[CANCER RESEARCH 60, 2864–2868, June 1, 2000]

b-Catenin Mutations and Protein Accumulation in All Hepatoblastomas Examinedfrom B6C3F1 Mice Treated with Anthraquinone or OxazepamColleen H. Anna, Robert C. Sills, Julie F. Foley, Patricia S. Stockton, Thai-Vu Ton, and Theodora R. Devereux1

Laboratories of Molecular Carcinogenesis [C. H. A., T. R. D.] and Experimental Pathology [R. C. S., J. F. F., P. S. S., T-V. T.], National Institute of Environmental HealthSciences, National Institutes of Health, Research Triangle Park, North Carolina 27709

ABSTRACT

The molecular pathogenesis of hepatoblastomas in the B6C3F1 mouse isunclear but may involve alterations in theb-catenin/Wnt signaling pathwayas was recently described for chemically induced hepatocellular neoplasmsand human liver cancers. The objective of this study was to characterize themutation frequency and spectrum ofb-catenin mutations and the intracel-lular localization of b-catenin protein accumulation in chemically inducedhepatoblastomas. In this study,b-cateninmutations were identified in all 19anthraquinone-induced hepatoblastomas and all 8 oxazepam-induced hepa-toblastomas examined. Although several hepatoblastomas had multiple dele-tion and/or point mutations, the pattern of mutations in the hepatoblastomasdid not differ from that identified in hepatocellular neoplasms. In a majorityof the hepatoblastomas (six of seven) examined by immunohistochemicalmethods, both nuclear and cytoplasmic localization ofb-catenin protein weredetected, whereas in hepatocellular adenomas, carcinomas, and normal liveronly membrane staining was observed. Our data suggest thatb-cateninmutations and the subsequent translocation ofb-catenin protein from the cellmembrane to the cytoplasm and nucleus may be critical steps in providinghepatocellular proliferative lesions with the growth advantage to progress tohepatoblastomas.

INTRODUCTION

An increased incidence of highly malignant hepatoblastomas hasbeen observed in mice following treatment with certain chemicals inNational Toxicology Program carcinogenesis studies (1, 2), whereasthese tumors are rare in untreated mice. It has been hypothesized thatthese tumors arise from or are a rare variant of hepatocellular carci-nomas (2). Diwanet al. (3) found that hepatoblastomas in micedeveloped after exposure to promoters such as phenobarbital in initi-ation-promotion protocols. Few studies of molecular analyses onchemically induced hepatoblastomas have been reported, althoughH-ras activation and p53 mutation have not been found (4, 5).

Hepatoblastomas are the most frequent malignant liver tumor foundin young children (6). There is also an increased incidence of thesetumors in patients with APC2 who carry a germline mutation in theAPC gene, and recently it was discovered that there is a high fre-quency ofb-cateninmutations andb-catenin protein accumulation insporadic hepatoblastomas (7).

b-catenin is a central and critical molecule in the Wnt signalingpathway. TheAPC gene product and the glycogen serine-threoninekinase-3b together targetb-catenin for degradation and modulate itsexpression (8, 9). In some cancers, including colon and hepatocellulartumors, mutations in eitherAPC or b-cateninlead tob-catenin proteinaccumulation and up-regulation of Wnt signaling, resulting ultimately incell proliferation and inhibition of apoptosis (10). During this process,cytoplasmicb-catenin may form a complex with Tcf transcription fac-tors, enter the nucleus, and target genes such asc-MYCfor transcription(9–11).

b-catenin mutations have been identified at high frequency inhuman hepatocellular carcinomas (12) and in hepatocellular adeno-mas and carcinomas of mice exposed to certain chemicals (13, 14). Inthis study, we examined hepatoblastomas, which developed in micetreated with anthraquinone or oxazepam for 2 yr, for mutations inb-catenin. Anthraquinone is used in the manufacture of dyes andpigment and in the pulp and paper industry, and oxazepam is acommonly prescribed tranquilizer. We were particularly interested inwhether theb-cateninmutation frequency or spectrum was chemicalspecific and whether the mutations in hepatoblastomas differed fromthose of hepatocellular adenomas or carcinomas. Therefore, we alsoassessed the mutation frequency and profile in hepatocellular neo-plasms from B6C3F1 mice treated with anthraquinone. Laser capturemicrodissection was used to collect cells from some of the smallerhepatoblastomas to avoid contamination with surrounding normaltissue or adjacent hepatocellular tumors. In addition, we compared theprotein expression and intracellular localization ofb-catenin proteinin hepatoblastomas and hepatocellular neoplasms.

