(CANCER RESEARCH 52. 978-982. February 15, 1992]

Chemically Induced Forestomach Papillomas in Transgenic Mice Carry MutantHuman c-Ha-ras Transgenes1

Kiyoshi Ando, Atsuhiko Saitoh, Okio Hiño,Ri-ichi Takahashi, Milioni Kimura, and Motoya Katsuki2

Department ofDNA Biology, School of Medicine, Tokai University, Bohseidai Isehara Kanagawa 259-11 (K. A., A. S., M. Ki., M. Ka.]; Department of Pathology, CancerInstitute, Tokyo ¡O.H./; and Central Institute for Experimental Animals, Kawasaki [R-i. T., M. Ka.], Japan

ABSTRACT

Forestomach papillomas and skin papillomas were induced very efficiently by a single dose administration of the chemical carcinogen meth-ylnitrosourea (MM ) in transgenic mice (rasH2 line) carrying humanhybrid c-Ha-ras genes, which encode the prototype p2l gene product.The incidence of forestomach papillomas was dose dependent; when 50mg/kg of MNU were administered i.p., all of the transgenic mice (56 of56) developed forestomach papillomas within 12 weeks after administration, whereas 5 and 0.5 mg/kg of MNU induced papillomas in 2 of 19and 1 of 19 mice, respectively. Nine of 56 transgenic mice (16%) alsodeveloped skin papillomas at sites wounded by bites or scratches. Only1 of 77 nontransgenic littermates developed forestomach papillomas afteradministration of 50 mg/kg of MNU, and no skin papillomas appearedwithin 12 weeks after MNU administration. The transgenes (integratedcopy number, 5-6) in the tumors developed in 55 of 56 affected transgenicmice (98%) contained at least 1 copy of the transgene that was activatedby somatic point mutation at the 12th codon, from GGC (Gly) to GAC(Asp). Because somatic point mutations at the 12th or 61st codon oftransgenes have never been detected in normal tissues of transgenic micethus far examined, these mutational activations of transgenes are tumor-specific events. RNA expression of these activated transgenes was alsodetected. From these results, it is suggested that somatic mutationalactivation of the human c-l IÜ-/Y/.Vtransgene plays a causative role in theoccurrence of forestomach and skin papillomas induced by MNU administration in these transgenic mice. This transgenic mouse provides aunique screening system for chemicals that induce or suppresspapillomagenesis.

INTRODUCTION

Members of the ras gene family (Ha-, Ki-, and N-ras) areassumed to play a role in signal transduction involved in theregulation of cell proliferation and differentiation in normaleukaryotic cells (1). These oncogenes are those most frequentlyfound to be activated in human cancers and appear to beimplicated in their development (2). They are known to acquiretransforming activity in in vitro cell culture systems such as theNIH3T3 cell line by somatic point mutation in the 12th or 61stcodon and less frequently in the 13th or 59th codon (1).However, the in vivo effects of these mutational changes of theras oncogenes in the development of animal and human cancersremain to be elucidated.

Many analytical systems have been devised to investigate thedevelopmental processes of tumorigenesis. Among them threetypes of animal model systems have shown the causative roleof the ras gene in the development of tumors and their tissue-specific oncogenic activity in vivo. The first system consists of

Received 7/18/91; accepted 12/3/91.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This research was supported in part by a Grant-in-Aid for Cancer Researchand a Grant-in-Aid for Scientific Research on Priority Areas from the Ministryof Education, Science and Culture, Japan: by a Grant-in-Aid from the Ministryof Health and Welfare for the Comprehensive 10 year Strategy for CancerControl, Japan; a grant from Science and Technology Agency, Japan (to M. Ka.);and a grant from the Waksman Foundation of Japan Inc. (to M. Ki.).

1To whom requests for reprints should be addressed. Present address: Depart

ment of Molecular and Cellular Biology. Medical Institute of Bioregulation,Kyushu University 69, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan.

a variety of carcinogen-induced animal tumor models in whichras oncogenes have been found to be reproducibly activated (1,3-5). For example, in rat mammary carcinomas induced by asingle dose of MNU3 during puberty, c-Ha-ras oncogenes were

found to be activated in 82% of treated rats (6). MNU is knownto cause GC to AT transition in DNA via the formation of (f-methylguanine (7). Mice with skin papillomas and carcinomasinduced by treatment with the initiator dimethylbenzanthracenefollowed by multiple applications of the promotor 12-O-tetra-decanoylphorbol-13-acetate show a very high frequency (90%)of activation of c-Ha-ras oncogenes (8). These models alsoprovide good systems for studying the mechanisms of pointmutational activation of raígenes. The nature of the mutationsin ras oncogenes reflects the chemical specificity of the carcinogen, suggesting that the carcinogen interacts with ras sequences (5).

