high yields of k-ras mutations in intraductal papillary mucinous

Carcinogenesis vol.17 no.2 pp.3O3-3O9, 1996

High yields of K-ras mutations in intraductal papillary mucinoustumors and invasive adenocarcinomas induced by iV-nitroso(2-hydroxypropyl)(2-oxopropyl)amine in the pancreas of femaleSyrian hamsters

Kenji Sugio1-4, Adi F.Gazdar1*2, Jorge Albores-Saavedra2

and Demetrius M.Kokkinakis3"5

'Simmons Cancer Center and Departments of 2Pathology and 3Neurology,University of Texas Southwestern Medical Center at Dallas, 5323 HarryHines Boulevard, Dallas, TX 75235, USA4Present address: Department of Surgery II, School of Medicine, Universityof Occupational and Environmental Health, Kitakyushu 807, Japan

^ o whom correspondence should be addressed

Ductal adenocarcinoma, the most common form of pancre-atic cancer in humans, is associated with activation of theK-ras oncogene in ~90% of cases. In contrast, K-rasmutations are found in <50% of the relatively rare intra-ductal papillary mucinous tumor (IPMT), which arises inthe main pancreatic ducts. Since both adenocarcinomasand IPMTs are believed to arise from ductal cells andprogress through similar sequences of morphologicalchanges (i.e. flat hyperplasia, papillary hyperplasia, atypiaand carcinoma in situ), it is clear that such progressionmay not always necessitate activation of the ras oncogene.Experimentally ductal adenocarcinomas of the pancreascan be induced in the hamster model by a brief treat-ment with Ar-nitroso(2-hydroxypropyl)(2-oxopropyl)amine(HPOP), while IPMTs can be induced by a combinedtreatment with HPOP and orotic acid (OA) in an initiation/promotion schedule. Since animals are exposed to thecarcinogen only once, initiated normal epithelium isexpected to give rise to a wide spectrum of neoplastic andpreneoplastic lesions, progression of which will depend onthe extent of mutagenesis induced at initiation in thetargeted cells. In order to investigate the role of K-ras inprogression of IPMTs as compared with adenocarcinomaswe have examined the presence of K-ras mutations in theabove two types of experimentally induced pancreaticcancers, as well as in associated and preneoplastic lesions.K-ras mutations at codons 12, 13 and 61 were determinedby a designed restriction fragment length polymorphismmethod using mismatched nested primers in 77 neoplasticand preneoplastic foci microdissected from 20 pancreases.Mutations were found in all foci of atypical hyperplasia,in carcinomas in situ and invasive cancer, whether suchlesions originated in lobular tissue or in the main pancreaticduct. Mutations were also found in papillary hyperplasiaand flat hyperplasia in small ducts and also in the mainduct at high frequency. With one exception, all rasmutations were G—>A transitions at the second base ofcodon 12. Mutations were occasionally accompanied byexcessive presence of the mutant ras allele or loss of thewild-type ras allele, events that were more frequent in

•Abbreviations: IMPT, intraductal papillary mucinous tumor, HPOP, N-nitroso(2-hydroxypropylX2-oxoDropyl)amine; OA, orotic acid; H&E, nema-toxylin and eosin; RFLP, restriction fragment length polymorphism; BOP, N-nitroso(2-oxopropyl)amine.

atypical hyperplasia (5/17), carcinomas in situ (5/14), IPMTs(2/5) and invasive adenocarcinomas (2/5) than in flat hyper-plasia (0/6) or papillary hyperplasia (2/18). Our resultsdemonstrate that: (i) K-ras mutations, predominantly G—>Atransitions, are present in all experimentally induced ham-ster tumors; (ii) the incidence of K-ras mutations in IPMTsis lower in humans than in the hamster model; (Hi)advanced lesions in both adenocarcinomas and IPMTs werefrequently associated with an excess of the mutant over thewild-type K-ras allele. It is likely that both adenocarcinomasand IPMTs induced chemically in the hamster model ariseby mechanisms which involve early activation of K-ras.Such a mechanism seems to be applicable only in a fractionof human IPMTs.

