[CANCER RESEARCH 64, 48–54, January 1, 2004]

Hepatocarcinogenesis in Mice with �-Catenin and Ha-Ras Gene Mutations

Naomoto Harada,1 Hiroko Oshima,1,2 Masahiro Katoh,1 Yositaka Tamai,1 Masanobu Oshima,1,2 andMakoto M. Taketo2

1Banyu Tsukuba Research Institute (Merck), Tsukuba, and 2Department of Pharmacology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

ABSTRACT

We have established previously a mouse strain containing a mutant�-catenin allele of which exon 3 was sandwiched by loxP sequences[Catnblox(ex3)]. In this mouse strain, a Wnt-activating �-catenin mutationalone is insufficient for hepatocarcinogenesis, but additional mutations orepigenetic changes may be required. Here we report that hepatocellularcarcinoma develops at the 100% incidence in mice with simultaneousmutations in the �-catenin and H-ras genes that are introduced by ade-novirus-mediated Cre expression. Although H-ras mutation alone rapidlycauses large cell dysplasia in the hepatocytes, these cells show no auton-omous growth within 1 week after infection of the Cre-adenovirus. How-ever, simultaneous induction of an additional mutation in the �-cateningene causes a clonal expansion of such dysplastic cells, followed by nod-ular formation and development of hepatocellular carcinoma. These re-sults indicate that �-catenin mutations play a critical role in hepatocar-cinogenesis in cooperation with another oncogene and that these miceprovide a convenient model to investigate early steps of hepatocarcino-genesis.

INTRODUCTION

Hepatocellular carcinoma (HCC) is one of the most common hu-man malignant tumors (1). Although the molecular mechanisms ofhepatocarcinogenesis are not fully elucidated, many mutations havebeen found that inactivate tumor suppressor genes and stimulateexpression of oncogenes or growth factors (2). Various transgenic orknockout mouse lines have been established as HCC models with longand various latency periods of hepatocarcinogenesis (3, 4), whichsuggests a requirement of multiple and successive genetic alterationsin these mice.

�-Catenin is one of the key downstream effectors in the Wntsignaling pathway that plays an important role in various humancancers (5, 6). Mutations at the serine and/or threonine residues nearthe NH2 terminus in the �-catenin gene prevent their phosphorylationby the APC-axin-glycogen synthase kinase 3� complex and subse-quent degradation through the ubiquitin-proteasome pathway (7–12).Stabilized �-catenin associates with Tcf/Lef transcription factors,translocates into the nucleus, and activates transcription of a new setof genes (13, 14). We established previously a mouse strain contain-ing a mutant �-catenin allele of which exon 3 was sandwiched by loxPsequences [Catnb lox(ex3)] (15). Although the Cre-mediated deletion ofthe APC-axin-glycogen synthase kinase 3�-phosphorylation sitesfrom �-catenin causes numerous adenomatous polyps in the intestines(15), the same deletion by itself does not form any neoplastic foci inthe liver (16).

The ras family genes encode small GTPase proteins that trans-duce signals from the transmembrane receptors to the nucleus (17).Mutations at the hot-spots (codons 12, 13, and 61) in Ras proteins

lead to defects in the GTPase activity and constitutive activation ofthe downstream signals (17). Such mutations were found in �30%of human tumors. Although Ras mutations are rare in human livertumors (18), elevated expression of transforming growth factor �or insulin-like growth factor II have been reported in some humanHCC, which causes activation of the Ras signaling pathways (19).In addition, Harvey-ras (H-ras) mutation is detected frequently inthe spontaneous and carcinogen-induced mouse liver tumors (20,21). Accordingly, we have constructed a new transgenic mousestrain [Tglox(pA)H-ras*] in which an activated H-ras (H-rasG12V)protein is expressed on Cre-mediated recombination and investi-gated whether stabilized �-catenin can cause HCC in cooperationwith activated H-ras.