The second type of system consists of transgenic animalmodels that carry activated ras genes devised under control ofvarious kinds of promoters and accomplish their targetedexpression in specific tissues (9,10) or with their own promoters(11). For example, transgenic mice expressing an activated c-Ha-rai oncogene from a suprabasal keratin promoter developedskin hyperkeratosis and papillomas (12).

Thirdly, in the previous report we demonstrated that transgenic mice (rasH2 and rasH7 lines) with hybrid human c-Ha-ras genes, which encode the prototype human p21 gene product,developed tumors spontaneously; these were angiosarcomas,skin papillomas, lung adenocarcinomas, and Harderian glandadenocarcinomas (13). We detected somatic point mutationalactivation and expression of the transgenes in the tumors butnot in the normal tissues. These results suggested that definitesomatic mutations of the transgenes in certain cell types werea causative event in tumorigenesis in these transgenic mice.However, with this approach the tumor incidence was about50% and the latent period was as long as 18 months, since themutational events were sporadic.

In this paper, we presented that the administration of MNU,which is known to cause GC to AT transition in DNA, to rasH2transgenic mice induced forestomach and skin papillomas atvery high frequency within short latent periods.

MATERIALS AND METHODS

Transgenic Mice and MNU Administration. The rasH2 foundermouse was generated from mouse strains C57BL/6 x BALB/c (B6C)F2 eggs as we reported previously (13). Forty-nine F3 and 171 F4offspring of this rasH2 transgenic mice were obtained after backcrossingbetween C57BL/6J females and rasH2 males. They were maintainedin air-conditioned isolators at a temperature of 22°C,and given a

commercial diet in pellet form and tap water ad libitum. One hundredone female mice were housed 3/cage and 119 male mice were kept atI/cage. Methylnitrosourea (Iwai Biochem. Co., Tokyo, Japan) wasdissolved in citrate buffer solution (pH 6.0) just prior to use and injectedonce into each mouse i.p. One hundred thirty-three received 50 mg/kg

' The abbreviations used are: MNU, methylnitrosourea; PCR, polymerase

chain reaction; cDNA, complementary DNA.

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of MNU, 44 of the mice were given 5 mg/kg, and 43 were given 0.5mg/kg at 5 weeks of age.

All animals used were handled in accordance with the guidelinesestablished by the Central Institute for Experimental Animals and bythe School of Medicine, Tokai University.

Histopathological Examination. All mice were sacrificed 12 weeksafter MNU administration by cervical dislocation and were submittedto autopsy. The stomach and duodenum were removed en bloc and splitalong the greater curvature. After the papillomas in the stomach werecounted, they were removed and frozen in liquid nitrogen for analysisof DNA and RNA. Some of the papillomas were fixed in a 4%formaldehyde solution and then embedded in paraffin, sectioned 4 /imthick, and subjected to hematoxylin and eosin staining. Skin papillomasand lymphomas as well as normal tissues including brain, thymus, lung,heart, liver, spleen, kidney, intestine, testis, and ovary were also frozenfor DNA and RNA analyses and fixed in formaldehyde solution forhistopathological examination.

Detection of Somatic Point Mutation of the c-Ha-ras Gene in Tumors.To detect somatic point mutation of human and murine c-Ha-ras genes,the PCR method followed by oligonucleotide hybridization was performed (14). Oligonucleotides, for use as primer for both human andmurine c-Ha-ras genes or as probes for murine c-Ha-raí,were synthesized by Applied Biosystems 381A synthesizer using the manufacturer's

protocols and reagents and were used after deprotection. Sequences ofprimers were the same as those reported by Verlaan-de Vries et al. (14)and Brown et al. (15).

