Proc. Nati. Acad. Sci. USAVol. 90, pp. 5539-5543, June 1993Medical Sciences

Inactivation of the NFI gene in human melanoma andneuroblastoma cell lines without impaired regulation of GTPRasMAUREEN R. JOHNSON*, A. THOMAS LOOKt*, JEFFREY E. DECLUE*, MARCUS B. VALENTINEtf,AND DOUGLAS R. LOWY*§*Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, MD 20892; tDepartment of Experimental Oncology, St. Jude Children's ResearchHospital, Memphis, TN 38105; and $Department of Pediatrics, University of Tennessee College of Medicine, Memphis, TN 38163

Communicated by Alfred G. Knudson, February 12, 1993 (received for review Novemlber 13, 1992)

ABSTRACT The NFI gene, which is altered in patientswith type 1 neurofibromatosis, encodes neurofibromin, a pro-tein whose GTPase-activating function can negatively regulateGTP Ras by accelerating its conversion to inactive GDPRas. Inschwannoma cell lines from patients with neuroffbromatosis,loss of neurofibromin was previously shown to be associatedwith impaired regulation of GTP Ras. Our analysis of otherneural crest-derived tumor cell lines has shown that somemelanoma and neuroblastoma cell lines established from tu-mors occurring in patients without neurofibromatosis containreduced or undetectable levels of neurofibromin, with concom-itant genetic abnormalities of the NFI locus. In contrast to theschwannoma cell lines, GTP Ras was appropriately regulatedin the melanoma and neuroblastoma lines that were deficient inneuroflbromin, even when c-H-ras was overexpressed in thelines. These results demonstrate that some neural crest tumorsnot associated with neurofibromatosis have acquired somati-cally inactivated NFI genes and suggest a tumor-suppressorfunction for neurofibromin that is independent of Ras GTPaseactivation.

Type 1 neurofibromatosis is a dominantly inherited diseasewhose principal manifestations involve tissues derived fromthe neural crest (reviewed in ref. 1). The abnormal geneticlocus in neurofibromatosis, NFI, is thought to be a tumor-suppressor gene. In addition to neurofibromas, patients withneurofibromatosis are at an increased risk of developingcertain tumors derived from neural crest cells, such as benignpheochromocytomas and malignant schwannomas. Consis-tent with the role of NFI as a tumor-suppressor gene,pheochromocytomas and malignant schwannomas arising inpatients with neurofibromatosis are associated with somaticinactivation of their normal NFl allele (2-4).The NFI-encoded protein, neurofibromin, has extensive

homology with two negative regulators of Ras in Saccharo-myces cerevisiae, IRAl and IRA2 (5-7). NFl and the IRAgenes share homology with the domain of the mammalianGAP gene that encodes its GTPase-activating function,which negatively regulates mammalian Ras protein by cata-lyzing the conversion of the active GTP-bound form of Rasto the inactive GDP-bound form (8, 9). The GAP-relateddomain of the NFl protein product (NF1-GRD) also pos-sesses a Ras-specific GTPase-activating function (GAP-likeactivity), and the NFl sequences encoding NF1-GRD cansubstitute for the IRA genes in yeast (7, 10, 11). Sinceactivating Ras mutations have been found in human tumorsand are implicated in the pathogenesis of many cancers (12),these observations suggest that neurofibromin may act as atumor-suppressor protein through its GAP-like activity, func-tioning as a critical negative regulator of Ras. Indeed, studiesof schwannoma cell lines from neurofibromatosis patients

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have shown that low levels of neurofibromin and the subse-quent reduction of GAP-like activity are associated with anincrease in GTP-Ras, resulting in the stimulated growth ofthese lines (13, 14). These observations have provided evi-dence for the hypothesis that the GAP-like activity of neu-rofibromin is responsible for the upstream negative regula-tion of Ras in these lines, defining a function for the tumor-suppressor activity of neurofibromin.As in neurofibromatosis, patients with other dominantly

inherited conditions that carry a mutation of a tumor-suppressor gene, such as Rb in familial retinoblastoma andp53 in Li-Fraumeni syndrome, have a characteristic spec-trum of tumors for which they are at an increased risk.Mutations ofp53 and Rb are also found in certain tumor typesnot associated with the familial cancer syndromes (15, 16). Todetermine if somatically acquired alterations of NFI arefound in other types of tumors, we have analyzed cell linesderived from neural crest tumors and other tumors estab-lished from patients without neurofibromatosis.