MATERIALS AND METHODS

Tumor Samples and DNA Isolation. Oxazepam treatment, liver tumorcollection, tumor incidence data of B6C3F1 mice, and H-rasoncogene muta-tion analysis for the studies have been reported previously (4). The incidenceof hepatoblastomas in male mice was 0%, 4%, 40%, and 24% at 0 ppm, 125ppm, 2500 ppm, and 5000 ppm oxazepam, respectively; and for female mice,0%, 2%, 16%, and 14%, respectively. For collection of liver tumors from theanthraquinone study B6C3F1 mice were fed diets containing 0, 833, 2500, or7500 ppm anthraquinone for 105 weeks. The incidence of hepatoblastomas inmale mice was 2%, 10%, 22%, and 43% at 0, 833, 2500, and 7500 ppmanthraquinone, respectively; and for female mice, only 2% at the 7500 ppmdose. A subset of the hepatocellular neoplasms and a small number of hepa-toblastomas with surrounding tissue were frozen in liquid N2 and stored at270°C. The remainder of the liver tissues was preserved in 10% neutralbuffered formalin, trimmed, and embedded in paraffin. Sections 5–6mm wereused for staining with H&E. Normal liver, hepatoblastomas, and surroundinghepatocellular neoplasms (adenomas and carcinomas) were either hand-micro-dissected or laser capture microdissected (4 of 19 of the anthraquinone-inducedhepatoblastomas) from 2–5 serial 10-mm paraffin sections for mutation anal-ysis. A PixCell-I (30-mm spot size and about 500 hits/sample) LCM System(Arcturus Engineering Inc., Mountain View, CA) was used for the lasercapture microdissection, as described previously (15, 16). Microdissectedsamples were digested overnight in 500ml of digestion buffer [50 mM Tris (pH8.5), 0.5% Tween 20, 1 mM EDTA, and 0.5 mg/ml proteinase K] at 55°C. DNAwas obtained by boiling the digested samples for 5 min. In addition, most ofthe mutation analysis on the hepatocellular neoplasms was performed withDNA isolated from frozen tumor tissue, as described previously (4, 16).

b-Catenin Mutation Screening and Identification. SSCP analysis wascarried out on PCR products of exon 2 (corresponds to exon 3 in human) of themouseb-catenin gene (12), which contains the glycogen serine-threoninekinase-3b targeted phosphorylation sites within residues 33–45. The se-quences of the intronic PCR primers flanking the borders of exon 2 were:BCAT-1F, 59-TACAGGTAGCATTTTCAGTTCAC-39; and BCAT-2R, 59-TAGCTTCCAAACACAAATGC-3 (12). Inner primers were BCAT-7F, 59-TAACATACTCTGTTTTTACAGCTG-39, and BCAT-8R, 59-ACATCTTCT-TCCTCAGGGTTG-39. For DNA from frozen samples primers 1F3 2R wereused for amplification. For DNA from fixed sections nested PCR was usedwith primers 1F3 2R for the outer reactions and both 1F3 8R and 7F3 8R

Received 9/3/99; accepted 4/4/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at MD D4-04, National Instituteof Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709.Phone: (919) 541-3241; Fax: (919) 541-7784; devereux@niehs.nih.gov.

2 The abbreviations used are: APC, adenomatous polyposis coli; SSCP, single-strandconformational polymorphism.

2864

Research. on June 11, 2020. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from

for the inner reactions. [33P]-dATP was incorporated into the inner PCRreactions for SSCP analysis, and two gel conditions were used to detectmutations: 6% acrylamide gels with 10% glycerol were electrophoresed at 40W for 6 h at 5°C, and 0.53mutation detection enhancement (AT Biochem,Malvern, PA) gels were electrophoresed at 3 W for 17 h at room temperature.DNA from normal liver and no-DNA controls were included with all ampli-fication experiments to confirm no cross-contamination of PCR products.

Point mutations and some small deletions were identified by reamplifyingsamples with altered bands on SSCP gels. Other deletions were identified byexcision and boiling of the altered band, followed by a fresh amplification. Theamplified bands were gel purified on Qiagen columns (Qiagen, Valencia, CA)before sequencing with a [33P]-Thermo-Sequenase kit (Amersham, Cleve-land, OH) The amplification primers also served as sequencing primers.Mutation identification was confirmed with at least two amplificationreactions from original DNA.

