Proc. Natl. Acad. Sci. USAVol. 92, pp. 4407-4411, May 1995Medical Sciences

Wild-type p53 protein undergoes cytoplasmic sequestration inundifferentiated neuroblastomas but not in differentiated tumorsUTE M. MOLL*t, MICHAEL LAQUAGLIAt, JEAN BENARD§, AND GUY RIOU§*Department of Pathology, Health Sciences Center, State University of New York, Stony Brook, NY 11794-8691; tDepartment of Surgery, MemorialSloan-Kettering Cancer Center, New York, NY 10021; and §Laboratoire de Pharmacologie Clinique et Moleculaire, Institut Gustave Roussy,94805 Villejuif Cedex, France

Communicated by Richard B. Setlow, Brookhaven National Laboratory, Upton, NY, January 11, 1995 (received for review March 28, 1994)

ABSTRACT Neuroblastoma (NB), a tumor arising fromthe sympathetic nervous system, is one of the most commonmalignancies in childhood. Several recent reports on the p53genotype found virtually exclusive wild-type status in primarytumors, and it was postulated that p53 plays no role in thedevelopment of NB. Here, however, we report that the vastmajority of undifferentiated NBs exhibit abnormal cytoplas-mic sequestration of wild-type p53. This inability of p53 totranslocate to the nucleus presumably prevents the proteinfrom functioning as a suppressor. Thirty of 31 cases (96%) ofundifferentiated NB showed elevated levels ofwild-type p53 inthe cytoplasm of all tumor cells concomittant with a lack ofnuclear staining. p53 immunoprecipitation from tumor tis-sues showed a 4.5- to 8-fold increase over normal proteinlevels. All of 10 tumors analyzed harbored wild-type p53 bydirect sequencing of full-length cDNA and Southern blot. Inaddition, no MDM-2 gene amplification was seen in all 11tumors analyzed. In contrast, no p53 abnormality was de-tected in 14 differentiated ganglioneuroblastomas and 1 be-nign ganglioneuroma. We conclude that loss of p53 functionseems to play a major role in the tumorigenesis of undiffer-entiated NB. This tumor might abrogate the transactivatingfunction of p53 by inhibiting its access to the nucleus, ratherthan by gene mutation. Importantly, our results suggest that(i) this could be a general mechanism for p53 inactivation notlimited to breast cancer (where we first described it) and that(ii) it is found in a tumor previously not thought to be affectedby p53 alteration.

Wild-type p53 plays a key role in the negative regulation of cellgrowth and tumor formation. p53, located on chromosome 17p13.1, is a nuclear phosphoprotein, and nuclear localization isessential for its growth-suppressing activity in late G1 (1).Normally, the level of p53 protein is tightly regulated and notdetectable by immunocytochemistry. p53 acts as a cell cyclecheckpoint after DNA damage and induces G1 arrest orapoptosis (2) and is required to maintain genomic stability (3,4). p53 also contributes to terminal differentiation (5, 6). Themechanism underlying its central growth suppression appearsto be that p53 regulates, directly or indirectly, gene expressionof crucial growth-inhibiting and growth-promoting genes andDNA replication. p53 is a potent transcription factor that bindsto a consensus-binding site present in regulatory sequences ofp53-dependent genes (30, 31). This DNA binding site mediatesthe p53-induced transactivation of downstream effectors suchas WAF 1, an inhibitor of cyclin-kinase complexes in exertingsuppression (7, 8). On the other hand, p53 represses transcrip-tion from promoters that lack p53-specific DNA binding sites,presumably by sequestering TATA binding protein (9).