MATERIALS AND METHODS

Transgenic Mice. Tglox(pA)H-ras* transgenic mice were generated as fol-lows. The 1.4 kb lox-polyA-lox cassette from the plasmid pBS302 (Invitrogen,Groningen, the Netherlands) was subcloned into the NotI site downstream ofcytomegalovirus (CMV) promoter of the plasmid pcDNA3.1 Hygro[�] (In-vitrogen), 2.6 kb PuvII fragment of which was replaced with a NruI linkerfragment. The 4.8 kb SmaI-BamHI fragment from human activated Ha-rasgene (22) was then inserted into the XbaI site of the resulting plasmid(pCMV-loxP-bpA). The 7.3-kb whole-insert fragment was excised by NruIand injected into FVB/N fertilized eggs. Offspring mice were genotyped byPCR with the tail DNA using primers H-ras-F1 (5�-GCTCCATGCTGAACT-GTGGG-3�) and bpA-R (5�-GTGGCACCTTCCAGGGTCAA-3�) at 94°C for30 s, 60°C for 1 min, and 72°C for 1 min, for 35 cycles. Nine independenttransgenic mouse lines were obtained, two of which were used to generatecompound heterozygous mutants. Construction of Catnblox(ex3) knockout micehave been described previously (15), and they were backcrossed to theC57BL/6N strain for five generations.

Cre-Mediated Recombination in Liver. Construction of the cre-express-ing recombinant adenovirus (AdCMV-cre) has been described previously (16).The diluted aliquots of AdCMV-cre (108 plaque-forming units/100 �l) wereinjected into the tail vein of 7-week-old mice.

Western Immunoblot Analysis. Tissue lysates were prepared by sonica-tion in lysis buffer [10 mM HEPES (pH 7.8), 10 mM KCl, 0.1 mM EDTA, and0.1% NP40] containing protease inhibitors (phenylmethylsulfonyl fluoride,aprotinin, pepstatin, leupeptin, and antipain.) Aliquots of 50 �g total proteinwere resolved by SDS-PAGE, transferred to a membrane, and detected byantibodies against �-catenin (Sigma Chemical Company, St. Louis, MO) orH-ras (Nihonkayaku, Tokyo, Japan) coupled with the ECL-detection system(Amersham-Pharmacia Biotechnology Inc., Piscataway, NJ).

Bromodeoxyuridine (BrdUrd) Labeling. BrdUrd labeling and detectionkit (Roche Diagnostics, Mannheim, Germany) was used. Mice were injectedi.p. with 250 �l of BrdUrd solution 4 h before they were killed. Tissue sampleswere fixed with 10% formalin in PBS at 4°C overnight, dehydrated, andembedded in paraffin wax. Deparaffinized sections were microwaved in 10 mM

citrate buffer (pH 6.0) for 10 min, denatured in 2 N HCl at 37°C for 2 min andneutralized in 0.1 M boric acid/NaOH (pH 8.5) at 37°C for 2 min beforeimmunostaining.

Histology and Immunohistochemical Analyses. Sections were preparedand stained with H&E as described previously (15). For immunohistochemis-try, sections were incubated with antibodies against �-catenin, H-ras, cyclinD1 (Oncogene Research Products, Boston, MA), or BrdUrd (Becton DickinsonImmunocytometry Systems, San Jose, CA), respectively. The primary anti-bodies were detected using the Elite ABC rabbit kit (Vector Laboratories,Burlingame, CA) as described (15).

Received 7/16/03; revised 9/21/03; accepted 10/17/03.Grant support: Supported in part by grants from the Ministry of Education, Culture,

Sports, Science, and Technology of Japan, and Organization of Pharmaceutical Safety andResearch of Japan.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Makoto M. Taketo, Department of Pharmacology, GraduateSchool of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo, Kyoto 606-8501, Japan.Phone: 81-75-753-4402; Fax: 81-75-753-4391; E-mail: taketo@mfour.med.kyoto-u.ac.jp.

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RESULTS

High Incidence of HCC in the Catnblox(ex3):Tglox(pA)H-ras* MiceInfected with AdCMV-cre. We constructed a new transgenicmouse strain [Tglox(pA)H-ras*] in which a human activated H-ras(H-rasG12V) protein is expressed on Cre-mediated recombination(Fig. 1A). Nine independent transgenic lines were established,carrying 4 –17 copies of the transgene determined by Southernhybridization analysis (Fig. 1B). Two independent transgenic lines,HR3 and HR7, were crossed with Catnblox(ex3) mice to generateCatnblox(ex3):Tglox(pA)H-ras* compound mutant mice. To introduceboth �-catenin and H-ras mutations into the hepatocytes by Cre-mediated recombination, AdCMV-cre (16) was injected into thecompound mutant mice through the tail vein at 108 plaque-formingunits/mouse (Fig. 1C), as well as into the single transgenic andwild-type littermate controls. Six months after the inoculations,mice were euthanized, and gross morphology of the liver wasexamined. As shown in Fig. 1D, large liver tumors were found inthe infected Catnblox(ex3):Tglox(pA)H-ras* (HR-3) mice, whereas liv-ers from the infected single mutant or wild-type littermates ap-peared normal. Many HCC lesions were also found in the othercompound mutant strain (HR-7) infected with AdCMV-cre (data