One ng of DNA samples was amplified for 30 cycles by PCR.Amplified DNA was electrophoresed on composite gels consisting of3% NuSieve and 1% Seakem agarose. After transfer to nylon membranes the filters were hybridized with each of the "P-labeled 19-baseOligonucleotides probes: 7 probes for the 12th codon of human c-Ha-ras (GTC-GGC-GCC-GGC-GGT-GTG-G:Gly, CGC:Arg, AGOSer,TGC:Cys, GAOAsp, GCCAla, and GTC:Val) (Dupon), 7 probes forthe 13th codon of human c-Ha-ras (GGC-GCC-GGC-GGT-GTG-GGC-AA:Gly, AGT:Ser, TGT:Cys, CGT:Arg, GAT:Asp, GCT:Ala,GTTjVal) (Clontech), 8 probes for the 61st codon of human c-Ha-ras(ACC-GCC-GGC-CAG-GAG-GAG-T:Gln. CAT:His, CACHis,AAGjLys, GAGjGlu, CTG:Leu, CCGjPro, CGG:Arg) (Dupon), 6probes for the 12th codon of murine c-Ha-raí(TG CGC GCT GGACGC GTG GG:Gly, GAA:Glu, GTA:Val, GCA:Ala, CGA:Arg.AGA:Arg) and 8 probes for thé61st codon of murine c-Ha-ras (ACÕOCA GGT CAÕGAA GAG TA:Gln, AAA:Lys, GAA:Glu, CGA:Arg,CCA:Pro, CTA:Leu, CACHis, CAT: H is), in hybridization buffer of 3Mtetramethylammonium chloride (Wako) (16). The filters were washedas reported previously (14, 15).

Transgene Expression with Somatic Point Mutation in the Tumors.To detect RNA expression of the transgenes with somatic point mutation we performed cDNA synthesis, PCR, and oligonucleotide probehybridization. Each cDNA was synthesized using a cDNA synthesis kit(Amersham) from total RNA isolated from tissues. PCR and oligonucleotide hybridization were performed as described above. To discriminate cDNA from genomic DNA, we used a primer pair correspondingto the different exons (5' primer for the 12th and 3' primer for the

61st codons). The length of the PCR product of cDNA was 216 basepairs, whereas that of genomic DNA was 483 base pairs.

RESULTS

Tumor Induction in Transgenic Mice Caused by MNU. TherasH2 transgenic mouse line was established as reported previously (13). This mouse line carried 5-6 copies of the humanhybrid c-Ha-ras genes, which encode the prototype p21 and areintegrated into the mouse genome in a tandem array. Theyexpressed human c-llu-ras transgene products, p21, as well asendogenous c-Ha-ros gene products. Therefore, the amount ofp21 that was detected was 2-3 times higher in transgenic micethan in non transgenic mice. We obtained 220 (49 F3 and 171F4) offspring of rasH2 transgenic mice, 94 of which weretransgenics.

To examine the dose-response effects of MNU on these mice,171 of them were divided into 3 groups; 43, 44, and 84 weregiven a single injection of 0.5, 5, or 50 mg/kg of MNU i.p. at8 weeks of age, respectively. Twelve weeks after administration,all mice were sacrificed for systemic pathological observation.Twenty-four of the F2 offspring of rasH2 not given injectionsof MNU were also examined as controls at 20 weeks of age.Although no early deaths immediately occurred after administration of MNU, several mice died by 12 weeks after MNUadministration due to intrathoracic expansion of thymomas.

Interestingly, forestomach papillomas developed in all transgenic mice when they received 50 mg/kg of MNU, even thoughthe introduction of MNU was carried out only once i.p. andnot directly into the stomach. This was confirmed by an additional experiment in which 49 rasH2 mice were given 50 mg/kg of MNU i.p. at 10 weeks of age. Total tumor incidences andtypes observed are shown in Table 1. The threshold dose oftumor occurrence seemed to be between 5 and 50 mg/kg. Theseresults clearly indicated that tumor incidence by MNU administration is dose dependent; when treated with 50 mg/kg ofMNU i.p., all 56 transgenic mice developed forestomach papillomas and the average number of papillomas counted macro-scopically per mouse was 5.38 ±2.53 (SD), whereas 5 and 0.5mg/kg of MNU induced papillomas in 2 of 19 and 1 of 19 miceand average numbers were 0.42 ± 1.30 and 0.05 ±0.23,respectively. The incidence of 56/56 was also much higher thanthat of 1 of 77 found in the nontransgenic littermates (P <0.0001). These results demonstrate that the development offorestomach papillomas was due to the effects of both trans-

genes and MNU.Skin papillomas were developed in 9 of 56 mice and the

incidence was clearly higher than the 0 of 77 in the controls (P< 0.0001). All these skin papillomas were developed at siteswounded by bites or scratches. These observations can be explained by the fact that the wounding or the wound-healingprocess is considered to promote skin papilloma formation(17). No papillomas developed at all in nontransgenic litter-mates used as controls. These results suggested that the wound-healing process plays an important role in skin papillomaformation in addition to the transgene function.