Inactivation of p53 is the single most common event inhuman malignancies and is present in at least 50% of all

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4407

cancers (10). Mutational inactivation is the most commonmechanism and is found in many cancers of, for example, thebreast, gastrointestinal tract, lung, and gliomas. Most aredeletions of one allele accompanied by missense mutations inthe remaining allele. Most point mutations alter the confor-mation and prolong the half-life of the p53 protein, leading tonuclear accumulation. Though less frequently, mutation-independent mechanisms are also utilized in human malig-nancies. Here, the common theme is sequestration of normalp53 protein by another protein that abrogates its suppressiveaction. Originally, this was discovered in mammalian models ofviral oncogenesis. The viral oncoprotein Simian virus 40 largetumor antigen and adenovirus type 5 E1B protein formstabilized complexes with p53 that functionally inactivate it(11, 12). In humans, the HPV-16/18 E6 oncoprotein forms acomplex with p53 in human papillomavirus-positive genito-anal cancers, resulting in the degradation of p53 (13). In softtissue sarcomas, p53 is apparently inactivated by binding to theprotein product of the amplified MDM-2 oncogene, whichinhibits p53-mediated transactivation in vitro (14, 15). Re-cently, we described a fourth mechanism involving alteredsubcellular localization with nuclear exclusion ofwild-type p53in breast carcinoma (16).Neuroblastoma (NB) is one of the most common childhood

tumors with a prevalence of 1 in 6000 (17). It derives fromneural crest cells that migrate during embryogenesis to giverise to the sympathetic nervous system. NBs form a spectrumof tumors that represent progressive neuroblast differentia-tion. This differentiation occasionally occurs spontaneously orcan be induced by chemotherapy. Undifferentiated NB gen-erally arises in the adrenal medulla but can occur anywherealong the sympathetic chain and consists of sheets of "small,round blue cells." Ganglioneuroblastoma shows intermediatedifferentiation and consists of both primitive neuroblasts andrather mature ganglion cells embedded in a matrix of nervefibers. Ganglioneuroma, the benign version, is composed offully mature ganglion cells embedded in nerve fibers andoccurs at an older age.

Genetic analysis identified low- and high-risk prognosticgroups (18). While N-myc amplification is clearly related toadvanced stage (19), additional genetic events are likely to playa role in onset and progression of NB. Although inactivationof suppressor genes play a major role in childhood tumors, noNB suppressor has been identified to date. Putative NBsuppressors are located on chromosome lp 36 and 14q, whereloss of heterozygosity (LOH) is frequently observed (18, 20).Trisomy for 17q paired with monosomy for 17p (due toisochromosome formation) also occurs in NB, suggesting asuppressor on 17p (21, 22). Three recent reports on the p53gene status showed absence of mutations in virtually all of 172

Abbreviations: LOH, loss of heterozygosity; H&E, hematoxylin/eosin;Alk Phos, alkaline phosphatase; HRP/DAB, horseradish peroxidase/diaminobenzidine; aa, amino acid; NB, neuroblastoma; PCNA, pro-liferating cell nuclear antigen.tTo whom reprint requests should be addressed.

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primary NBs (2 had missense mutations that were present onlyin metastasis) (17, 23, 24). This led to the conclusion that thep53 suppressor plays no role in NB. In a parallel study however,we have analyzed the p53 status in 46 primary tumors andfound striking abnormalities in protein localization, with cy-toplasmic sequestration of wild-type p53 in virtually all undif-ferentiated NBs but not in differentiated variants. Our resultsprovide further evidence for the ubiquitous importance of p53inactivation in the development of human tumors, even inlineages that were previously thought to be unaffected.

MATERIALS AND METHODSTissue. All 46 tumors were from primary sites (6 stage I, 11

stage II, 7 stage III, 18 stage IV, 4 unknown) and immediatelysnap frozen. Pathologic diagnosis determined 31 undifferen-tiated NBs, 14 ganglioneuroblastomas of the composite type,and 1 benign ganglioneuroma. Normal controls consisted ofnerves, sympathetic ganglia, and adrenal medulla.