not shown). The incidence of liver tumors for each genotype issummarized in Table 1 (Exp. 1). It should be noted thatCatnblox(ex3):Tglox(pA)H-ras* (HR-7) showed 100% incidence ofHCC and started to die with swollen abdomen 3– 4 months afterinfection (Fig. 1E). The incidence of HCC in the HR-3 line was78% at the same age, which could be explained by possibleinactivation of the transgene, because the HR-3 locus was mappedon X-chromosome, whereas HR-7 on an autosome (data notshown). We have not detected any gross morphological or histo-logical changes in other organs.

Fig. 1. Hepatocarcinogenesis in Catnblox(ex3):Tglox(pA)H-ras* mice. A, construct of the transgene for Tglox(pA)H-ras* mice. The lox-polyA-lox cassette and the H-rasG12V gene wereplaced downstream of the cytomegalovirus promoter. Red triangles and polyA indicate loxP and polyadenylation sequences, respectively. B, Southern blot analysis of NruI-digestedgenomic DNA from each founder mouse (HR1-HR9). The 7.3-kb NruI fragment containing the whole transgene construct was used as a probe. One copy-equivalent of the transgeneplasmid was applied on the “1 copy” lane. The arrow indicates the transgene band. Note that higher amounts of genomic DNA caused a slight delay in the transgene mobility thanthe control plasmid alone. C, Ad-CMV-cre mediated deletion of the �-catenin gene exon 3 and expression of the H-rasG12V gene in the Catnblox(ex3):Tglox(pA)H-ras* liver. Cre-mediatedrecombination events should delete exon 3 of the �-catenin gene in the Catnblox(ex3) and the polyadenylation sequence in the Tglox(pA)H-ras* transgene, respectively. D, gross morphologyof livers from the Catnblox(ex3):Tglox(pA)H-ras* (HR-3) mice (�ex3:ras*3) and littermate controls (WT:WT, WT:ras*3, and �ex3:WT) at 6 months after inoculations of 108 plaque-formingunits/mouse AdCMV-cre. E, survival curve of the Catnblox(ex3):Tglox(pA)H-ras* (HR-7) mice (�ex3:ras*7) and littermate controls (control) after the infection. F, Western analysis ofthe �-catenin protein (�-�-catenin) and the H-ras protein (�-H-ras) in the tumor (T) and adjacent normal (N) liver tissues from the compound heterozygotes. A lysate ofH-rasG12V-transformed cells were used as a positive control for the H-ras protein (C). The arrows on the left show the positions for wild-type (WT) and mutant (�ex3) �-catenin,respectively, whereas the arrow on the right shows that for H-ras protein. Each lysate with 50 �g total protein was loaded/lane.

Table 1 Incidence of liver tumors in the mouse strains

Genotype(Catnb�ex3:TgH-ras*)

Exp. 1 Exp. 2

6 months P.I.a 1–4 weeks P.I. 5–14 weeks P.I.

HCC (%) Dysplastic cell (%) Nodules/HCC (%)

�ex3:ras*3 7/9 (78) N.D. N.D.�:ras*3 0/8 (0) N.D. N.D.�ex3:ras*7 7/7 (100) 5/5 (100) 8/8 (100)�:ras*7 0/10 (0) 4/4 (100) 0/7 (0)�ex3:� 0/14 (0) 0/6 (0) 0/7 (0)�:� 0/8 (0) 0/3 (0) 0/7 (0)

a P.I., postinfection; N.D., not determined; HCC, hepatocellular carcinoma.