Introduction

The ras mutation is the most frequent of the known geneticchanges in ductal adenocarcinoma of the pancreas, with anincidence of 75-95% (1). The mutations, mainly in codon 12of the K-ras gene, occur at much higher frequency than thosefound in any other tumor type (2), suggesting that mutationsin the K-ras gene play an important role in pancreatic carcino-genesis. Furthermore, K-ras mutations are early events in thedevelopment of pancreatic cancer in man, since they havebeen found in hyperplastic changes associated with pancreaticadenocarcinomas (3). Recently K-ras mutations have beenreported in the development of mucous hyperplasia in patientssuffering from chronic pancreatitis (4). However, such involve-ment has not been generally accepted (3,5). Activation of Rasoncoproteins may not be the only mechanism for transformingductal cells of the pancreas into an over-populated hyperplastic,frequently papillary ductal epidielium. Examination of severalcases of a distinct form of ductal cancer of the pancreas,known as intraductal mucinous papillary tumors (IMPTs*),and by various other names (6—11), indicated that K-rasmutations are relatively infrequent in such tumors, regardlessof the developmental stage. Although the reported incidenceof K-ras mutations in IPMTs varies considerably, the overallK-ras incidence of 44% (12) is markedly lower than the 75-95% incidence reported for adenocarcinomas. In order to studythe role of ras mutations in the development of aggressiveadenocarcinomas and also of other ductal tumors we haveinvestigated the appearance of K-ros mutations in the ductaland ductular epithelium of the hamster pancreas followingexposure to pancreatropic nitrosamines. Continuous admin-istration of A/-nitroso-(2-hydroxypropyl)(2oxopropyl)amine(HPOP) for 9 days to female hamsters followed by 30 weeksof 20% protein synthetic diet results in a 67% incidence ofmultifocal induction of adenocarcinomas and preneoplasticlesions of ductular origin in the body of the gastric lobe ofthe pancreas (13). In contrast, a 28 week promotion of HPOP-initiated female hamsters with orotic acid (OA) 2 weeksafter initiation results in induction of a large incidence of

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preneoplastic lesions in the common and main pancreatic ductsand less frequently in the lobules (14). These two regimenshave been employed to induce both ductular adenocarcinomasand IPMTs. Since carcinogen administration is limited to abrief time span, mutations needed to initiate various lesionsare directed toward the normal epithelium of the hamsterpancreas and not toward already transformed cells from previ-ous carcinogenic treatments, as may be the case with repeatedexposure to carcinogen in other models for pancreatic carcino-genesis (15,16). Variations in the severity of lesions, therefore,are expected to be the result of the extent of mutations inflictedon cells before clonal expansion to yield transformed foci andlesions, rather than to the age of the lesion. We consider thatthe hamster-HPOP model is most suitable for the study ofmutations required to induce various stages of tumor progres-sion. In this communication, processes leading to both ductaladenocarcinomas and IMPTs are characterized in terms ofacquisition of ras mutations.

Materials and methodsTissues

We used paraffinized blocks of pancreas and liver obtained from a previousstudy (14) as a source of seemingly normal and also neoplastic tissue. In thatstudy female hamsters were treated with 19 mg/kg/day HPOP over a periodof 9 days and fed: (i) a synthetic diet containing 20% casein for 30 weeks, atwhich time they were killed; (ii) a synthetic diet containing 20% casein for 2weeks after HPOP treatment, followed by a similar diet supplemented with1% OA for 28 weeks before death. Serial sections 5 |lm thick were cut fromparaffinized blocks. All slides in the series were stained with hematoxylinand eosin (H&E). Every first slide was covered with a cover slip and examinedto identify preneoplastic and neoplastic lesions. Other slides lacking coverslips were used for microdissection.

