Proc. Nadl. Acad. Sci. USAVol. 84, pp. 6317-6321, September 1987Neurobiology

Activated neu oncogene sequences in primary tumors of theperipheral nervous system induced in rats bytransplacental exposure to ethylnitrosourea

(neurogenic tumor/chemical carcinogenesis/transforming gene)

ALAN 0. PERANTONI*, JERRY M. RICE*, CARL D. REED*, MASAHIRO WATATANI*, AND MARTIN L. WENKt*Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD 21701-1013; and tMicrobiologicalAssociates, Inc., Bethesda, MD 20816

Communicated by Howard A. Bern, June 1, 1987 (received for review February 11, 1987)

ABSTRACT Neurogenic tumors were selectively inducedin high incidence in F344 rats by a single transplacentalexposure to the direct-acting alkylating agent N-ethyl-N-nitrosourea (EtNU). We prepared DNA for transfection ofNIH3T3 cells from primary glial tumors of the brain and fromschwannomas of the cranial and spinal nerves that developedin the transplacentally exposed offspring between 20 and 40weeks after birth. DNA preparations from 6 of 13 schwan-nomas, but not from normal liver, kidney, or intestine oftumor-bearing rats, transformed NIH 3T3 cells. NIH 3T3clones transformed by schwannoma DNA contained rat repet-itive DNA sequences, and all isolates contained rat neuoncogene sequences. One schwannoma yielded a transformantwith rat-specific sequences for both neu and N-ras. A pointmutation in the transmembrane region of the putative proteinproduct of neu was identified in all six transformants and in theprimary tumors from which they were derived as well as in 5of 6 schwannomas tested that did not transform NIH 3T3 cells.Of 59 gliomas, only one yielded transforming DNA, and anactivated N-ras oncogene was identified. The normal cellularneu sequence for the transmembrane region, but not themutated sequence, was identified in DNA from all 11 gliomassurveyed by oligonucleotide hybridization. Activation of theneu oncogene, originally identified [Schechter, A. L., Stern,D. F., Vaidyanathan, L., Decker, S. J., Drebin, J. A.,Greene, M. I. & Weinberg, R. A. (1984) Nature (London) 312,513-516] in cultured cell lines derived from EtNU-inducedneurogenic tumors that by biochemical but not histologiccriteria were thought to originate in the central nervous systemin BD-IX rats, appears specifically associated with tumors ofthe peripheral nervous system in the F344 inbred strain.

The occurrence of dominant transforming genes in chemi-cally induced neoplasms of rodents invites the hypothesisthat these genes play a role in the development of the tumorsin which they occur. In those systems (1-4) in which onespecific activated gene is consistently found-for example,the Harvey-ras gene (Ha-ras-1) in mammary carcinomasinduced by methylnitrosourea (MeNU) in rats (1) and inchemically induced skin papillomas in mice (4), the Kirsten-ras gene (Ki-ras-2) in chemically induced renal mesenchymaltumors in rats (2), and the N-ras gene in chemically inducedthymic lymphomas in mice (3)-it is likely that activation ofthe cellular protooncogene to a transforming gene within thetumor cells is an important event in the pathogenesis of theseneoplasms. When exposure to the carcinogen is a single,transient event (1, 2, 4) and the distinguishing feature of theactivated gene is a mutation (5) that is consistent with theknown chemical reactivity of the carcinogen (6), activation of

the oncogene may be a direct result of reaction with thecarcinogen and constitute the initial event in carcinogenesis.

In this report we demonstrate activated neu oncogenesequences specifically in EtNU-induced tumors of the pe-ripheral nervous system but not in tumors of the centralnervous system. Activation is believed to result from thesame point mutation in the transmembrane domain of the neugene product described by Bargmann et al. (7) for neurogenictumor cell lines because DNA preparations from the schwan-nomas hybridize preferentially with an oligonucleotide se-quence of this region that contains a T-- A transversion. Thisobservation in primary peripheral nervous system tumorsdiffers from the study by Schechter et al. (8), who reportedthe activation of neu in established tumor cell lines describedas originating in the central nervous system but characterizedonly by biochemical criteria and not by histologic evaluationof the neoplasms (9).