Immunohistochemistry. Tumor tissues were fixed in 10% neutral buff-ered formalin, processed routinely, and embedded in paraffin. Localizationof b-catenin protein expression was investigated using a polyclonal goatanti-b-catenin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at adilution of 1:100 on serial 6-mm sections. Slides were deparaffinized andrehydrated through xylene and ethanol washes into 13 Automation buffer(Biomeda Corp., Foster City, CA). Antigen unmasking was accomplishedby heating in 200 ml of citrate buffer in a microwave oven at 50% powerfor 5 min. Following a 1-min break, the cycle was repeated, and the slideswere then allowed to cool for 20 min. Endogenous peroxidase activity wasblocked with 3% H2O2 for 15 min. After rinsing in 13 Automation buffer,sections were blocked with 5% normal goat serum for 30 min. The primaryb-catenin antibody was then applied to sections for 1 h at room tempera-ture. Nonimmune rabbit IgG (Jackson ImmunoResearch Laboratories, WestGrove, PA) was used as the negative control at equivalent conditions inplace of the primary antibody. The bound primary antibody was visualizedby streptavidin-biotin-peroxidase detection using the goat Immunocruzstaining system (Santa Cruz Biotechnology), according to the manufactur-er’s instructions, and with 3,39-diaminobenzidine as the color-developingreagent. Slides were counterstained with Harris hematoxylin, dehydratedthrough a graded series of ethanol washes to xylene, and coverslipped withPermount (Fisher, Springfield, NJ).

Western Analysis for b-Catenin Protein Expression. A small number(5) of frozen tumor samples containing hepatoblastomas from the anthraqui-none study were obtained for this study. The samples were sectioned with acryostat, and a single section was quick stained to identify the hepatoblasto-mas. Then, the unstained hepatoblastomas were dissected away from the restof the liver tumor and normal tissue to isolate protein. Frozen samples ofnormal liver, hepatocellular adenomas, and carcinomas from this study wereused for comparison. Cellular protein was extracted from these samples forWestern blot analysis of protein expression, as described previously (13).

RESULTS

Mutation Analysis. A high incidence of malignant hepatoblasto-mas resulted from anthraquinone or oxazepam treatment of B6C3F1mice, whereas this tumor type is found only rarely in control mice. Inthis study, we examined hepatoblastomas induced by these two chem-icals for mutations in exon 2 (corresponds to exon 3 in humans) of theb-cateningene. Mutations were identified in all 19 anthraquinone-induced hepatoblastomas and all 8 oxazepam-induced hepatoblasto-mas examined (Table 1 and Fig. 1,A andB). Multiple point mutationsand/or deletions in exon 2 were detected in many hepatoblastomasfrom both chemical treatment groups.

Point mutations ofb-cateninwere detected in hepatoblastomas atcodons 29–45 (Table 1). Three large hepatoblastomas that had beendissected into two or more parts contained more than one pointmutation or deletion found in different parts of the tumor. Two smallhepatoblastomas had the same mutations in different parts of thetumors. Fourteen of 19 (74%) anthraquinone-induced hepatoblasto-mas and 7 of 8 (88%) oxazepam-induced hepatoblastomas containeddeletions within exon 2 ofb-catenin. Approximately half of the

deletions detected in tumors from each chemical treatment groupstarted at codon 5, some affecting the splicing site for exon 2. Thedeletion mutants we identified removed 3–44 amino acids, and someof these included critical residues in the NH2-terminal phosphoryla-tion region necessary for degradation of theb-catenin protein.

Among the point mutations, any of the three bases of the codoncould be mutated, and specific patterns of G to A or G to T mutationswere not apparent for either chemical treatment. There did not appearto be a chemical signature mutation pattern in that the spectrum ofb-cateninmutations did not differ between the hepatoblastomas fromthe two different chemicals. Moreover, the pattern of point mutationsdid not differ from those found in hepatocellular neoplasms inducedby chronic treatment with other chemicals (13).