Microdisscction of materials from stained slides and DNA extractionWe utilized a modification of a previously described microdissection technique(17) to collect precisely identified groups of cells under direct microscopicobservation, using an inverted microscope with a joystick-operated hydraulicmicromanipulator (Nikon-Narishige). We microdissected 50-300 cells ineach area. Dissected foci included preneoplastic and neoplastic lesionsand histologically normal ductal, acinar, islet and stromal lymphocytes.Lymphocytes were also dissected from each slide and used as internal controls.Preneoplastic and neoplastic lesions from the liver, gall bladder and bile duct,which are also induced by HPOP at a low incidence in hamsters, were alsosampled (14). The dissected cells were allowed to adhere to the jaggededge of the microcapillary tip and were collected in 0.5 ml siliconizedmicrocentrifuge tubes. The materials were digested in 10-20 nl 20 mM Tris,pH 8.0, 1 mM EDTA, 0.5% Tween 20, 200 ng/ml proteinase K buffer for24-36 h at 42°C and then incubated for 15 min at 95°C to inactivate proteinaseK. Five microliters of the digested samples, which included at least 50 cells,were used for each PCR reaction as template DNA. Mixing experimentsusing cell lines having mutant and wild-type forms (data not shown) have

demonstrated that the assay is sensitive enough to detect one mutant cell per10- to 20-fold excess of wild-type cells in a total cell population >50.

PCR and designed restriction fragment length polymorphism (RFLP) analysisto detect K-ras mutationsExons 1 and 2 of the K-ras gene were amplified by hemi-nested PCR methodssimilar to those developed for analysis of the human ras gene (17). The assayswere performed in two steps: (i) a screening designed RFLP test to detectpoint mutations in codons 12, 13 and 61 of the K-ras gene; (ii) furtherdesigned RFLP tests to identify specific point mutations indicated by thescreening test. Primers used for the first step are shown in Table I. Celllines previously identified as having ras mutations were used as positivecontrols (17,18).

The first PCR reaction was performed in a 50 fll reaction mixture with 5 \x\template DNA in a thermal cycler (Perkin-Elmer Cetus 9600) with an initial94°C denaturation step for 4 min followed by 35 cycles of denaturation at94°C for 20 s, annealing at 55°C for 20 s and extension at 72°C for 20 s,with a final extension of 7 min at 72°C. The first PCR product was diluted1:100 in dH2O and 1 u.1 of the dilution was used as template for the secondPCR reaction. The latter was performed using a mismatched sense primer in20 (il reaction mixture with the same contents as the first PCR and 30 cycleswere used. The inner mismatched primers introduce a new restriction site intothe PCR product derived from a normal allele. The restriction site is BstN\for codon 12, BgR for codon 13, flc/I for the first two bases of codon 61 andEarl for the third base of codon 61. Ten microliters of the second PCRproduct were digested in a volume of 100 |il with 7-10 U of one of therestriction enzymes, under conditions recommended by the suppliers. ThenDNA was electrophoresed with //aelll-digested <{iX 174 RF DNA size markersand the bands visualized under UV light after ethidium bromide staining andphotographed. Wild-type alleles were digested by the enzyme and yielded asmaller product than the digestion-resistant mutant bands. Digestion wasalways complete, as determined from negative internal controls (lymphocytes)run simultaneously in each experiment. Whenever the internal standardsshowed evidence of incomplete digestion all samples were discarded and theexperiment was repeated.

If a mutant band was identified by the first screening step, the second stepwas performed to identify the specific base substitution. The primers andsteps utilized were identical to those used for identification of mutations inthe human ras gene (17).

