included in theassay. The results show that MDM2 protein levels in MTT-mdm2 clones were comparable to those in cells naturallyoverexpressing MDM2 (24).

We next quantified the negative effect on cell growth bydetermining the growth rate of MTT cells transfected witheither mdm2 or the control vector. Cells were seeded, and thenumber of viable cells was determined for 4 consecutive days(Fig. 3C). The results demonstrated a significant growth re-tardation induced by MDM2. After 4 days in culture, the totalcell number of MDM2 transfectants was up to 40% lowerthan that obtained for the parental cell line. This inhibitoryeffect, although variable, was observed in both individualclones and pooled cultures.

MDM2 promotes apoptosis in MTT cells

To analyze the cellular mechanisms responsible for MDM2interference with MTT cell growth, we analyzed cell cycledistribution of MTT cells transfected with mdm2. Asynchro-nous cultures from those clones positive in the Western blotwere collected and analyzed by flow cytometry. Histogramsfrom two individual clones and one pool are shown (Fig. 4),and data summarized in Table 2. Cell cycle distribution ofMTT cells, transfected with the control vector, showed valuessimilar to the previously described histograms for the pa-rental, untransfected MTT cells (3). MTT-mdm2 clones con-sistently showed a fraction of hypodiploid cells (rangingfrom 35–49%), with a DNA content below 2N (sub-G0/G1).This distribution is characteristic of apoptotic cells (25) andtherefore suggests that MDM2 promotes cell death in thesecells. It is remarkable that apart from this sub-G0/G1 fraction,the remaining cells are distributed along the cell cycle almostnormally, although G2-M values were slightly lower thanthose measured in MTT cells.

We further confirmed that hypodiploid cells detected by flow

TABLE 1. Effect of mdm2 expression on MTT colony formation

Vector Colonies

pCDNA3 400 6 17p19ARF 190 6 16a

pC-mdm2 155 6 23a

pZ-p53 sense 20 6 5a

MTT cells were transfected with 10 mg of the different plasmidsusing the lipofection technique. G-418-resistant cells were allowed togrow for 3 weeks, then fixed and stained with crystal violet to deter-mine colony number. Experiments were performed in triplicate. Av-erage values and SEs are shown.

a Statistically significant vs. pCDNA3 (P # 0.01).

FIG. 2. Morphological appearance ofoutgrowing MTT (A) and MTT-mdm2colonies (B). Phase contrast pictures ofasynchronous cultures derived after se-lection with G418 of MTT cells trans-fected with control vector (C) and ex-pression vectors for MDM2 (D), p53 (E),and p19ARF (F).




cytometry corresponded to cells undergoing apoptosis. For thatpurpose, MTT and MTT-mdm2-c1 cells, which exhibited thehighest sub-G0/G1 fraction, were collected and treated withannexin V. This protein, which specifically interacts with phos-phatidylserine exposed in the outer layer of the plasma mem-brane, is a valuable marker for detection of apoptotic cells (19).

Data obtained from fluorescence detection of MTT and MTT-mdm2-c1 together with the phase contrast microscopic imagesof the same fields are shown (Fig. 5). Apoptosis was clearlydetected in MTT-mdm2 cells (Fig. 5, A and B), whereas it wasvirtually absent in MTT samples (Fig. 5, C and D). Staining withpropidium iodide indicated that necrotic cells were almost ab-

FIG. 3. Constitutive expression of MDM2 results in growth retardation of MTT cells. A, Detection of mdm2 expression by Northern blot. RNAfrom MTT cells, those transfected with the control vector (pC-MTT), and those transfected with pCMDM2 (MTT-mdm2) were extracted,electrophoresed, and hybridized with a mdm2 cDNA probe. After stripping, the same blot was hybridized with a b-actin probe. The mobilitiesof the 28S and 18S ribosomal RNAs are indicated. B, Immunodetection of MDM2 in MTT-mdm2 clones. Nuclear extracts were separated bySDS-PAGE and probed with MDM2 antibodies. Mol wt markers are shown on the right. MCF-7 cell nuclear extracts were used as a positivecontrol. C, Growth profiles of MTT cells and MTT-mdm2 clones. The average values of viable cell number are represented. Experiments wereperformed in triplicate.