Immunocytochemistry. p53 was detected on frozen sectionswith four different human specific monoclonal antibodies (16):PAb 1801, DO-1, PAb 240 (all 500 ng/ml) (Santa CruzBiotechnology, Santa Cruz, CA), and DO-7 (1:100) (VectorLaboratories). PAb 1801, DO-1, and DO-7 recognize N-terminal epitopes between amino acids (aa) 32 and 79 (PAb1801), 17 and 26 (DO-1), and 37 and 45 (DO-7). PAb 240recognizes a central epitope between aa 210 and 219 and reactsequally well with mutant and wild-type p53 in fixed tissue (D.Lane, personal communication). In all cases, both streptavi-din-coupled horseradish peroxidase/diaminobenzidine (HRP/DAB) (Zymed) and alkaline phosphatase (Alk Phos)/fast red(BioGenex, San Ramon, CA) systems were used for detection.Normal mouse IgG at 500 ng/ml served as negative control.Also, specificity controls were carried out by preincubatingPAb 240 with 100-fold excess of the epitope peptide NTFRHS-VVVP (SynPep, Dublin, CA). Control tumors with nuclearp53 overexpression were included in most runs. Adjacenthematoxylin/eosin (H&E) sections served to evaluate tumormorphology. Nuclear integrity was verified by staining fornuclear antigen Ki-67 and proliferating cell nuclear antigen(PCNA) (AMAC, Westbrook, ME).

Immunoprecipitation. Frozen tissues were crushed withmortar and pestel in liquid N2 and 0.5 ml of buffer (10mM Tris,pH 7.5/250 mM sucrose/160 mM KCl/2 ,ug of aprotinin perml/2 ,tg of leupeptin per ml/1 mM phenylmethylsulfonylfluoride/0.5% Nonidet P-40/1 mg of bovine serum albuminper ml) and sonicated for 40 s. Samples were precleared withprotein G-agarose (GIBCO/BRL) and 1 mg of total proteinwas incubated with 1.5 ,tg of PAb 421 and precipitated with 30

Table 1. Cytoplasmic overexpression of wild-type p53 inundifferentiated NBs

No. of p53 Full-length Amplificationcases antibodies cDNA* N-myct MDM-2

4 Cyto Wild-type Yes No2 Cyto Wild-type Yes ND5 Cyto Wild-typet No No1 Cyto ND No No

14 Cyto ND No§ ND4 Cyto ND ND ND1 -ND No ND

Cyto, cytoplasmic staining by PAb 1801, PAb 240, DO-1, and DO-7with moderate (C+) to marked (C++) intensity. Normal mouse IgGserved as negative controls. ND, not determined.*For the sequenced cases, the % of tumor cells within the tissue fromwhich RNA samples were prepared was assessed in adjacent H&Esections. Five cases were 100%, two were 80%, and the remainderwere 95%, 90%, 60%, and 50%, which is still well within the sensi-tivity of detection for point mutations (16).tOne case showed 30-60 copies, one case 25-50 copies, one case20-40 copies, one case 6-12 copies, and two cases 3-6 copies.tOne case is incomplete due to scarcity of material; codons 336-394only.§One case showed overexpression of N-myc protein by immunocyto-chemistry.

,Il of protein G-agarose. Beads were washed, run on an 8%SDS/PAGE gel, and Western blotted using rabbit CM-1(1:1000; NovoCastra, Newcastle, U.K.) and the AmershamECL system.DNA Sequencing. RNA was extracted and reverse tran-

scribed (16). Full-length p53 cDNA was PCR amplified fromcodons 1-148, 118-353, and 253-393. Both strands of eachamplicon (400 ng) were directly sequenced with the fluores-cent DyeDeoxy sequencing kit (Applied Biosystems) and runon an automated DNA sequencer (Applied Biosystems model370 A).

Southern Blotting. Genomic contents from 11 individualswere probed on 6 jg of HindIII-restricted DNA (16) using[32P]dCTP-labeled MDM-2 cDNA (15) and [32P]dCTP-labeled human p53 cDNA. Gene dosage was measured bydensitometry and defined as amplified when -2.5 times that ofnormal testis, breast, and cervix (MDM-2), SK-N-SH (p53), orglobin pseudogene (N-myc). EcoRI-restricted DNA (6 ,tg)from 39 specimens was analyzed using [32P]dCTP-labeledN-myc or exon 2 pNbl probes. Blots were reprobed forcollagen to ensure equal loading.