Results

Seventy seven separate neoplastic lesions from 20 femalehamsters were examined. Tissues were dissected from fiveductal adenocarcinomas and four carcinomas in situ arisingfrom small ducts of the pancreases of the five hamsters treatedwith HPOP alone. Tissues from cholangiocarcinomas andcholangiomas were also collected from the same animals.Samples of IMPTs and precursor lesions, ranging from fiathyperplasia to papillary tumors, were obtained from 15 ham-sters treated with HPOP and OA in an initiation-promotionregimen previously described (14). The IPMTs induced in thehamster model bear an uncanny resemblance to human IPMTs,

Table I. Sequence of the primers for PCR reaction of the first (screening) step for codons 12, 13 and 61 of the hamster K-ras gene

Exon PCR step Primerorientation

Primer sequence* Productsize (bp)

123

9996

151

6969

Restrictionenzyme

BstNXBgR

BcREarl

Fragment sizeafter digestion

79 + 2076 + 20

50 + 1951 + 18

Exon 1

Codon 12Codon 13Exon 2

Codon 6I b

Codon 61C

1st PCR

2nd PCR

1st PCR

2nd PCR

SenseAntisenseSenseSenseSenseAntisenseSenseSense

5'-GGCCTGCTGAAAATGACTGA5'-CTCTATCGTAGGATCATATT5'-AAACTCGTGGTAGTTGGACC7"ss5'-CTCGTGGTAGTTGGCCCTGGr««c5'-CTGTAATAATCCAGACTGTG5 '-TCCCCAGTTCTC ATGTACTG5' -GGACATTCTCGACACAGCrG/t 7ca5'-GGACATTCTCGACACAGCAGG7TG/l«a«

•Primer sequences are capitalized and mismatched bases in the primer are in bold. The sequences recognized by the restriction enzymes are in italic. Theantisense primer for the second PCR is the same as the primer used for the first PCR in each exon.bFirst two bases.•Tliird base.

304


Role of K-ras in pancreas cardnogenesis

Fig. 1. IPMTs in human pancreas (A) and in hamster pancreas (B). The tumor in the hamster pancreas was induced by an HPOP-OA initiation-promotionregimen as described in Materials and methods. H&E, X250. Note histological similarities between the human and hamster tumors.

as shown in Figure 1A and B. In addition to the above, 51samples of seemingly normal duct, acinar, islet and lymphtissue were dissected from the 15 hamsters treated with HPOPand OA and analyzed for ras mutations. Such tissue sampleswere both adjacent to and distant from neoplastic lesions ortumors. A smaller number of samples were dissected from theliver and gall bladder of animals which, in addition to pancreaticcancer, also had tumors in these sites.

Incidence of K-ras mutationsMicrodissected foci were screened for the presence ofmutations in codons 12, 13 and 61 using the designed RFLPmethod. All microdissected materials provided suitable tem-plate DNA for use in PCRs, resulting in amplification of a 99bp product in the second PCR specific for codon 12 of the K-ras gene. Wild-type forms were digested, resulting in a band79 bp in size. Mutant alleles resulted in BstHl digestion-resistant bands 99 bp in size (Figure 2). Screening for mutations

M 1 2 3 4 5 6 7 8 9 NC PC

IMut

Fig. 2. Determination of K-ras codon 12 mutations in HPOP-OA-treatedhamsters using the designed RFLP method. The wild-type allele was cut byrestriction enzyme BstHl, yielding a fragment of 79 bp. A mutation in thiscodon abolishes the restriction site, leaving an intact PCR product of 99 bpafter digestion. DNA from normal hamster pancreas was used as a negativecontrol (NC) and cell line NCI-H358 (K12 mutation, GGT-VTGT) as apositive control (PC). The size marker (M) is Waelll-digested 4>X174 RFDNA. Lane I, lymphocytes dissected from the same slide; lane 2, acinarcells; lanes 3-5, fiat hyperplasia; lanes 6-9, papillary hyperplasia. The wild-type form (WT) is present in lanes 1-2, while all hyperplastic foci containmutant (Mut) and wild-type bands at similar intensities (pattern I).