MATERIALS AND METHODSCells and Probes. NIH 3T3 mouse cells (clone 5611) were

the gift of S. Sukumar (BRI, Frederick, MD). Cells weremaintained in Dulbecco's modified Eagle's medium (DMEM)(4.5 g of glucose per liter) with 10% calf serum. The v-erbBprobe (Lofstrand Labs, Gaithersburg, MD) was derived froma BamHI fragment of the avian erythroblastosis viral ge-nome. The plasmid neuc(t)/sp6400 from which we obtaineda 420-base-pair (bp) BamHI neu-specific fragment was pro-vided by R. Weinberg (Massachusetts Institute of Technol-ogy). Rat repetitive sequences were contained in a Pst Ifragment from p3B5 provided by A. Furano (National Insti-tute of Arthritis, Diabetes, and Digestive and Kidney Dis-eases). The ras oncogene probes (Lofstrand Labs) includeda 2.9-kb Sac I/Sac I fragment of c-Ha-ras-1 from human fetalliver, a 1-kb Sst I/HindIII fragment from the human c-Ki-ras-1 gene, and exons I and II (1 kb) from the human N-rasgene. Oligonucleotide probes were synthesized using aBiosearch model 8600 DNA synthesizer by L. Lee (ProgramResources, Frederick Cancer Research Facility). Sequenceswere confirmed by dideoxynucleotide chain-termination se-quencing. The 20-mer oligonucleotides were end-labeled withT4 polynucleotide kinase.Tumor Induction. N-ethyl-N-nitrosourea (EtNU) was se-

lected for use because, in most strains of rats, a singletransplacental exposure rapidly and selectively induces tu-morigenesis in the central and peripheral nervous systemswhile most other tissues remain unaffected (10, 11). Timedpregnant F344/NCr rats (Animal Production Area, FrederickCancer Research Facility) were anesthetized and given 0.2mmol ofEtNU per kg of body weight by i.v. injection into the

Abbreviations: EtNU, N-ethyl-N-nitrosourea; MeNU, methylnitro-sourea; Ki-ras-2, Kirsten-ras gene.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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saphenous vein on day 15 of gestation. A total of 55 male and56 female offspring were nursed by their natural mothers andseparated by sex at 4 weeks of age. Offspring that developedstupor, irritability, paresis/paralysis, or palpable masseswere killed, and the cranial, thoracic, and abdominal cavitieswere opened and examined. A portion of every abnormalmass and of grossly normal brain, liver, kidney, and smallintestine was fixed in Bouins' solution and processed forhistology; the remainder of each mass and each grosslynormal organ was frozen individually on solid CO2 and storedat -800C. The spinal cord was fixed in situ and processed forhistology only. Rats that died were necropsied, and selectedtissue was processed for histology, but no tissue sampleswere frozen for DNA extraction.

Establishment of Transfected Cell Lines. High-Mr DNA wasisolated as described by Sukumar et al. (1). Thawed mincedtissues were dissociated and digested overnight in 10 mMTris.HCl/2 mM EDTA, pH 7.8, containing 0.5% NaDodSO4and Pronase (400 ,ug/ml), then extracted with phenol/chlo-roform/isoamyl alcohol (25:24:1) and precipitated with abso-lute ethanol (-20'C). Precipitates were suspended in 10 mMTris.HCl/1 mM EDTA, pH 7.5. Transfection assays weredone as described (12, 13). DNA preparations (20 ,tg per100-mm dish) were precipitated in a 125 mM calcium/0.7 mMphosphate solution for 1 hr, then applied to cultures of NIH3T3 cells (1.5 x 105 cells per 100-mm dish) for 22 hr. Cultureswere washed 3 x with DMEM containing 5% calf serum, andmedium was replaced every 2-3 days thereafter. Foci exhib-iting a morphologically transformed phenotype were clonedin agar to demonstrate anchorage-independent growth and togenerate a relatively homogeneous transformed cell popula-tion.