FIG. 4. Flow cytometric analysis ofasynchronous MTT and MTT-mdm2clones. Cells were fixed with ethanol,stained with propidium iodide, and an-alyzed by FACScan. Histograms fromthe parental cell line, two individualmdm2 transfected clones (c1 and c4),and one pool (p1) are represented.




sent in both preparations (not shown). Quantification of dif-ferent fields indicated that the fraction of apoptotic cells wasabout 40% of the total cell population, thus providing a goodcorrelation among growth retardation profiles, cell cycle his-tograms, and apoptosis.

MDM2 induction of apoptosis is partially reverted bywild-type p53

In an attempt to understand whether MDM2 induction ofapoptosis is related to the p53 defect of MTT cells, a series oftransient transfection experiments was performed. BothMTT and MTT-mdm2 (clone c1) cells were seeded and thentransfected with an expression vector for wt p53. A parallelexperiment was also performed using an expression vectorfor the p19ARF, and finally, both vectors were also cotrans-fected. In all cases, cells were collected 72 h after transfectionto determine the percentage of apoptotic cells and cell cycledistribution by flow cytometry.

Control experiments with an empty expression vector gavesub-G0/G1 values similar to those obtained previously (Table1). After transfection with p53, the percentage of the sub-G0/G1was significantly lower, indicating that the tumor suppressorpartially reverts MDM2 induction of apoptosis (Table 3). Thisobservation parallels the increase in the percentage of cells inG0/G1 phase. Similar data were obtained after transfection withp19ARF, although in this case, cells were not clearly arrested inG0/G1. When both constructs were contransfected, results werecomparable to those obtained with p53 alone, indicating that theeffect of those genes is not additive.

Tumors derived from mdm2-expressing cells show anincreased number of apoptotic nuclei

We next evaluated whether the negative effect induced byMDM2 on cell growth and the ability to promote apoptosiswere extended when MTT cells were allowed to form tumorsin vivo. MTT and MTT-mdm2 (clone 1) cells were injected sc

TABLE 3. Cell cycle distribution of MTT-mdm2 cells transfected with p53, p19ARF, and p53/p19ARF

Sub-G0/G1 G0/G1 S G2/M

MTT-mdm2-c1 46.29 6 0.70 35.06 6 0.83 12.54 6 1.09 6.11 6 0.79MTT-mdm2/p53 33.48 6 0.87a 42.42 6 0.46 12.44 6 0.83 11.44 6 0.64MTT-mdm2/p19ARF 36.47 6 1.21a 37.37 6 0.63 15.73 6 0.86 10.64 6 0.99MTT-mdm2/p53 1 p19ARF 32.74 6 1.01a 43.93 6 0.64 12.10 6 0.73 11.33 6 0.82

Samples were collected and fixed, and cell cycle was analyzed by FACSscan as described in Materials and Methods. Experiments wereperformed in triplicate. Average values and SEs are represented.

a Statistically significant vs. MTT-mdm2-c1 (P # 0.01).

TABLE 2. Cell cycle distribution of MTT cells transfected with mdm2

Sub-G0/G1 G0/G1 S G2/M

MTT 1.57 6 0.17 47.34 6 0.20 27.08 6 0.43 24.01 6 0.30MTT-mdm2-c1 49.63 6 0.87a 34.94 6 0.54 9.25 6 0.13 6.18 6 0.24MTT-mdm2-c4 46.54 6 0.72a 35.21 6 0.95 11.65 6 0.37 6.60 6 0.19MTT-mdm2-p1 33.06 6 0.25a 41.75 6 1.18 14.32 6 0.29 10.87 6 0.26

Samples were collected and fixed, and cell cycle was analyzed by FACSscan as described in Materials and Methods. Experiments wereperformed by triplicate. Average values and SEs are represented.

a Statistically significant vs. MTT (P # 0.01).

FIG. 5. Detection of MDM2-inducedapoptosis in MTT cells. Apoptosis wasmonitored by annexin V and fluores-cence microscopy. Phase contrast mi-croscopy pictures of MTT-mdm2 (A) andMTT cells (C) were analyzed by fluores-cence at 515 nm (B and D, respectively).




into nude mice, and tumor formation scored after 4 weeks.As shown in Table 4, both MTT and MTT-mdm2 cells gaverise to tumors in 100% of the cases. Tumors derived fromMTT-mdm2 cells emerged later, although this difference wasnot significant, indicating that the negative interference ofMDM2 with cell growth does not reverse the transformedphenotype of these cells.