Lymphomas including thymomas were observed in 4 of 56transgenic mice and 11 of 77 nontransgenic littermates. Thedifference of the incidences was not significant. Therefore, itwas concluded that lymphomas were not due to the effects ofthe transgene.

These forestomach and skin papillomas were diagnosed as

Table 1 Tumor types and incidences in transgenic ras mice observed afteradministration of MNU

MNU was administered i.p. only once to 10-week-old transgenic mice andtheir nontransgenic littermates. Twelve weeks after administration, each mousewas subjected to systemic observation. No tumors other than papillomas andlymphomas have been detected.

MNUdose Tg.°or No. of Forestomach Skin

(mg/kg) non-Tg. mice papillomas papillomas Lymphomas

00.55.050Tg.Non-Tg.Tg.Non-Tg.Tg.Non-Tg.Tg.Non-Tg.1113192419255677001

(5.3%)02

(10.5%)056(100%)I

(1.3%)0000009(16.1%)00000004(7.1%)11(14.3%)1Tg., transgenic mice carrying human c-Ha-ras genes (rasH2 line).

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Fig. 1. Histológica! appearance of forestomach papilloma (I) and skin papil-loma (B). Bars: A, 200 urn; B, 400 »m.

Codoni2

GGC(Gly)

abcdefghljkli i i i i i i i i i i i

--63bp

GAC(Asp) -63

Fig. 2. Idenlincalion of somatic point mutation of the transgenes in tumorsinduced by MNU administration. A 63-base pair DNA fragment was amplifiedwith exon 1 amplimers, subjected to Southern blot analysis, and probed witholigonucleotides specific for mutations at the 12th codon. DNA samples were asfollows: Lane a, amplified DNA sample from human hybrid c-Ha-ras geneintegrated in rasH2 transgenic mice: Lanes b-i, normal tissues in the rasH2transgenic mouse as in Lane j; Lane h, brain; Lane c, lung; Lane d, heart; Lane e,stomach; Lane f, spleen; Lane g, liver; Lane h, kidney; Lane i, testis; Lane j,forestomach papilloma; Lane k, normal skin of the same rasH2 mouse as in LaneI: Lane I, skin papilloma. The activated transgenes were detected only in tumorsand not at all in normal tissues.

well differentiated papillomas, because the tumor cells showedregular outgrowth of the squamous epithelium in the form ofpapillary fronds with hyperkeratosis and the basement membrane remained intact (Fig. 1). Morphological abnormalitieswere not observed in any other tissues examined, includingtumor-free areas of the skin and forestomach. No carcinomawas found among the ton-stomach and skin papillomas within

the observation period.Somatic Point Mutational Activation of the Transgene in Pap

illomas. In the previous report we demonstrated that spontaneous tumors in rasH2 mice contained somatically activatedtransgenes (13). In the MNU-induced mammary tumor modelof the rat, it was shown that the c-Ha-ras gene in tumor cellswas specifically activated by MNU-induced GC to AT transitions (6). To determine whether the transgene in the tumorsbecame activated, we used the PCR method followed by oligo-nucleotide hybridization (14). Fifty-six pooled samples of fore-stomach papillomas, nine skin papillomas, and four lymphomasthat developed in rasH2 mice receiving 50 mg/kg of MNU wereanalyzed. We demonstrated that all of the 10 independentpapillomas from 3 affected transgenic mice (2, 3, and 5 papil-

lomas/mouse) had somatic mutations at the 12th codon of thetransgenes from GGC to GAC (data not shown). Therefore,forestomach papillomas (average of 5.38 ±2.53/mouse) fromone mouse were pooled and treated as one sample. Normaltissues of the rasH2 mice were also analyzed as controls at thesame time.