Clinical Assessment. The patients' clinical stages rangedfrom 1 to 4 and 4 S (25), with ages from 1 to 70 months, except

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FIG. 1. Cytoplasmic overexpression of p53 in NB (case 28). (A) PAb 1801. (B) DO-1. (C) Mouse IgG control. Staining is limited to the cellsoma (see B). Ring (A) and cap (B) patterns are present within the same tumor and with both antibodies. The different patterns probably haveno additional significance but might simply represent cytoplasmic shape. (Alk Phos/fast red with nuclear hematoxylin as counterstain; x270.)

Proc. Natl. Acad. Sci. USA 92 (1995)

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FIG. 2. Cytoplasmic overexpression of p53 in NB (case 15). (A) DO-1 with nuclear counterstain. (B and C) PAb 1801 (B) and mouse IgG (C)without counterstain. Cap and ring patterns are present as in Fig. 1. (HRP/DAB; x270.) (Inset) The nuclear integrity of frozen tissue is verifiedby staining nuclear proliferation antigen Ki-67. PCNA gives identical results. (x130.)for 1 patient of age 17 years. Patients with unresectable stage3 and 4 tumors generally had undergone preoperative chemo-therapy. Twelve patients died of disease, 1 is alive with disease,29 are in complete remission, and 4 patients are lost forfollow-up.

RESULTS

Cytoplasmic Sequestration of Overexpressed Wild-Typep53 in Undifferentiated NB. We investigated the possibility ofnonmutational inactivation ofp53 in NB. Sections of 31 tumorswere analyzed by immunocytochemistry using four differentp53-specific monoclonal antibodies and two enzyme-linkeddetection systems. A remarkably homogeneous phenotype wasobserved that was in accord with the molecular genetics (Table1). In 30/31 cases, exclusive cytoplasmic staining as perinu-clear ring and cap patterns was seen (Figs. 1-3). The intensityvaried from moderate to marked and there was concordancebetween all antibodies, excluding nonspecific crossreactivitywith cytoplasmic proteins. Moreover, staining by PAb 240 wascompletely abrogated by preincubation with its epitope pep-tide (Fig. 3A Inset). To control for potential nuclear leakageduring freezing, nuclear integrity was verified with antibodiesagainst nuclear proliferation antigens Ki-67 and PCNA (Fig.2C Inset). p53 overexpression was limited to the cytoplasm and

was not present within the neuropil formed by tangles ofneuritic processes (e.g., Figs. 1B and 2A). None of the casesshowed any nuclear p53 (e.g., Figs. 2B and 3A). Nonimmunecontrols showed no detectable staining (Figs. 1 C, 2C, and 3B).Normal sympathetic paraganglia,. nerves, and adrenal tissueshowed undetectable p53. Some cases showed diffuse back-ground staining that was identical for nonimmune IgG and p53antibodies. As an independent proof of p53 overexpression, weperformed immunoprecipitations from tissue (Fig. 4). Allundifferentiated tumors showed marked overexpression ofp53compared to barely visible bands from benign ganglioneu-roma, normal ganglion, and breast. The densitometric increasewas 4.5-fold for tumors 16 and 24 (both C+), 7-fold for tumor1 (C++), and 8-fold for tumor 30 ("sh", C++) (Table 1).Molecular Correlations. Full-length sequences of p53

cDNAs from 10 cases were exclusively wild-type (Table 1).Remarkably, 8 of these 10 cases were advanced stage 3 or 4,and 5 patients died from their disease. Therefore, this tumor,in contrast to many other human malignancies, shows noselection for p53 mutations even as the disease progresses. Theoverexpression is not due to deregulated p53 expressionsecondary to gene amplification or rearrangement (Fig. 5).Nine of the 10 cases and an additional unsequenced case werealso analyzed for the MDM-2 oncogene (Fig. 5 and Table 1).The gene copy number was between 0.7 and 1.5 and thedetected bands were of the expected size, ruling out MDM-2

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FIG. 4. p53 immunoprecipitation from NBs. Lanes (left to right):1, IgG only; 2-5, tumors 24, 16, 1, and 30 ("sh"); 6, ganglioneuroma;7, normal sympathetic ganglion; 8, normal breast; 9, breast cancer lineMDA 468 (overexpresses nuclear p53 273 Arg -> His).