To rule out the possibility that the lack of effect couldbe due to a loss of mdm2 expression during tumor devel-opment, samples were analyzed for the presence of MDM2protein by immunohistochemistry. Slide preparationsfrom MTT and MTT-mdm2 tumors were fixed and incu-bated with MDM2 antibodies. As expected, tumors de-rived from the parental cell line did not show immuno-staining. However, MTT-mdm2 tumors showed positivestaining, indicating that MDM2 is efficiently expressed inthe tumor (data not shown).

We next examined whether mdm2, expressed in the tumorsderived from MTT-mdm2 cells was also able to promoteapoptosis in vivo. For this purpose, tumor sections were an-alyzed for the presence of apoptotic nuclei by TUNEL assay(Table 3). Tumors derived from the MTT cell line showed avery low percentage of TUNEL-positive cells (0.3%). How-ever, in those tumors derived from MTT-mdm2 cells, thepercentage of apoptotic nuclei increased almost 20-fold(5.3%). These results unambiguously confirm the ability ofthe MDM2 protein to induce apoptosis in MTT cells, both invivo and in vitro.

Bcl-2 and caspase-2 participate in MDM2-induced apoptosis

Molecular mechanisms underlying MDM2-mediated ap-optosis in MTT cells were explored. We reasoned that ifMDM2 is able to induce apoptosis in MTT cells, specificantiapoptotic pathways operating in the parental cell lineshould be shut down in those cells transfected with mdm2. Totest this hypothesis, we measured protein levels of Bcl-2, aprotein that suppresses programmed cell death in many celllines (26). Using specific antibodies for Bcl-2, we detected animmunoreactive band in the parental MTT cells (Fig. 6). Inthose clones transfected with mdm2, Bcl-2 protein levels werealmost undetectable. Only after long exposure of the auto-radiographs could a faint band be visualized, indicating astrong down-regulation of Bcl-2 induced by mdm2. We alsomeasured protein levels of Bcl-x. The bcl-x gene is related tobcl-2, although proteins encoded by this locus can functionindependently of Bcl-2. Two products, generated by alter-native splicing, arise from the bcl-x gene: Bcl-xL and Bcl-xS.Whereas the former also inhibits apoptosis in some cell sys-tems, Bcl-xS promotes cell death (27). In MTT cells, Bcl-x was

present in asynchronous cultures and, upon transfectionwith mdm2, no consistent modifications of Bcl-x were ob-served. In some clones, a slight up-regulation of Bcl-x wasdetected, whereas in most cases no major differences werefound.

It was recently shown that Bcl-2 regulates apoptoticcascades mediated by caspase-3 and caspase-2 (28), so weanalyzed whether any of these cystein proteases could bedetected in MTT-mdm2 cells (Fig. 7). We used antibodiesfor caspase-2 and caspase-3 and probed membranes con-taining total protein extracts from MTT and MTT-mdm2

FIG. 6. MDM2 down-regulates protein levels of Bcl-2. Protein ex-tracts from MTT cells and MTT cells transfected with mdm2 (clonesc1, c4, and p1) were separated by SDS-PAGE and probed with specificantibodies for Bcl-2 and Bcl-x. Equal loading of the samples wasassessed using an actin antibody. Mol wt marker migration is indi-cated.

TABLE 4. Effect of mdm2 expression on tumorigenicity andpopulation of TUNEL-positive cells from the tumors

Vector Tumors in nude mice % Apoptotic cells

pCDNA3 6/6 0.3pcMDM2 6/6 5.3

Tumorigenicity is expressed as the number of tumors/sites injectedand was scored 4 weeks after transfection. The percentage of apo-ptosis was determined by TUNEL assay.

FIG. 7. Caspase-2 is up-regulated in MTT cells transfected withmdm2. Protein extracts from MTT cells and MTT cells transfectedwith mdm2 (clones c1, c4, and p1) were separated by SDS-PAGE andprobed with specific antibodies for caspase-2, caspase-3, RIP, PARP,and actin. Mol wt marker migration is indicated.




cells. In the parental cell line, caspase-2 was undetectable.However, it was clearly up-regulated in cell lines trans-fected with mdm2. We next determined protein levels ofcaspase-3. As shown, we were unable to detect the pres-ence of this protease in extracts from either MTT or MTT-mdm2 cell lines, although it was clearly visualized inextracts from Jurkat cells (not shown). To further rule outa participation of caspase-3, we determined the proteolyticcleavage of PARP, an enzyme involved in DNA repair, anda common substrate of caspase-3 (29). Total extracts weresubjected to Western blot analysis with an antibodyagainst PARP. As expected, only the uncleaved, 115-kDaisoform was observed in both MTT and mdm2 transfectedcells.