PCR products were analyzed by Southern blot hybridizationusing mutation-specific oligonucleotide probes (Fig. 2). TheMNU-induced point mutations of the transgenes were detectedonly in the papillomas and not at all in normal tissue samples.Prototype as well as activated transgenes were detected inpapillomas, since the transgenic mice carried 5-6 copies oftransgenes.

The frequency and position of the mutation of the transgenesare shown in Table 2. All positive samples, 55 of the 56forestomach papillomas, 9 of the 9 skin papillomas, and I ofthe 4 lymphomas, contained GC to AT transition at the 12thcodon in the human c-Ha-ras gene from GGC (Gly) to GAC(Asp). Mutation at the 12th codon from GGC (Gly) to GTC(Val) was detected only in one skin papilloma, coexisting withthe mutation from GGC (Gly) to GAC (Asp). We rarely detected mutations at the first letter G of the 12th codon. In aseries of the same kind of experiment not included in this paper,only one mutation from GGC (Gly) to AGC (Ser) has beendetected thus far. We did not examine mutations at the silentletter. We never detected the mutational activation at the 61stcodon in the transgene that was detected in angiosarcomasoccurring spontaneously in this rasH2 line.

Nineteen samples of forestomach papillomas and eight skinpapillomas were further examined using oligonucleotide probesspecific for mutations at the 13th codon of human c-Ha-ras andthe 12th and 61st codons of murine c-Ha-ras genes. Mutationsat the 13th codon from GGT (Gly) to GAT (Asp) coexisted inone case of forestomach papillomas that also contained a mutation at the 12th codon from GGC to GAC. Mutations at the12th and 61st codons in the murine c-Ha-ras gene have neverbeen detected at all. The forestomach papillomas from oneaffected transgenic mouse did not show any mutations at the12th, 13th, and 61st codons of the transgene and murine c-Ha-ras gene.

From these results, it was shown that mutational activationsof the transgenes were detected in almost all papillomas butnot in normal tissues, and mutations induced by MNU occurredalmost exclusively at the 2nd letter of the 12th codon of thetransgene and substituted Gly (GGC) to Asp (GAC).

Expression of Activated Transgenes in Papillomas. To determine the RNA expression of the point mutaiiunally activatedhuman c-Ha-ras gene, we used reverse transcription/PCR (18)followed by oligonucleotide hybridization as described in "Materials and Methods" (Fig. 3).

Two of the skin papillomas (Fig. 3, Lanes h and j), two of

Table 2 Somatic point mutational activation oftransgenesPoint

mutationsatTumorsForestomach

papillomas

Skin papillomasLymphomasSpontaneous an

giosarcomas"Tested56941612th

codon55

(98%)9(100^)1

(25%)06

1stcodon000

16(100";,)Type

ofmutationGGC

-»GACGGC

—¿�GACGGC —¿�GACCAG

-. CTG

°Data from the previous experiment (Saitoh et al., 1990). No point mutations

at 12th and 61st codons of the transgenes were detected at all in normal tissues.Somatic point mutations were examined by PCR and oligonucleotide-probehybridization methods.

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a b c d e f g h i jkl

Codon12 i i i i i i i i i i i i* » .»

GGC(Gly)

Lf̂

^r

V

GAC(Asp)

-483

-216

Fig. 3. Detection of RNA expression of the transgenes with somatic pointmutation in the tumors induced by MNU administration. A 216-base pair DNAfragment was amplified from cDNA using a primer pair corresponding to 5'region of the I2th codon and 3' region of the 6lst codon of the human c-Ha-rasgene. Using the same pair of primers, a 483-base pair DNA fragment wasamplified from genomic DNA. PCR, Southern blot analyses, and oligonucleotidesprobe hybridization were performed as described in "Materials and Methods."