In National Toxicology Program studies, hepatoblastomas are oftenfound within a hepatocellular adenoma or carcinoma. It has been hypoth-esized that this tumor may represent a more malignant hepatocellularneoplasm. We, therefore, examined six samples from anthraquinone-treated mice in which a hepatocellular adenoma or a hepatocellularcarcinoma was microdissected from the same tissue area as a nearbyhepatoblastoma. Two of three adenomas and two of three carcinomas hadb-cateninmutations in exon 2 as compared with nearby hepatoblastomasthat all had mutations. However, all of the point mutations or deletions inthe hepatoblastomas were different from those found in the correspond-ing hepatocellular neoplasms (data not shown). These results suggestedthat mutations found in hepatoblastomas may be different and lead to amore malignant phenotype than those in the hepatocellular tumors.

To further address this question, we examined DNA from 63frozen hepatocellular neoplasms for mutations inb-catenin. Ten of32 adenomas (31%) and 13 of 31 carcinomas (42%) exhibitedb-cateninmutations (Table 2). Although there were more deletionsand more multiple mutations in the hepatoblastomas than in the

Table 1 Summary ofb-catenin mutations in hepatoblastomas from B6C3F1 micetreated with anthraquinone or oxazepam

Tumor group Frequency Codon mutation (amino acid)

Anthraquinone 19/19 (100%)1 Codon 29 TCT to TCC (silent)1 codon 31

TTG to GTG (Leu to Val)1 codon 33TCT to TAT (Ser to Tyr)1 Del. codons 34–37

2 Codon 32 GAT to AAT (Asp to Asn)3 Codon 32 GAT to GTT (Asp to Val)4 Codon 32 GAT to AAT (Asp to Asn)

1 Del. codons 32–385 Codon 32 GAT to AAT (Asp to Asn)

1 Del. codons 15–366 Codon 34 GGA to GAA (Gly to Glu)7 Del. codons 5–238 Codon 35 ATC to GTC (Ile to Val)9 Codon 37 TCT to TTT1 Del.

codons 5–7, 5–13, and 21–4310 Codon 45 TCC to TTC (Ser to Phe)11 Del. codons 5–712 Codon 33 TCT to TTT1 Del.

codons 5–713 Del. codons 5–2314 Del. codons 12–5115 Codon 32 GAT to TAT1 Del.

codons 15–3616 Del. codons 18–4817 Del. codons 21–4318 Del. codons 34–3719 Del. codons 21–43 and 25–69

Oxazepam 8/8 (100%)1 Codon 32 GAT to GGT (Asp to Gly)

1 Del. codons 5–82 Del. codons 5–73 Del. codons 36–484 Del. codons 23–495 Codon 34 GGA to GTA (Gly to Val)6 Del. codons 5–137 Del. codons 16–368 Del. codons 21–43

2865

b-CATENIN MUTATIONS IN CHEMICALLY INDUCED HEPATOBLASTOMAS

Research. on June 11, 2020. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from

hepatocellular neoplasms, the spectrum of mutations was not extensivelydifferent among the different tumor histotypes. We also analyzed sixnormal liver tissue samples from mice in the high-dose anthraquinonegroup and found no mutations in these samples (Fig. 1). The frequency ofmutations in the hepatoblastomas (27 of 27) is statistically higher(P , 0.0001 by Fisher’s exact test) than in hepatocellular neoplasms (23of 63 anthraquinone induced and 18 of 41 oxazepam induced (4), pro-viding evidence that hepatoblastomas evolve from preexisting hepatocel-lular neoplasms withb-cateninmutations.

Protein Expression. In addition to mutation analysis, proteinexpression was performed on a subset of the samples from the

anthraquinone study. We evaluated five microdissected hepato-blastomas from frozen samples and eight hepatocellular neoplasmsfor b-catenin protein expression by Western blotting. In general,the tumors expressed similar or slightly greater levels of the mutantnormal-sized or truncatedb-catenin protein than was observed fornormal liver (Fig. 2). That we did not observe significantly greaterexpression in the hepatocellular neoplasms and hepatoblastomasmay be due to heterogeneous accumulation of protein in parts ofthe tumors and/or contamination with normal tissue relative tob-catenin protein.

To examine the intracellular localization ofb-catenin in the tumors,we also evaluated some of the samples including seven hepatoblas-tomas by immunohistochemistry. Immunohistochemical staining ofthe hepatoblastomas revealed strong membrane, cytoplasmic, andnuclear reactivity (Fig. 3). This was in striking contrast to normal liverand hepatocellular adenomas and carcinomas from the same sections,in which only membrane staining was observed (Fig. 3).