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K^ugio et at

Table II. Incidence of K-raslesions induced by HPOP in

Diagnosis

Pancreas

mutations in neoplastichamster pancreas

K-ras lesion

Lesion/total

Histologically normal tissueAcinar cellsIslet cellsStromal lymphocytesDuctal cellsTotal

Flat hyperplasiaCommon ductMain ductDuctule proliferationTotal

Papillary hyperplasiaCommon ductMain ductTotal

Atypical hyperplasiaCommon ductMain ductDuctulesTotal

Carcinoma in situMain duct/sub-branchesLobularTotal

CancerIPMTsAdenocarcinomas

0/170/100/110/130/51

2/44/61/47/14

10/108/10

18/20

5/58/84/4

17/17

10/104/4

14/14

5/55/5

10/10Metastasis to lymph nodes 2/2Total

LiverCholangiomasCholangiocarcinomas

Gall bladderAdenocarcinomas

Bile ductPapillary hyperplasia

2/2

2/44/5

0/22/22/2

(%)

(0)(0)(0)(0)

(50)(67)(25)

(100)(80)

(100)(100)(100)5/17

(100)(100)

(100)(100)

(100)

(50)(80)

(0)(0)(100)

and preneoplastic

Pattern 11

Pattern Il/no.of lesions

0/20/40/10/7

1/101/82/18

2/52/81/429

3/102/45/14

2/52/54/120/2

onon4/4

0/20/2

%

0

12

35

40

40

M 1 2 3 4 5 NC PC

in codons 13 and 61 was performed by a similar techniqueusing appropriate primer pairs and restriction enzymes. Theresults of the designed RFLP analysis are summarized in TableII. All microdissected foci of stromal lymphocytes, islet cells,acinar cells and normal ducts had a wild-type K-ras gene. Fourfoci of ductular proliferation were identified and microdissectedfrom two pancreases and one of these four foci was positivefor a codon 12 mutation. Ten foci of flat hyperplasia wereobtained from three animals. Four samples were collectedfrom the common duct and six from the main duct. Six ofthese 10 lesions had a codon 12 mutation, with mutantfrequency being greater in the main than in the common duct.Twenty foci of papillary hyperplasia were obtained from sevenhamsters with the lesion. Eighteen of the 20 foci were positivefor codon 12 mutations. The mutation was present in all fociof papillary hyperplasia of the common pancreatic duct, whileit was absent in the main pancreatic duct of one of the animalsin which cell proliferation was focal. All foci of atypicalhyperplasia, carcinoma in situ and invasive adenocarcinomahad a codon 12 mutation. These included five cases of ductaladenocarcinoma from five animals, five cases of papillaryinvasive cancer arising from the main pancreatic duct fromfive animals, four cases of carcinoma in situ arising in small

306

Fig. 3. Determination of K-ras codon 12 mutations in the pancreas ofHPOP-OA-treated hamsters using the designed RFLP method. Lane 1,lymphocytes; lane 2, acinar cells; lanes 3-4, atypical hyperplasia; lane 5,IPMT. The wild-type form (WT) is present in lanes 1-2. One focus ofatypical hyperplasia (lane 3) contains mutant (Mut) and wild-type bands(pattern I) and one focus of atypical hyperplasia and invasive cancer has anexcess mutant band (lanes 4 and 5) (pattern II).

ducts in three animals and 10 cases of intraductal carcinomain situ arising in the main pancreatic duct from five animals.No mutations in codons 13 and 61 were found.