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FIG. 5. Southern blot analysis of genomic DNA from NB tumors1-4 and 12 for MDM-2 (a), p53 (b), and N-myc (c) genes.

amplification or rearrangement. Therefore, cytoplasmic p53sequestration due to overexpressed MDM-2, a nuclear protein,appears unlikely. No correlation was seen with N-myc ampli-fication (Table 1), clinical stage, DNA ploidy, karyotype,survival, and preceding chemotherapy.Lack of p53 Overexpression in Differentiated Tumors. We

then analyzed 15 tumors with ganglionic maturation (Table 2).Fourteen cases were "composite" ganglioneuroblastomas con-sisting of scattered mature ganglion cells within a spindle cellbackground that histologically closely resembles normal gan-glia. Abruptly interspersed islands of primitive neuroblastswere present in other parts of the resected tumors. One casewas a fully mature benign ganglioneuroma. In sharp contrastto the undifferentiated NBs, virtually all cases lacked detect-able levels of p53 within the cells (Fig. 6). As seen in normalsympathetic tissue, background staining was present consistentwith the close morphologic resemblance of these tumors totheir normal counterpart. The reason for this background,which is not present in undifferentiated tumors, is unclear butis possibly due to high endogenous enzyme levels that cannotbe quenched (Alk Phos and HRP). Two cases did showincreased diffuse staining for p53 but lacked a clear cytoplas-mic pattern. Again, no MDM-2 and p53 gene amplification wasseen in the one case analyzed and none of them showed N-mycamplification (Table 2).

DISCUSSIONNBs stand out by a conspicuous absence of p53 mutations inprimary tumors as well as in tumor-derived cell lines. In a totalof 172 primary tumors reported so far, 170 cases harbor p53wild-type genes (17, 23, 24). Likewise, 10 of 11 human NB linesstudied were wild-type (24, 26). This "resistance" to p53 gene

I

Table 2. Lack of aberrant p53 expression in differentiatedganglioneuroblastomas and benign ganglioneuromasTumor No. of AmplificationTumor No. oftype cases p53 antibodies N-myc MDM-2

GNB 1 -No NoGNB 8 No NDGNB 2 Diffuse punctate No NDGNB 3 - ND NDGN 1 -No ND

GNB, ganglioneuroblastoma; GN, ganglioneuroma; ND, not deter-mined.

alterations is even more remarkable in the NB lines, since, ingeneral, cells in culture have a high susceptibility to p53mutations and deletions, probably due to a growth advantagethat is acquired by the loss (27). In addition, no LOH in thevicinity of the p53 gene was seen in 20 primary NBs, excludingany large rearrangements (20). This led to the hypothesis thatthe p53 gene plays no role in NB tumorigenesis. However, it isalso possible that a mutation-independent mechanism of p53inactivation is involved, as we had previously described in asubset of breast carcinomas (16). In particular, this idea wassupported by data on human NB cell lines that showed greatlyelevated levels of immunoprecipitable wild-type p53 from celllysates in 4/5 cases (26). No analysis of cellular compartmen-talization of p53, neither in NB tumors nor in cell lines, hadpreviously been reported. We therefore undertook a detailedlocalization study and correlated the data with p53 andMDM-2 gene analysis.