MATERIALS AND METHODSCell Lines. The ST-8814 schwannoma cell line was estab-

lished from a neurofibromatosis patient (17). The nine mel-anoma cell lines used to study NFI gene expression wereobtained from the American Type Culture Collection CellRepository. The childhood solid tumor cell lines (all derivedat St. Jude Children's Research Hospital) included sevenrhabdomyosarcoma cell lines, eight Ewing sarcoma cell lines,and three brain tumor cell lines, some of which have previ-ously been described (18-21). Fourteen neuroblastoma celllines were analyzed: NB1, NB2, NB3, NB4, NB5, NB6,NB7, NB8, NB9, NB10, NB13, NB14, NB16, and NB19[NB6, NB8, NB2, and NB3 lines have been reported asN2307L, N1108L, N0303L, and N1003L (22), and NB5, NB7,NB14, and NB16 were reported as NJF, NCG, NCC, andNKP (23)]. All of the neuroblastoma cell lines have N-MYCgene amplification, except NB1, NB3, NB9, and NB16,which have single copy N-MYC levels (24). The cell lineshave been passaged 60-170 times. NIH 3T3 cells infectedwith a retrovirus that expresses v-H-ras and neomycin re-sistance (neor) (pBW1423) (25) were used as a positivecontrol in the in vivo guanine nucleotide-binding experi-ments.

Cell Extracts and Western Blot Analyses. Cell lysates weremade in 20 mM Tris'HCl, pH 8.0/100 mM NaCl/5 mMMgCl2/0.5% Nonidet P-40, containing aprotinin at 17 .g/ml,leupeptin at 10 ug/ml, and 1 mM dithiothreitol. After deter-mination ofprotein concentration by the BCA assay (Pierce),50 &g of each cell lysate was loaded onto an SDS/8%polyacrylamide gel (GAP) or an SDS/6% polyacrylamide gel(neurofibromin). Western blot analyses were carried out as

Abbreviation: FISH, fluorescence in situ hybridization.§To whom reprint requests should be addressed.

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previously described (13), using a 1:1000 dilution of theanti-GAP RH6-2A antibody (supplied by F. McCormick,Onyx Pharmaceuticals, Richmond, CA), or 1 Mg/ml of theanti-neurofibromin 3' antibody (26).Northern and Southern Blot Analyses of Neuroblastoma Cell

Lines. RNA and DNA purified from the cell lines wereanalyzed by Northern and Southern blotting techniques,following standard procedures (27). Twenty micrograms ofmRNA from each cell line was hybridized with either the NFlcDNA probe FB50 (6), generously provided by R. M. Caw-thon and R. White, or a f-actin probe. Five micrograms ofDNA from each cell line was digested with HindIII restrictionendonuclease and was hybridized with the same NFl cDNAprobe. DNA was also obtained from a mouse-human hybridcell line that has retained only an intact human chromosome17 (NA10498; Coriell Institute for Medical Research, Cam-den, NJ).