Identification of specific K-ras mutationsWith one exception, all lesions had a G—»A transition at thesecond base of codon 12, resulting in a glycine to aspartic acidsubstitution. One focus of flat hyperplasia had a GGT—»GTTmutation in codon 12, which leads to a glycine to valinesubstitution.The pattern of K-ras mutationsThe designed RFLP method made possible simultaneous visu-alization of both mutant and normal alleles in heterozygouscell populations (Figure 3) and permitted an estimation of theratio of mutant to wild-type alleles. The microdissectionmethod also permitted precise collection of cells from histo-pathologically identified foci without contamination by othercell types. Thus the ratio of mutant to wild-type bands wasnot skewed by the presence of stromal or other cells presentin analyses of gross tumor samples. The patterns present inmutant ros-containing human tumors and cell lines have beenpreviously classified into three groups (17). In pattern I thePCR products corresponding to the wild-type and mutant formsare of similar intensities. In pattern Ha the mutant band is ingreat excess (relative increase). In pattern lib the wild-typeband cannot be identified (absolute increase). This latter patternwas rare in hamsters, shown only in two out of 77 identifiedmutations. Therefore, in the hamster model patterns Ila andlib were classified as pattern II, denoting excess of the mutantover the normal allele. Pattern I was the most common patternseen, including all foci of ductular proliferation and flathyperplasia with the mutation. Pattern II was seen mostly inhistopathologically more advanced lesions. This pattern wasmore frequent in atypical hyperplasia or invasive cancer thanflat or papillary hyperplasia (Table II).

Tumors in other sitesIn the hamster-HPOP model lesions were induced primarilyin the pancreas. There was a low incidence of liver tumors(cholangiomas and cholangiocarcinomas), gall bladder tumors(adenocarcinomas) and bile duct proliferative changes (hyper-plasia). These lesions were also examined for K-ras mutations(Table II). The K-ras mutations at these sites were only incodon 12 and were characterized as G—»A transitions. Thefrequency of mutation in cholangiocarcinomas was greaterthan in cholangiomas. Mutations were absent in gall bladderadenocarcinomas in the two cases examined. Bile duct hyper-plasia was associated with K-ras mutation in the one casestudied. All cholangiocarcinomas showed pattern II mutation.


Role of K-ras in pancreas cardnogenesis

Table IlL Review of K-ras mutations in human pancreatic intraductal papillary mucinous neoplasms

Tumor Degree ofdysplasia

Mutation Codon Incidence Reference

IPN1

IMHNIMCHTotalIPMAIPMAIPNIPHIMHNTotalIPMCIPMCIPMCIPNIPNIPNIMHNIMCCIMCCTotal

NoneNoneNone

MildMildMildMildMild

Mod/sevMod/sevMod/sevMod/sevMod/sevMod/sevMod/sevMod/sevMod/sev

GAT

GAT

GTTGATGTT

my

GATGATGTTGTTCGT

NDGATGTT

12

12

121212

12

1213121212

121212

1/10/23/54/8(50)b

1/41/41/10/93/86/22 (28)2/131/132/131/11/30/58/101/31/3

17/45 (37)

21224

1212215

22

12121221215

2244

•IPN, intraductal papillary neoplasm; IMHN, intraductal mucin hypersecreting neoplasm; IMCH, intraductal mucinous cystic hyperplasia; IPMA, intraductalpapillary mucinous adenoma; IPH, intraductal papillary hyperplasia; IPMC, intraductal papillary mucinous carcinoma; IMCC, intraductal mucinous cysticcarcinoma.''Percent incidence.'Not done.

Discussion

The frequent presence of K-ras mutations in pancreatic ductaladenocarcinomas and accompanying preneoplastic lesions(3,19) underlines the importance of this oncogene in pancreaticcarcinogenesis as an early stage event. In contrast to adeno-carcinomas, a group of pancreatic neoplasms arising in themain duct, known by various names such as intraductalpapillary carcinoma, mucus hypersecreting carcinoma, mucin-ous ductal ectasia or papillary cystadenoma (6—11), dependingon the stage of their development (20), have been shown tohave a markedly lower incidence of K-ras mutations. Theexact incidence of K-ras mutations in such tumors, collectivelyknown as IMPTs (12), cannot be estimated from the reportedstudies (4,5,12,21,22), but appears to be no greater than 50%of all the cases tested (Table III). Furthermore, the presenceof K-ras mutations in human IPMTs is random, with noapparent correlation between morphology, stage of malignancy,site of tumor, sex or age of patients (12).