DNA samples were as follows: Lane a, amplified DNA samples from humanhybrid c-Ha-ras gene integrated in the rasH2 transgenic mice; Lane b, cDNAfrom the human activated c-Ha-ras gene with single point mutation at the 12thcodon isolated from a bladder tumor (T24); Lane c, cDNA from normal brain ofnontransgenic littermate: Lanes d-g, normal samples from a transgenic mouse,Lane d, brain; Lane e, thymus; Lanef, spleen; Lane g, stomach; Lanes h-l, tumorsamples from transgenic mice; Lane h, skin papilloma developed in the samerasH2 mouse as in Lane i: Lane i, forestomach papilloma; Lanej, skin papilloma;Lane k, forestomach papilloma; Lane I, lymphoma.

forestomach papillomas (Fig. 3, Lanes i and k), and one of thelymphomas (Fig. 3, Lane /), which had somatically mutatedtransgenes, expressed activated transgenes. Endogenous murinec-Ha-ras cDNA could never be hybridized with human c-Ha-ras DNA used as a specific probe to human c-Ha-ras cDNA, asshown in Fig. 3, Lane b (brain sample of nontransgeniclittermate).

DISCUSSION

In rasH2 transgenic mice carrying human c-Ha-ras trans-genes that encode the prototype p21, a single i.p. injection ofMNU (50 mg/kg) induced forestomach papillomas at a veryhigh incidence (100%) within 12 weeks. This is a rather hightumor incidence when compared with the results when nontransgenic C57BL/6 x C3H FI mice given a single i.p. injectionof 50 mg/kg MNU developed forestomach tumors in 66% offemales and 36% of males at 60 weeks of age (19). It wassuggested that forestomach papilloma development in nisi 12mice occurs only with a single MNU administration becausewe rarely observe spontaneous forestomach tumor developmentin these mice within 18 months and as described later, the typesof tumors and mutational activations of transgenes differedfrom those of spontaneous tumors in rasH2 mice (13). Becausewe observed only 1 forestomach tumor in 77 MNU-treatednontransgenic littermates, activation of transgene by MNUplayed a causative role in forestomach papillomagenesis. Thetiming of mutational activation of the ras gene in the multistepprocess of tumorigenesis is also of interest. Since MNU isknown to have a very short half-life (about 20 min) in vivo,activation of the transgene was assumed to be an initiating stepin papillomagenesis in our system. In fact, Kumar et al. (20)showed that activated c-Ha-ras genes were present in normalmammary glands only 2 weeks after MNU treatment and atleast 8 weeks before the onset of neoplasia.

Compared with spontaneous tumors in rasi 12 transgenicmice, there are some differences in the nature of MNU-induced

tumors. The first is the pattern of tissue-specificity of tumordevelopment. In contrast with forestomach papillomas, angio-sarcomas were most frequently observed as spontaneous tumorsbut never detected as MNU-induced tumors within 12 weeks.Although skin papillomas were detected as both spontaneousand MNU-induced tumors, they were more frequently observedas MNU-induced tumors within a shorter latent period. MNUadministration seemed to promote tumorigenesis in the fore-stomach epithelium and skin epidermis and show suppressiveeffects in the vascular endothelium. If activation at the 61stcodon is important in the development of angiosarcomas, thepossible mutation from CAG to TAG or CAA which is inducedby MNU in the 61st codon would be the nonsense codon orwould make no substitution of the amino acid sequence, exceptfor inducing somatic mutational activation.

The second difference is the pattern of mutation in thetransgene. In the spontaneous tumors, the most frequent mutation occurred at the 61st codon with transversion from CAG(Gin) to CTG (Leu). However, in MNU-induced tumors, mutations exclusively occurred at the 12th codon, with transitionfrom GGC (Gly) to GAC (Asp), which is in accordance withother MNU-induced tumor models (1,4). MNU is assumed toinduce GC to AT transition by producing an t^-methylguanineadduct followed by mispairing with thymine during DNA replication (7).

It is also of interest to know why we rarely detect mutationsat the first guanine of codon 12 or either of the codon 13guanine residues but detect these at the second guanine ofcodon 12. Further studies such as sequencing the transgenes intumors and searching for other mutations than codons 12, 13,and 61 will be required. One explanation given by Topal et al.(21) is that the sequence around the 12th codon shares 75%homology with the consensus sequence in the ampicillinasegene, which cannot be efficiently repaired. Another possibilityis the difference in tumorigenicity among various types ofmutated c-Ha-ras gene.

The reason why mutational activation was found only in thetransgene and not in the murine c-Ha-ras gene is not clear. One

possibility is that the transgene is more susceptible to alkylationby MNU or less efficiently repaired after alkylation than themurine c-Ha-ras gene. Another possibility is that tumors produced by the activated transgene grow more rapidly than thoseproduced by activated murine c-Ha-ras. We observed only theformer type of tumors 12 weeks after MNU administration.Further study is needed to answer this question. It is also ofinterest to observe what kind of tumors develop in rasH2transgenic mice when carcinogens are used that induce A to Ttransversion at the 61st codon, such as dimethylbenz-anthracene.