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To confirm expression of the mutant H-ras and �-catenin proteinsin HCC, we prepared cell lysates from the tumor and adjacent normalliver tissues, and analyzed by Western analysis. As shown in Fig. 1F,expression of the mutant �-catenin and overexpression of H-ras wasdetected only in the tumor tissues, but not in the normal liver or othertissues (data not shown). Weak expression of H-ras in the normal liveris likely derived from the endogenous mouse H-ras protein, cross-reacting with the antibody for human H-ras.

Upon histological analysis of the HCC developed in the AdCMV-cre-infected Catnblox(ex3):Tglox(pA)H-ras* mice, most HCC tissues werewell-differentiated with a compact, and occasionally trabecular pat-tern. The lobular architecture was lost in these tumors. Representativesections of the HCC with “compact” and “trabecular” types are shownin Fig. 2, A and B, respectively. The trabecules consisted of multiplecell layers in which large dysplastic hepatocytes were found with ahigh nucleocytoplasmic ratio, numerous mitotic figures, and necrosis.Tumor cells often compressed the adjacent normal liver tissue (Fig. 2,C and D), and occasionally showed intrahepatic invasions (Fig. 2A).These histological features are very similar to those commonly foundin human HCC.

Upon immunohistochemical analysis, overexpression of �-catenin(Fig. 2E) and H-ras (Fig. 2F) were detected only in the tumor cells,but not in the normal hepatocytes. These results are consistent withthose of the Western analysis. Cyclin D1, one of the target genes ofthe Tcf/Lef/�-catenin transcription factor (23), was also overex-pressed in the tumor cells (Fig. 2G). Consistent with the result, thelabeling index with BrdUrd-uptake was increased in the tumor, witha high proliferation rate of the tumor cells (Fig. 2H). These dataindicate that simultaneous mutations in the �-catenin and H-ras genesare sufficient to induce HCC in the mouse liver.

Early Processes of Hepatocarcinogenesis in the Catnblox(ex3):Tglox(pA)H-ras* Mice. Because Catnblox(ex3):Tglox(pA)H-ras* (HR-7)mice infected with the AdCMV-cre showed a very high incidence ofHCC (100% at 6 months postinfection), we then investigated earlierprocesses of hepatocarcinogenesis in these mice. We infected anothergroup of the compound mutant mice and controls (Table 1, Exp. 2),and euthanized them at various time points from 1 to 14 weeks afterinfection. At 4 weeks, some small foci (�1 mm ø) were visible undera dissection microscope on the surface of the liver only in thecompound mutant mice (Fig. 3A). At 2 months, many large nodules(2–3 mm ø) were found macroscopically (Fig. 3B), whereas liversfrom the control mice all appeared normal (data not shown). Theincidence of HCC in the Catnblox(ex3):Tglox(pA)H-ras* (HR-7) mice wasagain 100% between 5 and 14 weeks after infection.

As early as 1 week after infection, dysplastic hepatocytes with largenuclei and high nucleocytoplasmic ratio were detected in the liversections of the compound mutant mice, although no gross morpho-logical abnormalities were observed (Fig. 3E). Such cells were ob-served in all of the compound mutant mice at 1–4 weeks afterinfection. Interestingly, these dysplastic hepatocytes were surroundedby inflammatory cells (Fig. 3, D and E, insets) including lymphocytes,neutrophils, and macrophages (Kupffer cells), which were not re-ported in other HCC models (24–26). Hardly any inflammatory cellswere observed in the AdCMV-cre-infected �-catenin mutant mice(Fig. 3C) or wild-type mice (data not shown), ruling out the possibilitythat the inflammation was caused by the adenoviral infection itself(see “Discussion”). As the acute inflammation subsided by the fourthweek after infection (Fig. 3, G and H), the dysplastic hepatocytespersisted and started to grow, and formed multifocal dysplastic nod-ules by the fifth week (Fig. 3K), followed by formation of HCC(Fig. 3N).

Proliferative Activation of the Dysplastic Hepatocytes by�-Catenin Mutation. Interestingly, these large dysplastic hepato-cytes were detected not only in livers from the compound mutant micebut also in the Tglox(pA)H-ras* mice (i.e., without �-catenin mutation;Fig. 3D), whereas they were not detected in the Catnblox(ex3) (Fig. 3C)or wild-type mice (data not shown). These data suggest that newlyexpressed activated human H-ras should be sufficient to cause amorphological change of hepatocytes in the mouse liver. Importantly,however, dysplastic cells in the Tglox(pA)H-ras* mice did not show anyproliferative changes (Fig. 3, G, J, and M), and neither dysplasticnodules nor HCC developed in the Tglox(pA)H-ras* transgenic mouselivers even at 17 months postinfection of AdCMV-cre, although wecannot exclude the possibility that HCC might develop much later likein some HCC models (27).

