Proc. Natl. Acad. Sci. USAVol. 92, pp. 4482-4486, May 1995Medical Sciences

Loss ofApc heterozygosity and abnormal tissue building innascent intestinal polyps in mice carrying a truncated Ape gene

(gene targeting/colon cancer/microadenoma/familial adenomatous polyposis)

MASANOBU OSHIMA*, HIROKO OSHIMA*, KYOKO KITAGAWA*, MASAHIKO KOBAYASHI*, CHITOSHI ITAKURAt,AND MAKOTO TAKETO*$*Banyu Tsukuba Research Institute (Merck), Tsukuba 300-33, Japan; and tDepartment of Comparative Pathology, Faculty of Veterinary Medicine, HokkaidoUniversity, Sapporo 060, Japan

Communicated by P. Roy Vagelos, Merck & Co., Whitehouse Station, NJ, January 4, 1995

ABSTRACT Mutations in the APC (adenomatous polyp-osis coli) gene appear to be responsible for not only familialadenomatous polyposis but also many sporadic cases of gas-trointestinal cancers. Using homologous recombination inmouse embryonic stem cells, we constructed mice that con-tained a mutant gene encoding a product truncated at aa 716(Apc^716). Mendelian transmission of the gene caused mosthomozygous mice to die in utero before day 8 of gestation. Theheterozygotes developed multiple polyps throughout the in-testinal tract, mostly in the small intestine. The earliest polypsarose multifocally during the third week after birth, and newpolyps continued to appear thereafter. Surprisingly, everynascent polyp consisted of a microadenoma covered with alayer of the normal villous epithelium. These microadenomasoriginated from single crypts by forming abnormal outpocketsinto the inner (lacteal) side of the neighboring villi. Wecarefully dissected such microadenomas from nascent polypsby peeling off the normal epithelium and determined theirgenotype by PCR: all microadenomas had already lost thewild-type Ape allele, whereas the mutant allele remainedunchanged. These results indicate that loss of heterozygosityfollowed by formation of intravillous microadenomas is re-sponsible for polyposis in Apc^7A6 intestinal mucosa. It istherefore unlikely that the truncated product interacts di-rectly with the wild-type protein and causes the microadeno-mas by a dominant negative mechanism.

Molecular genetic studies of familial adenomatous polyposis(FAP) kindreds led to the discovery of theAPC (adenomatouspolyposis coli) gene on human chromosome 5q21 (1-4).Mutations inAPC appear to be responsible for not only FAPbut also many sporadic cancers of the colorectal axis, stomach,and esophagus (5-7). While most FAP cases have mutations inthe upstream half of the last exon (8), mutations near the 5'end of the coding region cause an attenuated form withrelatively few colonic polyps (9). Another form of FAP, whichassociates congenital hypertrophy of the retinal pigment epi-thelium, contains mutations downstream of exon 9 (10).APCconsists of 15 coding exons and several noncoding exons,various combinations of which generate many isoforms byalternative splicing (3, 11, 12). The gene encodes a hugeprotein, of about 2840 aa (1, 4). The protein contains regionsthat may form an a-helical coiled-coil structure, and a subdo-main of the first 55 aa form a stable, parallel helical dimer (13).Truncated APC proteins can associate with the wild-typeprotein in vivo (14). Antibody studies showed that the wild-type, but not mutant, APC protein associated with the micro-tubule cytoskeleton (15, 16). The predicted structure of APC,its localization, and its interaction with p-catenin (17, 18)suggest its involvement in cell adhesion.

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

4482

A dominant mouse mutation, Min (multiple intestinal neo-plasia), which had been generated by chemical mutagenesisand causes polyposis in the digestive tract, has been located inApc, the mouse homologue of the humanAPC gene. It causestruncation of the gene product at aa 850 and multiple polypsin the intestinal tract (19, 20).To investigate the molecular mechanism of polyp formation

followed by carcinogenesis in the digestive tract, we employedhomologous recombination in embryonic stem (ES) cells andgenerated mice containing a mutant gene encoding a producttruncated at residue 716 of 2845 aa (Apc^716). Here we reportthe results of a systematic study using this experimental system.