In the hamster model a high incidence of papillary neoplasmswith a morphology very similar to human IPMTs has beeninduced with an initiating regimen of HPOP followed bypromotion with OA (14). In contrast to the A'-nitroso(2-oxopropyl)amine (BOP) model, preneoplastic lesions, particu-larly in large ducts of the pancreas, are not extensions of largetumors and most probably represent the site of tumor initiation.Various degrees of growth of papillary neoplasms in the largeducts of the hamster pancreas result in a variety of preneoplasticlesions informative of the overall carcinogenic process. Theheterogeneity of preneoplastic lesions in this model is probablydue to the different capacity of component cells of the lesionto progress through malignant transformation, rather than thevariable times of initiation, as may be the case in humans.This is concluded from the general failure of such lesions togive rise to cancer, regardless of the time the hamster is keptalive over a 30 week period after initiation (D.M.Kokkinakis,

unpublished observation). The action of OA is not wellunderstood (23,24), but it is theorized that it probably inhibitsgrowth of normal ductal epithelium, allowing mutated cells toexpand and give rise to focal hyperplasia and subsequently toextensive papillary lesions (14). The K-ras mutation couldpossibly render the cell resistant to the mito-inhibitory effectof OA and allow growth of tumors which subsequently carrythe K-ras mutant phenotype. This could explain the presence ofK-ras mutations in all intraductal tumors invading surroundingtissue and in all dysplastic lesions. It does not, however, meanthat all lesions having the K-ras mutation can progress tocancer, as is evident from the wide variety of early lesions, inspite of the presence of the mutation.

The incidence of ras mutations found in both neoplasticand preneoplastic lesions in this study is considerably higherthan that reported in the BOP model by other investigators(25,26). The higher incidence may be due to: (i) the use ofHPOP instead of BOP; (ii) the use of female hamsters insteadof the male animals employed in previous studies; (iii) the useof a promoting regimen which may be specific in selecting K-ros mutants. The difference between BOP and HPOP is thatthe former is activated to yield a methylating agent, while thelatter yields both methylating and 2-hydroxypropylating species(27). Although BOP is converted to HPOP in a reactioncatalyzed in many tissues, including the pancreas and its ductalcomponents (28), the ratio of methyl to hydroxypropyl adductsis 10 times higher in the pancreas of hamsters treated withBOP than in those treated with HPOP (29). A similar situationshould be true in the ductal component of the pancreas, sincein duct cell cultures the ratio of CAmethylguanine to O6-(2-hydroxypropyl)guanine following treatment with BOP andHPOP is seven and two respectively (28).

Hydroxypropylation of DNA results in G->A mutationsvia formation of die O6-(2-hydroxypropyl)guanine adduct (30).Furthermore, the yield of such mutations induced by the aboveadduct is at least four times greater than that induced by

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K.Sugio et at

C^-methylguanine under conditions which yield a similarconcentration of the above promutagenic lesions (30). Inthe hamster-HPOP model O6-(2-hydroxypropyl)guanine isrepaired more slowly and persists longer than O^methylguan-ine, allowing a greater window of opportunity for mutation inK-ras (31). The use of female hamsters instead of male animalsused in previous studies may also have affected the numberof K-ras-positive foci and the continuous growth of such focito sizable lesions. Growth of pancreatic tumors has been shownto be estrogen dependent (32) and can be inhibited by theestrogen competitor tamoxifen (33). Thus the effect of estrogenon selective growth of cells having a mutation in K-ras isprobable and should be investigated further. Another sourceof the high incidence of K-ras mutations in this study may beselection of K-ras mutants by the promoting regimen. Althougha direct relation between resistance to OA-induced mito-inhibition and K-ras mutation has not been demonstrated, ithas been observed that OA enhances the rate of appearanceof hyperplastic K-ras-positive lesions in the common and mainpancreatic ducts of the hamster pancreas as early as 3 weeksafter initiation of the promoting regimen (D.M.Kokkinakis,unpublished observations). However, the similar incidence ofK-ras mutations observed in HPOP-treated animals withoutOA promotion argues against the last possibility. Furtherexamination of the BOP-hamster model in parallel with themodels used here is needed in order to identify the mechanismsby which pancreatic preneoplastic lesions progress to invasivecancer without K-ras involvement. If, indeed, the triggeringof a ras-induced mitogenic pathway via ras mutation is notan absolute requirement for rapid growth of pancreatic cancers,the question arises whether or not other sites in that pathwayare affected by carcinogen-induced mutations resulting insimilar mitotic activity.