Fluorescence in Situ Hybridization (FISH). Colcemid (0.05p,g/ml) was added to tumor cells growing in culture, andslides containing fixed cells were prepared as previouslydescribed (28). The hybridization and fluorescence detectionconditions were as previously described (29), except that thehybridization solution contained 65% formamide and humanhighly reiterated competitor DNA at 100 ,ug/ml. DNAs fromtwo contiguous NFl cosmid clones [T315 and 7D5, gener-ously provided by R. M. Cawthon and R. White (30)] werenick-translated with digoxigenin-11-UTP and combined withthe biotinylated probe for the chromosome 17 centromere(D17ZJ, Oncor) in the hybridization mixture. Signals weredetected by incubating the slides with fluorescein-conjugatedsheep anti-digoxigenin antibodies (Boehringer Mannheim)and Texas red-conjugated avidin (Vector Laboratories), fol-lowed by signal amplification with fluorescein-conjugatedrabbit anti-sheep antibodies (Boehringer Mannheim), biotin-ylated anti-avidin, and then additional Texas red-conjugatedavidin and fluorescein-conjugated sheep anti-digoxigenin an-tibodies. Slides were counterstained with 4',6-diamidino-2-phenylindole (DAPI).

In Vivo Guanine Nucleotide-Binding Assays. The assayswere performed with subconfluent, serum-starved cells (31).Cells were plated at 0.4-1.2 x 104 cells per cm2. The followingday the cells were starved of serum for 6 hr, then labeled inserum- and phosphate-free medium with [32P]orthophosphate(Amersham) at 0.35-0.5 mCi/ml (1 Ci = 37 GBq) for 10 hr.Cell lysis, immunoprecipitation of p2lrms with the monoclonalantibody Y13-259 (32), and chromatography of the solubi-lized nucleotides were performed as previously described(13). Quantification of the chromatograms was performed onan AMBIS radioanalytic imaging system, and the resultswere normalized for phosphate content. In Fig. 4B, the cellswere infected with a retrovirus that expresses c-H-ras andneor (pBW1631) (25), and in vivo guanine nucleotide-bindingassays were performed as described above.

RESULTS

To determine if abnormalities of neurofibromin occur in otherneural crest-derived tumors obtained from patients withoutneurofibromatosis, Western blot analyses of lysates fromnine melanoma lines were studied (Fig. 1A). The level ofneurofibromin was reproducibly reduced in four lines: SK-MEL-24, SK-MEL-28, HT-144, and Hs695T. The slowermigration of neurofibromin in the Hs695T melanoma cell linewas not seen in subsequent gels. A fifth line, RPMI-7951,showed barely detectable levels of neurofibromin. The ST-8814 schwannoma line from a neurofibromatosis patientshowed no detectable neurofibromin under these conditions(Fig. 1A). In contrast to neurofibromin, the level of theproduct of the GAP gene, p120GAP, was similar in all of thelines. Lysates from eight Ewing sarcoma lines, three brain

A Melanomas

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FIG. 1. Analysis of GAP and neurofibromin (NF) protein levelsin various tumor cell lines. Western blot analysis of melanoma celllines (A) and neuroblastoma cell lines (B). Schwan. represents theST-8814 schwannoma cell line from a neurofibromatosis patient (17).In A all samples were run on a single gel, and in B samples NB14 andNB19 were run on one gel and all other samples were run on anothergel.

tumor lines, and seven rhabdomyosarcoma lines were alsoanalyzed and found to have normal levels of both neurofi-bromin and p120GAP (data not shown).We also examined a series of neuroblastoma cell lines

established from patients without neurofibromatosis. Thistumor does not appear to occur with an increased frequencyin patients with neurofibromatosis (1, 33, 34). Western blotanalysis of lysates from 14 lines showed 3 lines with severelydecreased or absent neurofibromin (NB5, NB10, and NB16),similar to the ST-8814 schwannoma line (Fig. 1B). The levelsof p120GAP were similar in all of the lines. Compared withlines that had normal levels of neurofibromin, the NF1mRNA levels were absent in line NB10 and decreased in linesNB5 and NB16, correlating with their extremely low levels ofneurofibromin (Fig. 2A). Southern blot analyses of genomicDNA hybridized with an NFI cDNA probe showed that NFlsequences from line NB1O were abnormal: NB10 DNAcompletely lacked the 3.8- and 2.1-kb HindIII fragmentspresent in the other lines examined (Fig. 2C). Both of theseHindIll fragments were present in a mouse-human hybridcell line that contains only human chromosome 17 (data notshown), indicating that these fragments contain coding se-quences of the authentic NFl gene, and not a pseudogene oran NFI-related gene. Similar results were obtained with theBgl II restriction enzyme, in which a human chromosome17-specific 4.7-kb NFI fragment was lacking from NB10 (datanot shown). These results suggest homozygous loss of thisportion of the NFl gene and explain the lack of detectableNFl mRNA (Fig. 2A) or protein (Fig. 1B) in the NB1O cellline.To further examine the neuroblastoma and melanoma cell