MATERIALS AND METHODS

Targeting Vector. A region of Apc was amplified by PCRusing primer D (5'-TGCATGTGGAACTTTGTGGA-3',cDNA bases 2031-2050; ref. 20) and primer C (5'-AAGAAGAGCTGGGCAATACCGTA-3', bases 2478-2500). This fragment was used as a probe, and four clones wereisolated from a 129/Sv genomic library (Stratagene). Twocontained the cDNA sequence encoding the C terminus of theApc protein. The PGKneobpA cassette (21) was inserted intothe Nco I site at the beginning of exon 15 (base 2150). Thebacterial diphtheria toxin a-subunit gene (22) driven by thephosphoglycerate kinase I gene promoter was inserted at anupstream Sac I site.ES Cells and Chimeric Mice. ES cell lines AB-1 and D3al

were cultured as described (21, 23). ES cells (2 x 107) wereelectroporated with the linearized targeting vector. Homolo-gous recombinant candidates were screened by PCR andverified by Southern hybridization. Chimeras were generatedby injecting the ES cells into C57BL/6J blastocysts and trans-ferred to MCH (CLEA Japan, Tokyo) foster mothers.

Chronological Analysis of Intestinal Polyp Formation. In-dividual heterozygous mice carrying the Apc^716 allele wereidentified by PCR analysis of the tail DNA at 3 weeks of age.At each time point, subject mice were sacrificed, and all polypswere scored at x30-60 magnification. The same investigator(M.O.) scored all samples.

Histologic Analysis. Tissue samples were fixed in 4% form-aldehyde in phosphate-buffered saline, embedded in glycol-methacrylate resin (Heraeus, Friedrichsdorf, Germany), sec-tioned at 3-,im thickness, and stained with 1% toluidine bluein 0.5 M phosphate buffer (pH 7.2).Microadenoma Dissection and PCR Analysis. The smallest

adenomatous polyps identifiable under a dissection micro-scope were in the range of 0.2 mm in diameter. The normalepithelium covering these nascent polyps was removed bypeeling it off very carefully under a dissection microscope.

Abbreviations: FAP, familial adenomatous polyposis; ES, embryonicstem; LOH, loss of heterozygosity; dpc, days postcoitum.ITo whom reprint requests should be addressed.

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Each microadenoma thus dissected was suspended in 30 \zl ofwater, and 0.5 /ul of 10 mM proteinase K (Boehringer Mann-heim) was added. After incubation at 55°C for 3 hr and boilingfor 5 min; 10-1ul aliquots were subjected to PCR analysis.Standard procedures (24) were used unless noted otherwise.

RESULTSGeneration of Mice with an Ape Gene Truncated at Codon

716 (Apc^7'6). A replacement-type targeting vector was used toisolate homologous recombinant ES cells (Fig. 1A). Of 156G418-resistant colonies, 7 were genuine homologous recom-binants (Fig. 1B). These recombinant ES cells expressed boththe truncated and the wild-type gene products at about equalmolar ratio (Western analysis data not shown). Two suchclones (nos. 45 and 110) were injected into blastocysts of theC57BL/6J strain, and nine founder chimeras were generated.All five chimeras derived from clone 110 were males, and theES cell contribution to their coats was almost 100%. Uponcrossing with C57BL/6J females, all of them gave birth toagouti offspring (without any black pups), 50% of whichcarried theApc^716 allele. When the F1 mice were intercrossed,only wild-type and heterozygous pups were born, withouthomozygous mutants (Table 1, full term). To investigate thetransmission of the Apc^716 mutation, heterozygous F1 femalesmated with heterozygous males were sacrificed at various

Table 1. Genotype analysis of interbred offspringNo. with Apc No. (no. of

Age of genotype No. unknown litters)embryos +/+ +/- -/- (resorbed) determined

8-8.5 dpc 8 14 1 9 23 (5)12-12.5 dpc 6 12 0 5 18 (3)Full term 10 16 0 nd 26 (6)(Total) 24 42 1 67 (14)

dpc, Days postcoitum; nd, not detectable for early resorptions.

stages of gestation, and embryos were analyzed by PCR. In all,67 embryos from 14 litters were genotyped (Table 1). Of 23embryos examined at 8.5 dpc (including the 9 shown in Fig. 1C),8 were wild type, 14 were heterozygous, and only 1 was homozy-gous. Of 44 embryos examined at 12.5 dpc or full term, 16 werewild type and 28 were heterozygous, without any homozygotes.Examination of intrauterine contents at several stages of gesta-tion revealed resorption, suggesting that most homozygous mu-tant embryos died in utero before day 8 of gestation.