Analysis of Chromosomal DNA. DNA preparations fromneurogenic tumors were analyzed for indicated sequences asdescribed by Southern (14). Ten micrograms of high-Mr DNAwas cleaved with appropriate restriction enzymes, and frag-ments were electrophoresed overnight at 25 V in 0.7% agaroseand blotted by capillary elution to nitrocellulose. Hybridizationwith 32P-labeled probes was done at 42°C with various concen-trations of formamide depending on the probe.

Analyses using oligonucleotides were done essentially asdescribed by Bargmann et al. (7). Ten micrograms of DNApreparations was digested with HindIlI, and fragments wereseparated in 1% agarose (5 mm) at 20 V overnight. Fragmentswere denatured in 0.5 M NaOH/0.6 M NaCl for 30 min andneutralized in 1 M Tris HCl, pH 7.4/1.5 M NaCl. Gels werethen dried onto 3MM Whatman paper with a Bio-Rad slab geldryer, removed from the paper by soaking the gel in distilledwater, and incubated for 2 hr in a solution of 2.5 x standardsaline phosphate EDTA (SSPE; 1 x SSPE = 0.18 M NaCl/10mM sodium phosphate, pH 7.4/1 mM EDTA)/10XDenhardt's solution (1x Denhardt's solution = 0.02% poly-vinylpyrrolidone/0.02% Ficoll/0.02% bovine serum albu-min)/500 ,ug of sonicated salmon sperm DNA per ml/0.1%NaDodSO4 at 57.5°C. Labeled oligonucleotides (6 x 106cpm/ml) were added to the gels in an identical solution andincubated at 57.5°C for 16 hr. Gels were subsequently washedtwice (10 min each) at room temperature in 6x standardsaline citrate (SSC; 1 x SSC = 0.15 M sodium chloride/0.015M sodium citrate, pH 7), once for 30 min at 50°C, and oncefor 30 min at 57.5°C. Labeling patterns were recorded onKodak X-Omat AR film using an intensifying screen.

RESULTSTumors of the central and peripheral nervous system werereadily induced in F344/NCr rats by transplacental exposureto EtNU (Table 1). Of 55 male and 56 female offspring thatdied or were killed by 74 weeks of age, only one, a male thatdied of leukemia at 29 weeks, had no neurogenic tumor.

Table 1. Transplacental neurotropic carcinogenesis by EtNU inF344 rats

AverageInduced tumor survival,

Type Origin No. Hosts, no. weeks

Glioma Brain 119 94 34Glioma Spinal cord 13 13 30Schwannoma Cranial nerves 16 16 30Schwannoma Spinal nerves 29 25 32Schwannoma Peripheral nerves 7 7 42

Between 20 and 40 weeks of age, 85% of males and 79% offemales were killed or died with neurogenic tumors. The mostfrequent neoplasms were gliomas of the brain that usuallydeveloped in the paraventricular regions of the cerebralhemispheres. Large tumors replaced virtually all of one orboth hemispheres, and these tumors provided sufficient DNAfor individual analysis by transfection into NIH 3T3 cells.Anaplasia was a constant feature of virtually all brain tumors;differentiated regions of oligodendroglioma or, less frequent-ly, astrocytoma contained multiple perivascular aggregatesof undifferentiated glial cells that tended to extend beyondthe apparent margins of the tumors. Mitotic frequency wasusually high in foci of anaplasia. Gliomas of the spinal cordwere too small for analysis by DNA transfection. Schwan-nomas (neurinomas) were found both intracranially, origi-nating from cranial nerves, usually the trigeminal, and inother locations. Of 52 schwannomas, 16 were intracranial; 12originated from thoracic spinal nerve roots and presented asmediastinal masses; 17 were intraabdominal and paraspinal inlocation; and 7 were found elsewhere. There was no differ-ence in incidence or latency by site between the sexes.Schwannomas were rapidly growing and aggressively inva-sive and presented the histologic picture typical of theseneoplasms with admixed regions of Antoni type A and Antonitype B tissue, the latter containing prominent cysts (15).Intracranial schwannomas were at least as large as gliomasand were often adherent to the overlying brain, but theseschwannomas were readily distinguished from gliomas byhistologic examination. Schwannomas that originated fromspinal nerve roots tended to generate large, highly cellularmasses within the mediastinum or abdomen, which providedabundant tumor tissue for DNA extraction.DNA of high Mr (.40 kb) was extracted from thawed