To additionally characterize the early changes in the compoundmutant mouse liver, serial sections of the dysplastic hepatocytes wereanalyzed immunohistochemically. Upon staining with an anti-H-rasantibody, a strong signal was detected in the membrane of the dys-plastic hepatocytes in both Tglox(pA)H-ras* transgenic mice (data notshown) and compound mutant mice (Fig. 4C). On the other hand,cellular �-catenin level and BrdUrd incorporation in the dysplastichepatocytes increased only in the compound mutant mice (Fig. 4, Band D), but not in the Tglox(pA)H-ras* transgenic mice (data not shown).As we reported previously (16), hepatocytes with the �-catenin mu-tation alone were nontumorigenic and quiescent despite the nuclearaccumulation of �-catenin (data not shown). Taken together, thesedata demonstrate that combination of the H-ras and �-catenin muta-tions are sufficient to activate proliferation of the dysplastic hepato-cytes, which is schematically summarized in Fig. 4E.

DISCUSSION

In the present study, we developed a new model for mouse HCC bysimultaneously introducing �-catenin and H-ras mutations in the liverof compound transgenic mice [Catnblox(ex3):Tglox(pA)H-ras*]. Althoughcooperation of two oncogenes, such as H-ras/c-myc, SV40 T-antigen/H-ras, and transforming growth factor �/c-myc have been reported toaccelerate hepatocarcinogenesis (24, 28), the present model is distinctfrom others in several features. In most other transgenic models,sequential development of HCC is observed, i.e., hepatic dysplasia,degeneration, nodular regeneration of hepatocytes, hepatocellular ad-enoma, and subsequent HCC with a “nodule-in-nodule” pattern (3). Incontrast, neither regenerative nor adenomatous lesions are observed inour model, but malignant neoplastic lesions appear rapidly after theclonal expansion of the dysplastic hepatocytes. Because transgenesare expressed ubiquitously in the liver in other models, it is stillunclear whether large dysplastic hepatocytes are the direct precursorlesion of HCC or they simply induce degeneration of hepatocytes,followed by a secondary regeneration and hyperplastic growth of thesurrounding cells. In our model, stabilized �-catenin and activatedH-ras are expressed simultaneously in the same hepatocytes infected withadenovirus, leading to generation of dysplastic cells. Accordingly, thedysplastic hepatocytes in the present model are the direct precursor lesionof HCC. Although it takes long latency periods before hepatocarcino-genesis in other transgenic models (4), our model develops HCC at a100% incidence within 2 months after the infection.

Although single transgenic mice, i.e., Catnblox(ex3) or Tglox(pA)H-ras*

did not develop any HCC on infection with AdCMV-cre, there was aninteresting difference between the two lines. As we reported previ-ously, �-catenin mutation alone did not cause any morphologicalchanges in the liver (16). In contrast, activated H-ras caused dysplasticchanges in hepatocytes immediately after the infection of AdCMV-cre. It is worth noting that mild hepatocyte dysplasia is found in H-ras

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transgenic mice in which the transgene product is expressed consti-tutively as one of the self-proteins (24). Appearance of large dysplas-tic hepatocytes is one of the earliest changes also in other mouse HCCmodels expressing SV40 T-antigen (24), c-myc (25), transforming

growth factor � (26), and so forth, although molecular mechanismsare not clear. In the hepatocyte transformation, it is conceivable thatsome common signals in the pathways of these oncoproteins areactivated by the H-ras mutation as well. Despite the rapid appearance

Fig. 2. Histological analysis of hepatocellular carcinoma (HCC) developed in Catnblox(ex3):Tglox(pA)H-ras* (HR-3) mice. A–D, representative H&E-stained sections of HCC in theCatnblox(ex3):Tglox(pA)H-ras* (HR-3) mice inoculated with 108 plaque-forming units/mouse Ad-CMV-cre. Arrows in A show intrahepatic invasion. E–H, immunohistochemical staining of thesections serial to that in C and D for �-catenin (E), H-ras (F), cyclin D1 (G), and bromodeoxyuridine (H), respectively. Bars, 100 �m for A and B; 500 �m for C; and 100 �m for D–H.