lines for loss of NFl sequences, we used two-color FISHtechniques (29). In this analysis, slide preparations of the celllines including both metaphase and interphase cells werehybridized with a mixture of two overlapping cosmid probescontaining coding sequences from the middle of the NFlgene, together with a probe specific for the chromosome 17centromere, which allowed detection of loss of NFI se-quences even in cases with chromosome 17 aneuploidy.

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CAU') ° (D . 'RI I- 0

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FIG. 2. Northern (RNA) and Southern (DNA) analyses of neu-roblastoma cell lines. (A and B) The RNAs (20 ,g) were hybridizedwith either an NFI cDNA probe (A) or a 3-actin probe (B). Positionsof ribosomal RNAs (28S and 18S) and the NFI mRNA (arrowhead)are shown. (C) The DNAs (5 p.g) were digested with Hindlllrestriction endonuclease and hybridized with the same NFl cDNAprobe. Arrowheads denote the restriction fragments missing in DNAfrom the NB1O cell line. Lane 1 contains DNA from a controllymphoblastoid cell line. The mobilities of bacteriophage A HindlIlfragments of known lengths (noted in kilobase pairs) are indicated.

Representative results with this technique in the NB5 cellline are shown in Fig. 3. Metaphase NB5 cells each containedtwo copies ofchromosome 17, as indicated by hybridization tothe chromosome 17 centromere probe (labeled red and indi-cated by arrows). The NFI probes (labeled green and indi-cated by arrowheads) hybridized to one ofthese chromosomesat the appropriate location adjacent to the centromere in theproximal region of the long arm ofchromosome 17. The otherchromosome 17, which appeared morphologically abnormalby trypsin-Giemsa banding, failed to hybridize to the NFIprobes in each of 15 metaphases examined. Similarly, eachinterphase cell contained two copies of the chromosome 17centromere, but only one copy of the NFl gene was detectedin each cell (arrowhead, Fig. 3).

Analysis of the other cell lines showed loss of NFl se-quences relative to the number of chromosome 17 cen-tromeres in one additional neuroblastoma cell line (NB1) andone of the melanoma cell lines (RPMI-7951). These dataprovide evidence for disruption of the NFl gene in two of thethree neuroblastoma lines (NB5 and NB10) and the mela-noma cell line (RPMI-7951) with low or undetectable levels ofneurofibromin. The third neuroblastoma line with low levelsof neurofibromin, NB16, did not show detectable abnormal-ities of the NFl gene when analyzed by Southern blotting orFISH, suggesting that a large deletion of the NFl gene is notresponsible for the lack of protein in this line. These resultsimply that somatically acquired NFl mutations leading toreduction of neurofibromin conferred a selective growthadvantage to the cells and may have contributed to thepathogenesis of these tumors.To determine if the reduced levels of neurofibromin were

associated with an increase in GTP-Ras, in vivo levels ofguanine nucleotides bound to Ras were analyzed in repre-sentative melanoma cell lines (Fig. 4A). The control cell lines,NIH 3T3 cells and v-H-ras-transformed NIH 3T3 cells,showed low and high levels of GTP-Ras, respectively. Aspreviously reported, a high proportion of the Ras protein was