tumor tissues and from liver, kidney, and small intestine oftumor-bearing animals and applied to monolayers ofNIH 3T3cells as a calcium phosphate precipitate. DNA preparationsfrom 6 of 13 schwannomas produced transformed foci withcriss-crossed refractile cells. Frequencies of transformationranged from 0.006 to 0.012 foci per ,ug of DNA (based upona minimum of 160 ,ug of DNA tested for each tumor) duringthe first cycle of transfection and increased to 0.06-0.17 fociper ,ug of DNA in the second cycle. No foci of similarmorphology have been seen using DNA preparations fromthe 21 grossly normal tissues examined from tumor-bearinganimals. DNA from only 1 of 59 gliomas yielded transformedfoci on NIH 3T3 cells.

Southern blot analysis of EcoRI-digested DNAs from alltransformed NIH 3T3 clones revealed the presence of ratrepetitive sequences in the mouse cell DNAs. Although nohomology was demonstrable with Ki-ras or Ha-ras probes,under conditions of low stringency a probe for erbB wasfound to hybridize selectively with a 34-kb restriction frag-ment of DNA from NIH 3T3 cells that had been transformedby rat schwannoma DNA. DNA from the only NIH 3T3transformant derived from a glioma preparation (designatedanaplastic glioma 7416) showed no homology to the erbBprobe, but this DNA was found to contain rat-specific N-rassequences. DNA from a single schwannoma yielded a trans-

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formed 3T3 focus that contained rat-specific DNA sequenceshomologous to erbB and to N-ras. We have yet to determineif both sequences represent transforming sequences in the3T3 cells.

Shih et al. (16) demonstrated in 1981 that DNA from celllines that had been established (9) from intracranial tumorsinduced in BD-IX rats by transplacental exposure to EtNUcould transform NIH 3T3 cells. Although these cultured cellswere characterized biochemically as originating in the centralnervous system, no histologic examination of primary tumortissue was reported. Interpretation of the tumor derivationwas therefore equivocal, because schwannomas also developintracranially. The transforming gene common to all theBD-IX cell lines, later named neu, was subsequently identi-fied (8) and shown to bear homology to erbB and to be atyrosine kinase-type receptor gene (17-19) distinct from andunlinked to the erbB gene that encodes the epidermal growthfactor receptor (20). To confirm that the oncogene in ourschwannoma-derived NIH 3T3 clones was neu, DNA from anormal rat tissue (kidney) or from transformed clones wasdigested with EcoRI plus BamHI. In Southern blots, a 1.8-kbfragment from normal rat DNA (Fig. 1, lane a) was found tohybridize under moderately stringent conditions with a420-bp BamHI fragment of neu, well separated from homol-ogous sequences of mouse origin that appeared as doubletscentered on 7.1 and 11.0 kb in DNA digests of both normal(Fig. 1, lane b) and transformed NIH 3T3 cells (Fig. 1, lanesc-i). A restriction fragment from schwannoma-derived trans-formant DNA that was identified by the neu probe migratedwith the normal rat sequence in agarose gel electrophoresisand appeared as a single band of 1.8 kb on Southern blots.This fragment was consistently identified and amplified in allschwannoma-derived transformant DNA preparations (Fig.1, lanes c-h). Similar results were obtained with the second