Fig. 1. b-cateninmutation identification in hepatoblastomas and normal liver from B6C3F1 mice treated with anthraquinone.A, SSCP analysis. Shown is a 6% acrylamide/10%glycerol gel with samples amplified with primers 1F3 8R. Lanes 1–8, DNA samples from anthraquinone-induced hepatoblastomas, as shown in Table 1: tumor samples 3, 4a, 7, 9,11, 19a, 19b (a and b represent different parts of the same tumor), and 6;Lane 9, no DNA control;Lane 10, normal control B6C3F1 mouse liver DNA;Lanes 11–15, DNA samplesfrom five different normal livers from the high-dose anthraquinone group of mice.B, cycle sequencing ofb-cateninwithin exon 2 in four tumor samples. Thearrowspoint to mutations.Sequence 1, hepatoblastoma 4a with deletion of codons 31–38;sequence 2, hepatoblastoma 4b (different part of the same tumor) with mutation at codon 32, GAT to AAT (Asp toAsn); sequence 3, hepatoblastoma 12 with mutation at codon 33, TCT to TTT (Ser to Phe; a different smaller PCR product of this sample had a deletion mutation of codons 5–7, datanot shown);sequence 4, hepatocellular carcinoma 43 with no mutation.

Fig. 2. Expression ofb-catenin protein in anthraquinone-induced mouse liver tumorsby Western blot analysis. Equivalent amounts of protein from total homogenates of eachsample were electrophoresed and immunoblotted, as described in “Materials and Meth-ods.” The blot was cut horizontally and developed with anti-b-catenin (top) and anti-actin(bottom). Lane 1, normal liver;Lanes 2–6, hepatocellular neoplasms;Lanes 7–11,hepatoblastomas 2, 5, 13, 6, and 14a.

Table 2 Summary ofb-catenin mutations in hepatocellular neoplasms from B6C3F1mice treated with anthraquinone

Tumor type Frequency Codon mutation (amino acid)

Adenomas 10/32 (31%)1 Codon 33 TCT to TTT (Ser to Phe)2 Codon 37 TCT to GCT (Ser to Ala)3 Codon 45 TCC to TTC (Ser to Phe)4 Del. codons 23–351 Codon 16 CCG to CCT (silent)5 Del. codons 5–66 Del. codons 5–417 Del. codons 5–498 Del. codons 5–519 Del. codons 29–41

10 Del. codons 31–50Carcinomas 13/31 (42%)

1 Codon 32 GAT to TAT (Asp to Tyr)2 Codon 32 GAT to TAT (Asp to Tyr)3 Codon 32 GAT to GTT (Asp to Val)4 Codon 32 GAT to GTT (Asp to Val)5 Codon 34 GGA to GAA (Gly to Glu)6 Codon 37 TCT to GCT (Ser to Ala)7 Codon 41 ACC to ATC (Thr to Ile)8 Codon 41 ACC to ATC (Thr to Ile)9 Codon 45 TCC to TTC (Ser to Phe)

10 Codon 45 TCC to TTC (Ser to Phe)11 Del. codons 5–712 Del. codons 5–2313 Del. codons 16–36

2866

b-CATENIN MUTATIONS IN CHEMICALLY INDUCED HEPATOBLASTOMAS

Research. on June 11, 2020. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from

DISCUSSION

b-catenin is a central and critical component of the Wnt signalingpathway (17) and is up-regulated in many human cancers followingmutation of either theAPC gene or theb-cateningene (10, 12). Thestriking finding of somatic mutations of theb-catenin gene in allmouse hepatoblastomas induced by anthraquinone and oxazepam isimportant because these are the same mutations that have been ob-served in human childhood hepatoblastomas (7) and adult hepatocel-lular carcinomas (12), as well as in chemically induced rodent hepa-tocellular neoplasms (13, 14, 18). The high frequency ofb-cateninmutations and accumulation ofb-catenin protein in the mouse livertumors is consistent also with findings in human colon tumors (10),

suggesting that up-regulation of the Wnt signaling pathway is animportant step in their pathogenesis. Thus, the occurrence of chemi-cally induced hepatoblastoma formation in the B6C3F1 mouse and itsassociated molecular alterations seem to be relevant to the humancarcinogenic process in liver.