With the exception of a single focus, the specific mutationfound in preneoplastic and neoplastic lesions of both ductaland ductular origin was always a transition at the secondposition of codon 12 of the K-ras gene, specifically a GGT(Gly)—»GAT (Asp) change. An identical mutation has beendescribed in the BOP model (25,26). In human IPMTs a G-»Atransition is the most common mutation, reported in five outof eight cases by Sessa (12) and in four out of five cases byYanagisawa (4). A G—>A transition is also the most commonmutation in human ductal adenocarcinomas, found in 48% ofall cases according to Hurban (19). The almost exclusiveG—)A transition observed in the hamster model is in agreementwith the mechanisms of carcinogen action yielding C^-methyl-guanine and O6-(2-hydroxypropyl)guanine adducts, whichremain unrepaired for prolonged time periods (31).

A correlation between the ratio of mutant to wild-type K-ras alleles and the degree of neoplasia was evident in thesestudies. An increase in this ratio has been observed in hyperpla-sia associated with atypia, in carcinoma in situ and in cancer.Complete loss of the wild-type allele was observed in twocases of adenocarcinoma, but was never seen in IPMTs andrelated preneoplastic lesions. The above indicate that in thehamster model, as in humans (17), progression through thevarious stages of hyperplasia to carcinoma in situ and cancermay depend on the relative ratio of mutant to wild-type alleles.However, in the absence of further evidence, caution shouldbe exercised not to overestimate either the mutation itself orthe relative increase in the mutant allele as a requirement forinitiation and progression of pancreatic cancer in the hamstermodel. The guanine at the second position of K-ras codon 12

is particularly sensitive to carcinogen-induced mutation forseveral reasons, including enhanced adduct formation (34),diminished repair by C^-methylguanine-DNA alkyltransferase(35) and enhanced mispairing with thymidine during DNAreplication (36). Thus, in the absence of easily induced K-rasmutations, the level of other mutations possibly required forpancreatic carcinogenesis may be insufficient to result inmalignant transformation of targeted cells beyond the stage offlat or papillary hyperplasia. The progression may stop at thatparticular stage for the rest of the animal's life, providing thatthe frequency of spontaneous mutations is not sufficient tochange that status.

In conclusion, unlike human IPMTs, which seem to developwithout an absolute requirement for K-ras activation, in thehamster model the full progression from papillary hyperplasiato intraductal papillary tumors morphologically similar tohuman IPMTs appears to involve such activation and perhapspartial loss of the wild-type allele. In this respect, developmentof human and hamster IPMTs may not be mediated by exactlythe same mechanism, although the possibility of mutations inoncogenes downstream from ras in the same pathway ofactivation of cell proliferation cannot be ruled out at this time.Certainly the presence of K-ras mutations in a significantnumber of, but not all, human IPMTs may result from morethan one etiology or induction mechanism. A certain fractionof such tumors could arise by early activation of K-ras in afashion similar to that seen in the hamster model and couldbe related to the action of chemical carcinogens.

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Received on May 24, 1995; revised on September 26, 1995; accepted onNovember 2, 1995

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