a b c d e f g h ikb

cycle transformants, showing the necessity of the neuoncogene for maintenance of the transformed phenotype.Because neu sequences were amplified in NIH 3T3 trans-

formants and because the erbB gene has been reportedamplified in primary human tumors, we evaluated DNApreparations from primary tumors. DNA from three schwan-nomas with activated neu sequences and from three schwan-nomas that did not transform NIH 3T3 cells were included inthis study. A v-erbB probe at low stringency was used in thisexperiment so that we could simultaneously observe foramplification of erbB and neu sequences. No amplification ofeither erbB or neu sequences was seen in primary tumorswith or without transforming neu sequences.

Recently, a single point mutation in the transforming neugene, a T -> A transversion at nucleotide 2012 that changesa valine residue in the transmembrane domain of the pre-dicted protein product to a glutamic acid, has been demon-strated in multiple independently derived tumor cell lines thatoriginated from intracranial tumors of EtNU-treated BD-IXrats and that contain the activated neu gene (7). To determineif a similar mechanism is operative in the F344 rat neurogenictumors, we performed hybridization studies using oligonu-cleotide probes with sequences identical to those describedby Bargmann et al. (7): wild type, 5' ACGCCC(A)CTACA-GTTGCAAT; mutant, 5' ACGCCC(T)CTACAGTTGCAAT.DNA from each of the six NIH 3T3 transformants withtransforming neu sequences was incubated with oligonucle-otides of either wild-type or mutant sequence under stringentconditions. In Fig. 2A, a 5.2-kb HindIII restriction fragmentfrom normal rat kidney DNA (lane a) hybridized with thewild-type sequence, producing a signal of moderate intensity,whereas transformant DNAs (lanes b-g) showed only weaksignals that approximated normal NIH 3T3 intensity (data notshown). When a duplicate gel was incubated with the mutantsequence (Fig. 2B), no signal was apparent in normal ratDNA (lane a); however, an extremely strong signal wasobserved for a 5.2-kb fragment in each of the transformantDNAs (lanes b-g).

kb a b c d e f g

9.4 -6.6- _I*4.4 -

2.3-A

kb a b

9.4 -6.6-4.4-

2.3 -

B

FIG. 1. Demonstration of rat-specific neu sequences in NIH 3T3transformants generated by DNA preparations from neurogenictumors. DNA preparations from normal F344 rat kidney (lane a),normal NIH 3T3 cells (lane b), and transformants derived from F344rat schwannomas (lanes c-h) or anaplastic glioma 7416 (lane i) weredigested simultaneously with BamHI and EcoRI, electrophoresed in

0.7% agarose, blotted to nitrocellulose, and hybridized with a probefor neu (420 bp) under conditions of moderate stringency (30%6formamide, 420C).

FIG. 2. Demonstration of a point mutation of neu in NIH 3T3transformants generated by schwannoma DNAs. Hindfli-digestedDNA preparations from normal F344 rat kidney (lanes a) ortransformants derived from F344 rat schwannomas (lanes b-g) weresize-fractionated in 1% agarose. Dried gels were incubated witheither (A) wild-type oligonucleotides [5' ACGCCC(A)CTACAGT-TGCAAT] or (B) mutant oligonucleotides [5' ACGCCC(T)CTACA-GTTGCAAT] representing sequences from the transmembrane do-main of the protein product.

9.4 W-_~_

6.6 > al

2.3-2.0 __~~~4

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6320 Neurobiology: Perantoni et al.

Recent reports indicate that mutations can arise from theprocess of transfection itself (21, 22); thus, the transformantsfrom the schwannoma DNAs could conceivably result fromour experimental procedures. Although this is unlikely be-cause of the high frequency with which schwannoma, but notglioma, DNAs transformed NIH 3T3 cells, we analyzedDNAs taken directly from the primary schwannomas withour oligonucleotide probes to rule out this alternative. Thesepreparations also preferentially hybridized with the mutantsequence (data not shown), showing that the mutation waspresent in the original tumor DNAs and did not result fromthe transfection process. The signal strength, however, wasconsiderably weaker than that observed for the transform-ants, supporting our earlier observation with the neu frag-ment that amplification of neu had occurred in the transform-ants.