Ninety-six percent (30/31) of undifferentiated NBs repre-senting all clinical stages showed marked cytoplasmic p53accumulation with concommitant nuclear exclusion. All of 10such cases revealed wild-type coding sequences. The strikinghomogeneity of this aberrant localization is mirrored by theuniform absence of p53 gene alterations. Its significance lies inthe fact that nuclear exclusion of p53 completely inactivates itsfunction as a transcription factor and growth suppressor (1).This impressive prevalence makes cytoplasmic sequestration ofwild-type p53 the unique molecular abnormality of NB, farexceeding N-myc amplification, chromosome lp 36 LOH, andother cytogenetic abnormalities. Furthermore, since it is foundin the earliest stages, the alteration appears to occur early intumor progression in contrast to N-myc amplification, which isfound only in late stages. Cytoplasmic sequestration in NB isnot due to deregulated p53 expression secondary to p53 geneamplification nor to increased MDM-2/p53 complex forma-tion secondary to MDM-2 gene amplification. While geneamplification is the mechanism for MDM-2 overexpression

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FIG. 6. Lack of p53 overexpression in ganglioneuroblastoma (case 32). These differentiated tumors show areas of large ganglion cells embeddedin mature "neurofibromatous" spindle cell stroma. Ganglion cells show equal staining intensity with nonimmune IgG and p53. Normal gangliaappear identical. (A) Mouse IgG. (B) PAb 1801. (C) DO-1. (Alk Phos/fast red; x130.)

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found in vivo (15), increased translation of MDM-2 has beenfound in two choriocarcinoma cell lines (28) and our tumorsmust be tested for this remote possibility. While our datacannot formally rule out the possibility of coexisting nuclearp53 protein present below detectability, several reasonsstrongly argue for a true functional impairment of p53 in thesetumors. (i) These cells lost growth suppression and becametumorigenic. (ii) The loss of p53's genome guardian functionis emphasized by the presence of N-myc amplifications (3, 4).(iii) To directly test for p53 function in these tumors, weestablished an in vitro model for p53 in human NB cell linesthat mimic the phenotype of primary tumors. When these cellsare challenged with DNA-damaging agents, cell cycle analysisshows that they exhibit impaired p53-mediated G1 arrest(U.M.M., unpublished data).

In contrast, tumor areas that had undergone ganglionicmaturation lacked p53 overexpression. All differentiated re-gions of 14 malignant ganglioneuroblastomas and 1 benignganglioneuroma failed to show detectable p53, indicating thataberrant p53 sequestration in NB is intimately associated withfailure to differentiate and malignancy. The down-regulationof elevated p53 to normal (undetectable) levels seems to occurduring morphologic maturation. The differentiation-associ-ated conversion from abnormal to normal phenotype indicatesthat the p53 alteration is potentially reversible and suggestsreconstitution of functional p53. In this context it is interestingthat wild-type p53 is involved in pre-B-cell differentiation (6).Our tissue data are also supported by an in vitro model of NBdifferentiation (refs. 26 and 29; U.M.M., unpublished data).The abnormal p53 level in undifferentiated cell lines is mainlydue to increased protein stability (>8 hr) but also to increasedlevels of mRNA. No other stabilizing protein is detected byimmunoprecipitation (26). When the LA-N-5 line is induced todifferentiate with retinoic acid, the high level of p53 dropsbelow detection after 48 hr (refs. 26 and 29; U.M.M., unpub-lished data). In addition, the cells become growth arrested andlose their tumorigenicity in nude mice (29). Therefore, cyto-plasmic accumulation of p53 in undifferentiated NB cells mayreflect an aberrant nuclear translocation pathway due to ablock in maturation.

We thank A. Ostermeyer for technical assistance, Dr. L. Gelbert ofBristol-Myers Squibb, Princeton, for assistance in sequencing, and theInstitut Gustave Roussy Pediatric Department for clinical data. Thiswork was supported by National Cancer Institute Grant CA 60664-02and American Cancer Society Grant IFRA-477 (U.M.M.).1. Shaulsky, G., Goldfinger, N., Tosky, M., Levine, A. J. & Rotter,

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