Although the transgene was activated by MNU administration and expressed in various tissues, tumors developed in oursystem exclusively in the skin epidermis and forestomach epithelium, both of which consisted of keratinocytes. This suggested that all cell types are not equally susceptible to transformation by the activated transgene. Several reports demonstrated a causal role of activated c-Ha-ras and the process ofepithelial tumorigenesis (22-24). This fact is especially intriguing when compared with tumor development in suprabasalkeratin-producing cells of the epidermis, recently seen in transgenic mice. The targeting expression of activated c-Ha-ras genein suprabasal keratin-producing cells using the keratin 10 genepromoter results in hyperkeratosis of the skin and forestomach(12). Skin papillomas developed only at sites liable to be bittenor scratched, such as the base of the tail or behind the ears.

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which seemed to be required as second events of the tumorigen-esis. No tumors were reported to develop in the forestomach inthis system; only hyperkeratosis occurred. These results arecomplementary to ours. Both results indicate that at least insuprabasal keratin-producing cells, the c-Ha-ras gene may playa key role in regulating cell proliferation and differentiation,with dysregulation leading to tumorigenesis.

Skin papillomagenesis in our system also seems to requirethe wound-healing process; the sites where papillomas developed were restricted to the base of the tail and the dorsal skin,and when an incision was made, they developed along thewound line (data not shown). Wounding, or the wound-healingprocess, has been considered to be a promotional factor in skintumorigenesis (17, 25). Wound healing is a complicated process, including intercellular communication by means of cell-cellinteraction and humoral factors such as transforming growthfactor ßand epidermal growth factor (18, 26). It is not clearwhich processes function as promotional factors at this time.Local application of either of these humoral factors to the skinof rasH2 mice after MNU administration is a useful approachto elucidate the second step of skin tumorigenesis. Anotherongoing approach to reveal the mechanism of tumorigenesis byactivated c-Ha-ras in skin epidermis is the study of the biological character of skin papilloma cells in an in vitro culturesystem. Human epidermal keratinocytes that contain an introduced v-Ha-ras gene were demonstrated to be independent ofthe epidermal growth factor, which is normally essential forprogressive colony growth, but they secreted a factor possessingsome specific biological activities of fibroblast growth factors(27).

In the forestomach, targeted expression of activated c-Ha-rasgene in keratin 10-producing cells resulted in hyperkeratosisbut not papillomagenesis (12). We have not found any otherfactor for tumorigenesis in the forestomach similar to thefactors involved in the wound-healing processes in the skin.However, it seems likely that activation of the transgene itselfmight be sufficient for forestomach papillomagenesis. In thiscase, the activated transgene should be expressed in the fore-stomach keratinocytes, which were in a different state fromthose of the keratin-ras transgenic mice, because they did notdevelop tumors. Events required for papillomagenesis otherthan expression of the activated c-Ha-ras might be elicited byMNU administration. Even if the latter is true, the fact thattransgenic mice harbored 5.4 ±2.5 papillomas in the forestomach suggests that not so many steps are required fortumorigenesis.

Our results offer good evidence that somatic point mutationalactivation of the human c-Ha-ras gene is an initiating event intumorigenesis in transgenic mice. The very high incidence ofpapillomagenesis ( 100%) within a short latent period ( 12 weeks)in our system provides a good animal model for the study ofchemical carcinogenesis and the function of the c-Ha-ras gene

in tumorigenesis.

ACKNOWLEDGMENTS

We thank K. Katsuki, A. Morita, K. Nakamura, K. Nakao, K.Kaseda, T. Hasegawa, and M. Yokoyama for their excellent technicalassistance and animal care in this work and D. Havens, Dr. J. D.Griffin, and Dr. A. Knudson for preparing the manuscript. We also

acknowledge Drs. T. Nomura and K. Hashimoto for encouraging ourresearch.

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1992;52:978-982. Cancer Res   Kiyoshi Ando, Atsuhiko Saitoh, Okio Hino, et al.  

TransgenesrasMice Carry Mutant Human c-Ha-Chemically Induced Forestomach Papillomas in Transgenic

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