Heterozygotes Develop Intestinal Polyps. The heterozygousmice developed multiple polyps in the intestinal tract shortlyafter birth. Fig. 2 shows a chronological investigation of polypdevelopment in the second backcross (N2) generation. Mostmice (7 of 8) developed polyps by.5 weeks of age, and all by 7weeks. The number of polyps increased with age (Fig. 2A), and

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FIG. 1. Generation ofApc^716 knockout mice. (A) Targeting by homologous recombination in ES cells. Targeting vector pAPCa7l6 containedboth the neo gene and the diphtheria toxin a-subunit gene (DTA) driven by the phosphoglycerate kinase I promoter (P), but arranged in the oppositetranscriptional orientations. The former is for the positive selection, whereas the latter for the negative selection (21, 22). Box A indicates thesegment containing a polyadenylylation site. Arrowheads A-C indicate the PCR primers used for homologous recombinant screening and forgenotype determination. Note that the segment between primers A and C (shown in parentheses) in the targeted allele is too long to be amplifiedby PCR. HincII sites (Hc) relevant to the identification of homologous recombinant ES cell clones are shown together with the expected sizes ofthe fragments hybridizable to the Apc (filled bars) and the neo (bars with wavy lines) probes. (B) Southern blot confirmation of the homologousrecombinant ES cell clones. Clones 45 and 110 were derived from D3 ES cells (ref. 23; parental ES), whereas clone 38c was derived from AB-1(21). (Left) Hybridization with the neo probe showing a 3.3-kb band specific to the targeted allele. (Right) Hybridization with theApc probe showinga 5.0-kb band derived from the wild-type full-length allele (ApCFL) and, in the homologous recombinants, a 3.3-kb band specific to the targetedallele (Apc^716). (C) Transmission of the targeted allele (Apc^716) to the progeny as determined by PCR. Homozygotes are embryonic lethal. Nineembryos obtained from two intercrosses analyzed day 8 of gestation are shown. All embryos but one (E3, the only homozygote) showed a bandamplified from the wild-type allele [ApCFL (A-C)], whereas four (E4, E5, E7, and E8) showed an additional band specific to the targeted alleleApca716 (A-B)]. In addition to the nine embryos shown here, there were 5 resorptions. Bottom arrowhead indicates a band amplified even withprimer B alone and used as an internal control for the PCR.

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FIG. 2. 'Time course of polyp formation in Apc^716 heterozygousmice. (A) Numbers of polyps counted in the entire intestinal tract ofthe N2 backcross heterozygous mice (0). Some F1 hybrid (0) andMin/+ (Apc^850) heterozygote (o) data are also shown. (B) Sizedistribution of the intestinal polyps in the N2 backcross heterozygotesat 3, 5, 7, 9, 12, and 16 weeks of age. indicates the polyp diametermeasured under a dissecting microscope.

they varied in size and stage (Fig. 2B). Polyps were found fromthe duodenum to the rectum, mostly in the small intestine. Thepolyp number at week 16 was 254 ± 55 (mean ± SD), muchlarger than that of Min/+ mice, either reported (19, 20) orcounted by us (Fig. 2A). The polyp diameter ranged from 0.2mm, which could be identified only under a dissection micro-scope, to 5 mm. Histologically, the polyps were polypoid, pap-illary, or sessile adenomas. Interestingly, every polyp of the smallintestine had a layer of normal villous epithelium on the surfacecovering the tumor, although much of this layer was lost in largerpolyps (see below). Even in older mice, most of the tumorsremained in the lamina propria. While early-onset polyps becamelarger with age, new polyps continued to appear, as the smallestpolyps were always found even in later stages (Fig. 2B). Thissuggests a stochastic mechanism of polyp development (seebelow). The onset of the polyposis detectable under a microscopeoccurred during the third postnatal week, although the actualonset is likely to have been earlier. The chimeras (100% contri-bution by ES cells) and F1 hybrids developed few or no polyps inthe colorectal axis, even after 26 weeks, and showed no symptomsof either bleeding or resulting anemia, which was observed inmany N2 heterozygotes after 17 weeks (see Discussion). The polypnumbers in the N4 backcross generation were in the same rangeas in the N2 mice (data not shown).Nascent Polyps Consist of Microadenomas with Loss of