To date, the pathogenesis of hepatoblastomas in mice has notbeen well defined. Hepatoblastomas occur only rarely in untreatedmice of certain strains or following treatment only with tumorinitiators such asN-nitrosodiethylamine (19). However, followingchronic treatment of B6C3F1 mice with promoters such as pheno-barbital, a high incidence of hepatoblastomas has been observed(20, 21). Hepatoblastomas are usually observed within hepatocel-lular carcinomas and may evolve as a variant or more malignant

Fig. 3. Immunohistochemical analysis ofb-catenin expression in anthraquinone-induced hepatoblastomas and surrounding liver tumors and tissue.A, hepatoblastoma 14b (notanalyzed forb-cateninmutations) within a hepatocellular carcinoma; H&E (340).B, the same hepatoblastoma reacted withb-catenin antibody (340). Note marked membrane andcytoplasmic staining, compared with surrounding hepatocellular cells of carcinoma with only weaker membrane staining.C, higher magnification of hepatoblastoma inB (3132).D,hepatoblastoma 2 with strong nuclear and cytoplasmic staining (350).E, high magnification of area in hepatoblastoma showing some cytoplasmic and nuclear staining (3100).F, highmagnification of hepatoblastoma showing marked nuclear staining (3200).

2867

b-CATENIN MUTATIONS IN CHEMICALLY INDUCED HEPATOBLASTOMAS

Research. on June 11, 2020. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from

form of adenoma or carcinoma (3, 21). The finding of 31% ofanthraquinone-induced adenomas, 42% of carcinomas, and 100%of hepatoblastomas withb-catenin mutations suggests that thismolecular event may be critical for the development of hepato-blastomas within existing benign and malignant hepatocellularneoplasms. Previously, we showed that hepatocellular neoplasmsin untreated mice have only a low frequency ofb-catenin muta-tions (13). Thus, the accumulation ofb-catenin protein at the cellmembrane in the chemically induced hepatocellular neoplasmswith subsequent translocation to the nucleus, and leading ulti-mately to transactivation of growth promoting genes, may providethe oncogenic potential to develop into hepatoblastomas.

We first hypothesized that theb-cateninmutations in the malignanthepatoblastomas would be different from and/or more severe than thosein the hepatocellular neoplasms, thus leading to the different phenotype.However, whereas the hepatoblastomas sometimes had multipleb-cate-nin mutations and many of these were deletions, most of the specificmutations were found in each of the tumor types. Moreover, it could beargued that because of the high incidence of spontaneous hepatocellularneoplasms that lackb-catenin mutations, some of which are foundamong the tumors in the treated mice, the frequency ofb-cateninmuta-tions in the chemically induced hepatocellular neoplasms is actuallyhigher than that observed. These findings suggest that another molecularevent is needed for the development of the hepatoblastoma histotype orthat multiple mutations lead to a different or more severe phenotype.E-cadherin at the cell membrane and the APC protein in the cytoplasmare known to compete forb-catenin binding (22). The accumulation ofb-catenin along the cell membranes in the hepatocellular neoplasmssuggests that at least someb-cateninmutations lead to binding of mutantb-catenin to E-cadherin. It is possible that most of theb-cateninpointmutations and some of the deletions in these hepatocellular tumors leadto decreased binding efficiency of APC but not E-cadherin. In contrast,the hepatoblastomas had prominent cytoplasmic and nuclear localizationof the b-catenin protein, suggesting that E-cadherin expression may bedecreased in these tumors. E-cadherin is a putative tumor suppressorprotein for some human cancers (23) and is lost during progression inseveral human tumor types (24, 25). However, another possibility is thatsomeb-cateninmutations (especially larger deletions) alter or removeE-cadherin binding sites from the protein. This could permit unboundtruncated, but potentially still activeb-catenin protein to move from thecell membrane into the cytoplasm and nucleus. Ultimately, following thebinding of cotranscription factors such as Tcf-4 to the mutantb-cateninprotein and transactivation of genes such asc-Myc (11), this cascade ofevents may result in enhanced cell proliferation. Most studies on humantumors that containb-cateninmutations have demonstrated cytoplasmicand nuclear staining ofb-catenin protein (12, 26, 27), and the mousehepatoblastomas seem to follow this pattern.