Taken together these results point to a mechanism involv-ing caspase-2 and independent of caspase-3. These mecha-nisms have been described in some apoptotic pathways, suchas those mediated by tumor necrosis factor-a (TNFa) (30). Toexplore whether a similar mechanism could be acting inMTT-mdm2 cells, we determined protein levels of RIP (31),an adapter molecule involved in apoptotic pathways involv-ing caspase-2 independently of caspase-3. Total protein ex-tracts from the MTT cell line and from cells transfected withmdm2 were obtained. Western blot was carried out using anantibody against RIP. As shown in Fig. 7 an immunopositiveband corresponding to RIP (74 kDa) was observed. However,we detected the same amount of the immunoreactive bandin both MTT and MTT-mdm2 clones, suggesting that acti-vation of caspase-2 may occur through a different apoptoticpathway.

Discussion

The results presented in this study provide evidence for anovel function mediated by MDM2 and indicate for the firsttime that this oncoprotein promotes apoptosis in a humanMTC cell line characterized by the presence of a geneticrearrangement of the p53 locus (3). These results togetherwith those showing the ability of MDM2 to arrest the cellcycle of normal fibroblasts (16) indicate that the product ofthe mdm2 protooncogene may also interfere negatively withcell proliferation. Several studies have previously demon-strated that MDM2 promotes tumorigenesis when it is over-expressed. It has been shown to cooperate with ras in thetransformation of rat embryo fibroblasts (32) and to induceneoplastic conversion of murine immortalized cells (33).Likewise, MDM2 is able to prevent p53-mediated apoptosisin some tumor cell lines (34) as well as G1 cell cycle arresteven in the absence of p53 (35).

These opposite functions support the idea that, as previ-ously described for other genes such as c-myc (36), E1A (37),or cyclin D1 (38, 39), some regulatory proteins could beinvolved in both tumorigenesis and apoptosis depending onthe cellular environment. MDM2 should, then, be consideredas a multifunctional regulator of cell cycle progression,whose effect on cell growth may be dependent not only onp53, but also on other known or unknown regulatoryproteins.

Expression of mdm2 is found in the three follicular tumorthyroid cell lines tested. It is important to mention that ex-

pression of mdm2 is higher in FRO cells, in which no alter-ations of p53 have been described (40). Both ARO and NPAcarry a mutation of the p53 gene and would render proteinproducts for this tumor suppressor unable to trans-activatemdm2. Nevertheless, differences in mdm2 expression are notdramatic among the three cell lines tested, suggesting thatother regulatory genes, apart from p53, participate in mdm2transcription. Of special interest is the observation that mdm2transcripts are not detected in any of the four MTC tumorsamples analyzed. Whether there is a correlation betweenlack of mdm2 expression and this particular tumor type re-quires further investigation and is currently being studied.

Cell growth profiles and cell cycle histograms of MTT-mdm2 clones indicate that whereas in some cells expres-sion of mdm2 promotes apoptosis, others not only remainviable, but also progress along the cell cycle. The fact thatthe same results have been observed in pools and indi-vidual clones rule out the possibility of an artifact causedby an inappropriate integration of mdm2 during transfec-tion. Rather, the effects must be explained considering thatin MTT cells, MDM2 may be also activating some of thepreviously described pathways that favor cell growth (41).It is also possible that the threshold of MDM2 expressiondictates the decision of a given cell to either enter the cellcycle or commit programmed cell death. In this regard, itmay be important that a correlation was observed betweenmdm2 expression levels and the percentage of apoptosis inthe asynchronous cultures. Expression is maximum in theindividual clones, where a higher percentage of apoptosisis found.

The results reported here have been observed at both earlyand late passages. To date, no significant decrease in theexpression of mdm2 has been observed in our cultures. Thefact that MDM2 levels are maintained, and apoptosis is alsodetected at late passages rule out the possibility that thedeleterious effect of MDM2 may be limited to early events inthe transfection assays, where high amounts of the proteinare expressed inappropriately. Rather, we believe thatMDM2 physiologically regulates and promotes apoptosis inthese cells. Moreover, apoptosis mediated by MDM2 may bepartially reverted by exogenous expression of both p53 andp19ARF, as determined by transient transfection analysis. Inthe case of p53, we have previously demonstrated that thetumor suppressor gene causes a G1 arrest in these cells (3),and here we observed that even in the presence of MDM2,p53 partially arrest MTT cells in that phase of the cell cycle,thereby preventing them from undergoing apoptosis. On theother hand, the ability of p19ARF to partially reverse MDM2-induced apoptosis is in keeping with the observation that theproduct of the INK4a locus is able to bind to and promote thedegradation of MDM2 (14, 15).