If mutation of neu in the transmembrane region is neces-sary for neoplastic transformation of rat Schwann cells, itwould be expected in all 13 of the schwannomas studied,rather than the 6/13 identified by transfection. Becausetechnical complications, including the large size of the neugene, could have prevented us from finding transformantswith every schwannoma preparation, we examined nontrans-forming schwannoma DNAs for mutated sequences. In fiveof six preparations analyzed, a 5.2-kb restriction fragmenthybridized preferentially with the mutant oligonucleotidesequence. Four of these preparations are pictured in Fig. 3 (Aand B represent hybridizations with normal or mutant oligo-nucleotides, respectively). In Fig. 3B, lanes b-d, schwan-noma DNAs preferentially hybridized with the mutant oli-gonucleotide sequence, yielding a signal of moderate inten-sity and comparable to the level observed for normal rat DNAwith the normal oligonucleotide sequence probe (Fig. 3A,lane a). Slight reactivity of schwannoma DNA with normalsequence (lanes b-d) was also apparent and could result fromcross-reactivity ofprobes or from hybridization of probe witha normal sequence in the rat tumor genome. We noted,however, no reciprocal cross-reactivity, supporting the latterexplanation and suggesting that both alleles need not bemutated or the normal allele lost. Cellular heterogeneity inthe tumor, however, is another explanation that cannot beexcluded. In only one (Fig. 3A, lane e) of six DNAsexamined, a strong signal at 5.2 kb was seen when hybridizedwith normal sequence, whereas no signal was apparent with

kb

9.4-6.6-

4.4-

a b c d e

i"W :`, .~ ..... lw

2.3-A

a b c d ekb

9.4-

6.6- a s am

4.4-

2.3-

B

FIG. 3. Demonstration of a point mutation of neu in primaryschwannoma DNAs that did not transform NIH 3T3 cells. DNA fromnormal F344 rat kidney (lanes a) or primary schwannomas (lanes b-e)was processed as described in Fig. 2 and hybridized with (A)wild-type or (B) mutant oligonucleotides.

the mutant oligonucleotide sequence. Thus, 11 of 13 schwan-nomas contained the same point T -- A transversion muta-tion of the transmembrane sequence.To confirm that the absence of detectable transforming neu

sequences in gliomas was not due to technical consider-ations-i.e., the limited quantities of DNA available fromthese small neoplasms or the efficiency of the transfectionassay-we probed a series ofDNA preparations from gliomaswith the same oligonucleotides for normal or mutated neusequences as described above. DNAs from all 11 gliomastested, including 9 derived from animals that also hadschwannomas and another with an activated N-ras oncogene,contained a 5.2-kb HindIII restriction fragment that hybrid-ized with the normal but not the mutant oligonucleotidesequence. Band intensities were comparable to that ofnormalrat DNA, indicating that this sequence had not becomeamplified in these tumors.

DISCUSSIONSeveral studies now indicate that certain oncogenes, notablymembers of the ras gene family, are activated reproduciblyby point mutation in specific codons in at least some kinds ofchemically induced experimental tumors in rodents, and thatthe position of the mutation is determined by properties of thechemical applied. MeNU (23) invariably induces mammarycarcinoma with codon 12 mutations of the c-Ha-ras-I gene inrats, whereas 7,12-dimethylbenz[a]anthracene (24, 25) andN-hydroxy-2-acetylaminofluorene (26), which also activatethe same oncogene, consistently produce codon 61 mutationsin mouse skin tumors and hepatomas, respectively. Morerecently, the oncogene neu was found activated by pointmutation in established cell lines derived from EtNU-inducedneurogenic tumors from BD-IX rats (7). The mutation, a T --