Heterozygosity (LOH), Covered by Normal Epithelium. Toinvestigate the earliest event in polyp development, we dis-sected nascent polyps under a dissection microscope (Fig. 3Aand B) and examined them histologically. Every polyp con-

sisted of a microadenoma enveloped with a layer of the normalvillous epithelium (Fig. 3 C and D). Examination of serialsections revealed the origin of such microadenomas in singlecrypts; a contiguous layer of the epithelium extended from thecrypt into the microadenoma (e.g., Fig. 3D). These histologicalpictures suggest that the earliest microadenomas originatedfrom the proliferation zone of the crypts, grew toward the backside, forming outpockets, and expanded subsequently into theinner (lacteal) side of the neighboring villi. To investigate themolecular mechanism behind this phenomenon, we dissectedthe microadenomas from the nascent polyps by carefullyremoving the normal epithelium and determined their geno-type by PCR. All 39 microadenomas examined had already lostthe normal, full-length allele (ApCFL) but retained the mutantallele (Apc^716). None of the normal adjoining villi showedsuch allelic loss. Some examples are shown in Fig. 4.

DISCUSSION

Using homologous recombination in ES cells, we constructeda mouse strain that contains a truncation mutation at codon716 ofApc (Apc^716). While the homozygotes are embryoniclethal, the heterozygous mice form polyps in the entire intes-tinal tract, mostly in the small intestine. These features aresimilar to those of Min mice, in which the Apc gene waschemically mutated to cause a truncation at codon 850 (19, 20).However, the numbers of polyps are much larger in theApc^716/+ mice, although both mice are in the C57BL/6Jbackground. It is possible that the extra 134 aa in the Min/+mice are responsible for the difference. We noticed that ourfounder chimeras (in the 129/Sv background) and their F1 withC57BL/6J mice formed no or few polyps in the colorectal axis,although the total number of polyps was similar. Other mod-ifier genes in the 129/Sv background than Mom-1 (25) may beinvolved in this phenomenon, since this phenotype segregatedfrom the D4Mit16 locus (M.O., H.O., and M.T., unpublishedwork).Our results strongly suggest that the LOH is responsible for

microadenoma formation in the Apc^716 heterozygotes. Ac-cordingly, Knudson's "two-hit" theory (26) applies also to theformation of intestinal microadenomas, the earliest step inpolyposis. Earlier FAP studies suggested that polyps couldarise as a result of heterozygosity for anAPC mutation withoutthe need for a further genetic event (27). Subsequent studiesshowed no or few allelic losses of 5q markers in early adeno-mas, but a significant percentage of more advanced adenomasand carcinomas lost a chromosome 5 allele (e.g., ref. 28). Smalladenomas were examined in an FAP patient and five out ofseven samples showed loss of the normal allele (29). LOH inthe APC gene was involved in 77% of the human esophagealcancers tested (5). The discrepancy between these reports andour results may be explained by several reasons. (i) In theearlier studies, markers distant from theAPC gene were usedto test the LOH. (ii) It is difficult to show LOH by using DNAsamples isolated from whole tumors, because tumor tissuesalways contain normal cells. As we have demonstrated, earlypolyps are covered by normal epithelium, which was carefullyremoved in our study, whereas more advanced tumors oftencontain cells of mesenchymal origin due to secondary tissueremodeling. (iii) APC gene LOH is not the only mechanismin human FAP. Detailed studies of seven FAP cases showedvarious somatic mutations in the tumor tissues in addition tothe LOH (30). (iv) It is conceivable that the mouseApc LOHaccompanies loss of other mouse chromosome 18 geneswhose homologues are located on different human chromo-somes.We foundApc LOH in all 39 nascent microadenomas tested.