FIG. 3. FISH. Hybridizations to metaphase (A) and interphase(B) cells from the NB5 cell line were performed with fluorescein-labeled NFl cosmid DNA and a Texas red-labeled chromosome 17centromere probe. For each field, 4',6-diamidino-2-phenylindolestained DNA fluorescence is shown on the right and fluorescein andTexas Red fluorescence on the left. Arrows indicate chromosome 17centromeres. Arrowheads indicate hybridization of the NFl cosmidprobe.

in the active, GTP-bound form in the ST-8814 schwannomacell line, which lacks neurofibromin (Fig. 4A; refs. 13 and 14).In contrast, the proportion ofGTP-Ras in the melanoma lineswith low (HT-144 and SK-MEL-28) or absent (RPMI-7951)neurofibromin was similar to that in lines with normal levelsof neurofibromin (NIH-3T3 and SK-MEL-3). These resultsimply that neurofibromin might not be an important regulatorof GTP-Ras in the melanoma lines. Analyses of Ras/RaplAchimeras have also suggested that neurofibromin may not benecessary to maintain Ras in the inactive state in mouse NIH3T3 fibroblasts (35).To confirm that neurofibromin is not an important negative

regulator of GTP-Ras in the melanoma lines, we isolatedc-H-ras-overexpressing derivatives of representative celllines by infection with a retrovirus that encodes the normalc-H-ras gene product. Overexpression of c-H-ras in controlNIH 3T3 cells, which contain normal levels of neurofibrominand p120GAP, resulted in a decrease in the proportion ofGTP*Ras, as previously described (31), suggesting that in thisline the positive regulation of Ras is limiting relative to itsnegative regulation (Fig. 4B). In the control ST-8814 schwan-noma line, which contains barely detectable neurofibromin,overexpression of c-H-ras was associated with an increase inthe proportion of GTP-Ras, reflecting deficient negativeregulation associated with its lack of neurofibromin. Theresults obtained in the HT-144, SK-MEL-28, and RPMI-7951melanoma lines were similar to those obtained in the NIH 3T3cells: overexpression of c-H-ras resulted in a lower propor-tion of GTP-Ras, whether the lines had reduced (HT-144 andSK-MEL-28) or barely detectable (RPMI-7951) neurofibro-

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FIG. 4. In vivo guanine nucleotide-binding ofRas in tumor cell lines. (A) Melanoma cell lines. (B) Melanoma cell lines overexpressing c-H-ras.(C) Neuroblastoma cell lines. Neurofibromin (NF) status: NOR, normal; NEG, negative; LOW, low. In A and C, the data represent the mean± the standard deviation from at least three experiments. B depicts a representative experiment, which was repeated two additional times withqualitatively similar results.

min. These findings indicate that, in contrast to the schwan-noma cell line, in the melanoma lines neurofibromin is not amajor regulator ofGTP Ras. GAP, or other molecules, mustbe sufficient to keep Ras predominantly in the inactiveGDP-bound state in these cells.When in vivo levels of GTP-Ras were determined for three

representative neuroblastoma lines, no correlation was foundbetween the level of neurofibromin in a line and the propor-tion ofRas in the GTP-bound state (Fig. 4C). The proportionof GTP-Ras in the two lines analyzed that lacked neurofibro-min (NB5 and NB16) was similar to that in a neuroblastomaline with normal neurofibromin (NB7). Therefore, the resultssuggest that neurofibromin may have a function that isindependent of its GAP-like activity in the neuroblastoma aswell as the melanoma cell lines.

DISCUSSIONOur studies have shown that several melanoma (5/9) andneuroblastoma (3/14) lines contain abnormally low levels ofneurofibromin. Although it remains possible that these ab-normal levels of neurofibromin are an artifact of the cultureconditions, a large number of Ewing sarcoma, rhabdomyo-sarcoma, and brain tumor lines established and grown undersimilar conditions showed normal levels of neurofibromin(unpublished observations). Furthermore, reduced neurofi-bromin was associated with abnormalities of the NFI locus,as determined by Southern blotting and FISH analyses,suggesting that somatically acquired abnormalities that re-sulted in decreased neurofibromin expression contributed agrowth advantage to the cells. The fact that neurofibrominwas reduced, rather than absent, in some lines indicateseither that the protein that is present in these lines isnonfunctional or that the reduced level of functional proteinwas sufficient to stimulate growth.