In agreement with results published for the MTC cell lineTT and MTC tumors (42), we have clearly detected expres-sion of Bcl-2 in MTT cells, suggesting that the Bcl-2 onco-protein may contribute to the pathogenesis of these tumorsand transformed cells. Here we show that apoptosis inducedby MDM2 is accompanied by down-regulation of Bcl-2, thusallowing cell death to progress. This together with the acti-vation of caspase-2 suggest that, as previously described forother cell systems (43), both pathways interact. Nevertheless,




in our Western assays with caspase-2 antibodies, we detectimmunoreactive forms corresponding to the procaspaseform and have been unable to detect any band correspondingto any cleaved form of this protease. Results also show thatcaspase-3 is not activated in MTT-mdm2 cells, as 1) thisprotease in not detected in protein extracts; and 2) fragmen-tation of PARP, a well characterized substrate for caspase-3,is not cleaved in MTT-mdm2 cells. This points to an apoptoticcascade dependent on caspase-2, although caspase-3 inde-pendent, such as those described for cell death mediated byTNFa. It has been shown that TNF binding to its receptorresults in the clustering of receptor death domains. Then, theadapter protein RIP binds through its own death domain tothe clustered receptor death domain, and this complex joinsanother adapter molecule, RAIDD/CRADD (44, 45). Uponrecruitment by CRADD, caspase-2 drives its activationthrough self-cleavage. Two pieces of evidence suggest that adifferent pathway is acting in MTT cells transfected withMDM2. First, protein levels of RIP were similar in control andMDM2-expressing cells, and as mentioned, we have beenunable to detect a cleaved form of caspase-2.

Previous reports (8, 14) and the observation described heredefinitively indicate that the response to MDM2 overexpres-sion is cell specific. Therefore, it is important to determine thecellular environment in which MDM2 is able to induceapoptosis, and in this context, the medullary carcinoma cellline MTT constitutes an excellent system for these studies. Asthese cells are naturally devoid of p53 (3), other regulatoryproteins functionally related to MDM2 should be carefullyanalyzed. Potential candidates for this analysis include thep53 homolog p73 (46), an antiproliferative protein whosefunction is modified by MDM2 (47, 48).

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14th International Symposium of The Journal of Steroid Biochemistry& Molecular Biology

“RECENT ADVANCES IN STEROID BIOCHEMISTRY & MOLECULAR BIOLOGY”24–27 June 2000 — Quebec, Canada

The 14th International Symposium of The Journal of Steroid Biochemistry & Molecular Biology—“RecentAdvances in Steroid Biochemistry & Molecular Biology” will be held in Quebec, Canada, on 24–27 June 2000.The following topics will be considered:

1. Steroid Receptors, Structure, and Mechanism of Action2. Anti-Steroids3. Steroids and Cancer4. Control of Steroidogenesis, Including Defects in Steroid Metabolism5. Enzyme Inhibitors6. Steroids and the Immune System7. Steroids and the Menopause

Lectures (approximately 25–30) will be by invitation of the Scientific Organizing Committee and, in addition,there will be a poster section. All poster presentations will be subject to selection by the Scientific OrganizingCommittee and abstracts (maximum 200 words) must be sent to Dr J. R. PASQUALINI by Monday 14February 2000 (postmark) (ORIGINAL 1 12 copies). For further details, please contact:

General Scientific Secretariat: Dr. J. R. PASQUALINI, Steroid Hormone Research Unit, 26 Boulevard Brune,75014 Paris, France. Tel.: (33-1)145 39 91 09/45 42 41 21; Fax: (33-1)145 42 61 21; e-mail: [email protected].

Local Organizing Committee: Dr. F. LABRIE, MRC Group in Molecular Endocrinology, CHUL Laval, Centrede Recherches, 2705 Boulevard Laurier, Ste-Foy, Quebec G1V 4G2, Canada. Tel.: (418)1654 27 04; Fax:(418)1654 27 35; e-mail: [email protected].




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