The results indicate that LOH is one of the earliest changes inthe microadenoma formation, at least in theApc^716 heterozy-gous epithelium. It is therefore unlikely that a dominant

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FIG. 3. Morphology of nascent intestinal polyps in Apc^716 heterozygotes. (A) Three polyps (arrowheads) in different stages of polypdevelopment. The top one is a representative nascent polyp, whereas the middle and bottom ones are in more advanced stages. Polyps were viewedunder a dissection microscope. (x17.5.) (B) A nascent polyp viewed from the side. Most of the normal villi were removed for the photographicpurpose. Note the double-layer wall of the polyp with the inner layer extending from the crypt region. (x70.) (C andD) Two nascent polyps sectionedalong the axis of the crypts/villi. Note the normal villous epithelium covering the microadenomas that originated from single crypts. (x280.)

negative mechanism is directly responsible for microadenomaformation. The haploid amount ofApcFL appears to be enoughfor normal epithelial growth. In this context, transgenic micethat contain a third copy ofApc mutated to express a truncatedproduct (Apca716 orApc^1287) at very high levels do not developany intestinal polyps (M.O., H.O., and M.T., unpublishedwork). It remains conceivable, however, that a dominantnegative mechanism plays a role in causing the LOH itself.We have demonstrated that microadenomas are formed by

the crypt epithelium growing in an abnormal direction-i.e.,into the intravillous space. Should these cells expand directlyinto the intestinal lumen, they would be exposed to a negativegrowth factor, transforming growth factor 3 (TGF-13) (31). Itis also possible that many of them would be shed into the lumenbefore adenomas are formed. In theApc^716 heterozygotes, thenormal epithelium covering the surface will help retain mostmicroadenomas and keep them from exposure to TGF-3,allowing subsequent changes. Microadenomas in the nascentpolyps in the Min/+ mice are also covered by the normalepithelium (data not shown), which suggests the same mech-

anism also in these mice. In the colonic mucosa, which consistsof crypts but lacks villi, finding abnormal crypts with microad-enomas was rather difficult under a dissection microscope.Extensive serial sectioning analysis, however, revealed someoutpocketing epithelium forming microadenomas betweennormal crypts (M.O., H.O., and M.T., unpublished work),suggesting a similar mechanism as in the small intestine.Studies of colonic chemical carcinogenesis in mice showed twoorigins of atypical epithelial cells in the earliest stage: at thecryptal base or an outpocketing pouch (32). It is thereforepossible that Apc mutations cause the latter, whereas theformer is caused by mutations in other genes. Such outpock-eting of the epithelium may be caused by the failure of thetruncatedApc gene product to interact with 3-catenin (17, 18)properly. As shown by cell kinetic studies, the growth rate itselfis not much different between colonic adenomas and normalmucosa (33). It is not a faster growth rate but an abnormaltissue building (i.e., outpocketing) that appears to be the directoutcome of the Apc mutation, which leads to microadenomadevelopment.

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FIG. 4. Apc gene LOH in microadenomas isolated from nascentintestinal polyps in Apc^716 germline heterozygous mice, shown byagarose gel analysis of the PCR products of five microadenoma DNAsisolated from four Apc^716 mice. Lanes P, microadenomas dissectedfrom nascent polyps; lanes N, normal intestinal villi. Generation (N2or F1) and sex (M, male; F, female) are shown with identificationnumbers of individual mice. The polyps were 0.2-0.25 mm in diameter.Primers A-C and their positions and orientations are shown in Fig. 1A.ApcFL (A-C) indicates the band amplified from the wild-type full-length Apc allele (for 2845 aa), whereas Apc^716 (A-B) indicates theband from the knockout allele. Bottom arrowhead shows the bandamplified even with primer B alone (see Fig. 1C). Lane M, sizemarkers (A phage DNA digested with HindIII and EcoRI).We thank H. Maruyama for histology sample preparation, T.

Deutschman for ES cell line D3al, P. Soriano for the PGKneobpAcassette, T. Yagi for the DTA cassette, K. Sugihara for human FAPinformation, and K. Ochiai for discussion. We thank S. Nishimura, H.Ozeki, W. F. Dove, and C. Luongo for discussion and encouragement.

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