A transversion in a sequence that encodes the transmem-brane region of the neu gene product, was observed consis-tently in these cell lines. Because the derivation of the celllines remained equivocal, it became important to establishthe observation in EtNU-induced primary tumor tissues. Aswe have reported here, the neu oncogene is activated andmutated in primary schwannomas, tumors of the peripheralnervous system, but not in gliomas, tumors of the centralnervous system that had been thought to be the source of theBD-IX rat tumor cell lines (9). We have observed the same T-- A transversion in the transmembrane domain of neu in 11of 12 F344 rat schwannomas tested (of 13 studied), as hasbeen described for the BD-IX rat cell lines.While the direct-acting alkylating agents such as EtNU are

best known for their capacity to alkylate guanine residues atthe extranuclear oxygen atom at position 6 (6), resulting inpoint G -* A transition mutations such as those that activatethe c-Ha-ras-J oncogene during MeNU-induced mammarycarcinogenesis in rats (23), these agents also alkylate thymineresidues at the extranuclear oxygen atom at position 4 (27).The resulting products are not efficiently repaired (28, 29) bythe suicidal methyltransferase (30) that reverts 06-alkyl-guanine residues to guanine. Thymine O-alkylation productscan lead to transition mutations (T -* C) in vitro (31), and T-3 A transversions have been identified among mutationsproduced in mice by exposure to EtNU in vivo (32). It is thuspossible that the initial event in chemical carcinogenesis byEtNU in the peripheral nervous system of the rat is mutationof the neu protooncogene by O-alkylation of a specificthymine residue.DNA from one schwannoma described in our studies

reacted only with the oligonucleotide of wild-type sequence.Two independently extracted DNA preparations from thissame tumor both showed amplification of the gene but neithertransformed NIH 3T3 cells. Recently the human homologueto neu, c-erbB-2, was isolated (33) and shown to be amplified

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in a number of nonneurogenic human tumors including aprimary salivary gland adenocarcinoma (33), a gastric adeno-carcinoma cell line (34), and in a high proportion of humanmammary carcinomas (35, 36). Activation in human tumorsof c-erbB-2 to a gene capable of transforming NIH 3T3 cellshas not been reported. From studies performed with mem-bers of the ras gene family, the efficiency with which tumorDNAs transform NIH 3T3 cells appears to be somewhatdependent upon the position ofthe ras gene mutation (37, 38).It is thus conceivable that a mutation in neu outside thetransmembrane region would be missed by the inefficienttransfection assay. This one tumor might contain an alter-ation of neu more characteristic of the human tumors de-scribed. It seems unlikely that mere amplification of the geneis sufficient for transformation because Hung et al. (39) havealready shown that high expression of the normal neu gene incells does not produce a transformed phenotype. Therefore,we predict involvement of another heretofore undefinedalteration in this tumor. Because no gross neu gene polymor-phisms were apparent in the restriction fragment profiles withthe enzymes used in our studies, this predicted alterationprobably involves either a point mutation or small deletionnot detectable in agarose gels.The specific association of a transforming gene with a

single kind of neurogenic tumor has several precedents bothin experimental animals and in humans. Introduction of earlyregions of several papovavirus genomes including simianvirus 40 (40, 41) and JC (42) into transgenic mice causesselective development of choroid plexus papillomas (40, 41)and adrenal neuroblastomas (42), respectively. Malignantschwannomas are rarely seen in humans except in individualswith neurofibromatosis, a disorder transmitted as an auto-somal dominant trait (15). The specific association of acti-vated neu with malignant schwannomas in rats extends thispattern and adds a genetic locus independent of the ras genefamily to those that are apparently activated by single pointmutations and, by their consistent presence in specificchemically induced neoplasms, appear to play a role inchemical carcinogenesis in certain tissues.

We are indebted to Deborah Devor and John Henneman forcapable technical assistance.

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