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of large dysplastic hepatocytes, no HCC developed from H-ras trans-genic mice at least for 1 year after infection, which is different fromother H-ras transgenic lines reported previously (24, 27). The numberof hepatocytes expressing activated H-ras is much less in the presentmodel than those in other ubiquitous expression mice. The expressionlevel of H-ras in our transgenic mice may also be lower than in othermodels because of the CMV promoter. Additionally, the geneticbackground may also affect the incidence of HCC (29). It remains to

be seen whether HCC develops in our model after �1 year post-infection.

Activated H-ras alone is insufficient for clonal expansion of thedysplastic hepatocytes, but an additional mutation in the �-cateningene can cause HCC, suggesting that one of the roles of thestabilized �-catenin in hepatocarcinogenesis may be to stimulateproliferation of the dysplastic cells generated by activated H-ras.This interpretation is supported further by the fact that cyclin D1,

Fig. 3. Early stages of hepatocarcinogenesis in Catnblox(ex3):Tglox(pA)H-ras* (HR-7) mice. A, dissection micrograph of a small focus in a liver at 4 weeks after infection. B, gross morphologyof a liver at 9 weeks after infection. C–N, H&E-stained liver sections at 1 week (C–E), 4 weeks (F–H), 7 weeks (I–K), and 9 weeks (L–N) after infection. Insets show higher magnifications.Arrowheads and arrows in D and E show “dysplastic hepatocytes” and inflammatory cells, respectively, whereas � in K indicate multifocal nodules. Bars, 100 �m (20 �m for insets).

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one of the target genes of Wnt signaling, is overexpressed in HCCin the compound mutant mice (Fig. 2G). Although cooperation ofH-ras and c-myc, another target of Wnt signaling, in hepatocarci-

nogenesis was reported, we could not detect any overexpression ofc-myc in HCC (data not shown). As mutations in the �-cateningene are often found in HCC developed in transgenic mouse lines

Fig. 4. Immunohistochemical analysis of the dysplastic hepatocytes. A–D, serial liver sections containing the atypical hepatocytes at 11 weeks after infection stained with H&E (A),anti-�-catenin (B), anti-H-ras (C), and anti-bromodeoxyuridine (D) antibodies. E, schematic diagram showing the effect of �-catenin or H-ras mutation alone and compound mutationson the hepatocarcinogenesis. Bars, 100 �m.

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with c-myc as well as H-ras transgenes (30), stabilized �-cateninmay cooperate not only with H-ras but also with various oncogenesin hepatocarcinogenesis. Although no �-catenin mutations werefound in HCC of SV40 T-antigen transgenic mice (31), mutationsin or activation of other Wnt signaling genes may be contributingto the hepatocarcinogenesis (32).

We have observed inflammatory responses against dysplastic hepa-tocytes expressing activated H-ras, consistent with a previous reportthat overexpression of the H-ras gene enhanced inflammation in theliver (33). Regeneration of hepatocytes caused by this acute inflam-mation may help accumulate additional genetic mutations in otheroncogenes, although it remains to be determined what other muta-tion(s) are involved in the �-catenin and H-ras genes. Our investiga-tions can be extended further with helper-dependent adenovirus (34)or naked DNA (35) that should express Cre without causing muchinflammation.

In conclusion, we have established a new mouse HCC model with�-catenin and H-ras mutations simultaneously introduced by anadenovirus-mediated liver-specific Cre-expression system. We havedemonstrated that neither H-ras nor �-catenin mutation alone is suf-ficient for hepatocarcinogenesis, but both are when combined. Both�-catenin mutations and activation of H-ras signaling pathways, suchas overexpression of transforming growth factor � or insulin-likegrowth factor II, are often found in human HCC. In the present mousemodel, it takes only a short latency period of several weeks with 100%incidence. Accordingly, this HCC model provides an ideal system toinvestigate early steps of hepatocarcinogenesis, as well as to evaluateanticancer drug candidates.

ACKNOWLEDGMENTS

We thank Yukino Ohtake for excellent technical assistance.

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HCC IN MICE WITH BOTH �-CATENIN AND HA-RAS MUTATIONS

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