If the results obtained with the cell lines reflect changesthat were present in the tumors, the data presented heresuggest that reduced or absent neurofibromin may occur withsome frequency in melanomas and neuroblastomas. Activat-ing ras mutations have been described in melanomas (36-38),and the addition ofexogenous ras to melanoma cells has beenshown to act as positive growth signal (39). Consistent withthese observations, the melanoma lines overexpressing c-H-ras had increased growth rates (unpublished observations).

In contrast to the situation in melanomas, activating rasmutations are not a frequent event in neuroblastomas (40,41), and they may actually be associated with a good prog-nosis (42). Although the role of ras in neuroblastomas has notyet been elucidated, the effects of activated ras in otherneural crest-derived cells are complex. In the rat PC12pheochromocytoma cell line, ras signals differentiation,rather than growth, inducing neurite outgrowth (43, 44). InSchwann cells, ras alone induces growth arrest, but it istransforming when coexpressed with a cooperating nuclearoncogene (45).

It is noteworthy that patients with neurofibromatosis arenot at an increased risk of developing melanomas or neuro-blastomas, possibly because NFl inactivation does not ini-tiate malignant transformation in melanocytes or neuroblastsbut contributes to later steps in the tumorigenic pathway.Analogous observations have been made with other tumor-suppressor genes associated with heritable forms of cancer:p53 mutations have been found in colon cancers, althoughpatients with Li-Fraumeni syndrome do not have an in-creased risk of forming colon cancer (46), and Rb mutationshave been found in small cell lung cancers, although patientswith familial retinoblastoma do not have an increased risk ofdeveloping this tumor (47, 48). Li et al. (49) found a mutationin the NFI gene in 1 out of 22 colon adenocarcinomas and in1 out of 28 peripheral blood samples from patients withmyelodysplastic syndrome, two conditions which do nothave an increased incidence in neurofibromatosis patients.The inactivation of tumor-suppressor genes at certain stagesof development, in the proper context of other activatedoncogenes, growth factors, or hormones, could confer agrowth advantage to form tumors in tissues not associatedwith the inherited disease.Our studies demonstrate that there is no increase in the

levels of GTP-Ras in the melanoma and neuroblastoma celllines that have reduced or absent neurofibromin, even in themelanoma lines that overexpress c-H-ras. Neurofibromin isnot functioning in these lines as a major negative regulator ofRas, in marked contrast to schwannoma cell lines derivedfrom neurofibromatosis patients (13, 14). Therefore, thepostulated growth inhibitory function of neurofibromin inthese lines appears to be independent of its GAP-like activity.This putative function would presumably be distinct fromthat proposed by Li et al. (49), who suggested that the amino

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acid substitution they found in the neurofibromin GAP-related domain contributed to a loss ofGAP-like activity, andtherefore a loss of negative regulation of Ras.The mechanism by which neurofibromin negatively regu-

lates the growth of the melanoma and neuroblastoma celllines remains to be elucidated. Neurofibromin may act as acompetitor, inhibiting the interaction of Ras with positivedownstream targets. Alternatively, the protein may functionas a downstream target of Ras that normally inhibits cellgrowth (50), although our findings do not rule out a functionof neurofibromin that is independent of Ras.

We thank W. C. Vass, J. Kim, K. Jolly, B. Jones, S. Nooner, andS. Rowe for technical assistance. A.T.L. is supported by NationalInstitutes of Health Grants CA-23099 and CA-21675 and by theAmerican Lebanese Syrian Associated Charities of St. Jude Chil-dren's Research Hospital.

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