The dominant nature and high frequency ofb-cateninmutationsidentified in the mouse hepatoblastomas in this study suggest thatalteration in the stability and regulation ofb-catenin expression is animportant event in the formation of these tumors in the B6C3F1mouse. Moreover, theb-catenin mutations are the same as thosefound in human hepatoblastomas, suggesting that similar carcinogenicpathways exist in the two species. Studies in our laboratory are nowinvestigating the consequences ofb-catenin protein accumulation andinteraction with other proteins in the development of hepatocellularneoplasms and hepatoblastomas to further understand the mechanismsof chemical induction of liver carcinogenesis in this model.

ACKNOWLEDGMENTS

We thank Drs. David Malarkey and James R. Hailey for critical reading ofthe manuscript and helpful comments.

REFERENCES

1. NTP Toxicology and carcinogenesis studies of oxazepam in Swiss-Webster andB6C3F1 mice. NTP TR #443 U.S. Dept. HHS, PHS, NIH, RTP, NC, 1993.

2. Maronpot, R. R., Haseman, J. K., Boorman, G. A., Eustis, S. E., Rao, G. N., and Huff,J. E. Liver lesions in B6C3F1 mice: The National Toxicology Program, experienceand position. Arch. Toxicol.,10 (Suppl.):10–26, 1987.

3. Diwan, B. A., Ward, J. M., and Rice, J. M. Promotion of malignant “embryonal” livertumors by phenobarbital: increased incidence and shortened latency of hepatoblasto-mas in (DBA/2 x C57Bl/6)F1 mice initiated withN-nitrosodiethylamine. Carcino-genesis (Lond.),10: 1345–1348, 1989.

4. Devereux, T. R., White, C. M., Sills, R. C., Bucher, J. R., Maronpot, R. R., andAnderson, M. W. Low frequency of H-ras mutations in hepatocellular adenomas andcarcinomas and in hepatoblastomas from B6C3F1 mice exposed to oxazepam in thediet. Carcinogenesis (Lond.),15: 1083–1087, 1994.

5. Calvert, R. J., Tashiro, Y., Buzard, G. S., Diwan, B. A., and Weghorst, C. M. Lackof p53 point mutations in chemically induced mouse hepatoblastomas: an end-stage,highly malignant hepatocellular tumor. Cancer Lett.,95: 175–180, 1995.

6. Weinberg, A. G., and Finegold, M. J. Primary hepatic tumors of childhood. Pathol-ogy, 14: 512–537, 1983.

7. Koch, A., Denkhaus, D., Albrecht, S., Leuschner, I., von Schweinitz, D., and Pietsch,T. Childhood hepatoblastomas frequently carry a mutated degradation targeting boxof the b-cateningene. Cancer Res.,59: 269–273, 1999.

8. Munemitsu, S., Albert, I., Souza, B., Rubenfeld, B., and Polakis, P. Regulation ofintracellularb-catenin levels by the adenomatous polyposis coli (APC) tumor sup-pressor protein. Proc. Natl. Acad. Sci. USA,92: 3046–3050, 1995.

9. Rubinfeld, B., Albert, I., Porfiri, E. F. C., Munemitsu, S., and Polakis, P. Binding ofGSK-3b to the APC-b-catenin complex and regulation of complex assembly. Science(Washington DC),272: 1023–1026, 1996.

10. Morin, P. J., Sparks, A. B., Korinek, V., Barker, N., Clevers, H., Vogelstein, B.,and Kinzler, K. W. Activation ofb-catenin-Tcf signaling in colon cancer bymutations inb-catenin or APC (see comments). Science (Washington DC),275:1787–1790, 1997.

11. He, T-C., Sparks, A. B., Rago, C., Hermeking, H., Zawel, L., da Costa, L. T., Morin,P. J., Vogelstein, B., and Kinzler, K. W. Identification of c-MYC as a target of theAPC pathway. Science (Washington DC),281: 1509–1512, 1998.

12. de La Coste, A., Romagnolo, B., Billuart, P., Renard, C. A., Buendia, M. A.,Soubrane, O., Fabre, M., Chelly, J., Beldjord, C., Kahn, A., and Perret, C. Somaticmutations of theb-catenin gene are frequent in mouse and human hepatocellularcarcinomas. Proc. Natl. Acad. Sci. USA,95: 8847–8851, 1998.

13. Devereux, T. R., Anna, C. H., Foley, J. F., White, C. M., Sills, R. C., and Barrett, J. C.Mutation of b-catenin is an early event in chemically induced mouse hepatocellularcarcinogenesis. Oncogene,18: 4726–4733, 1999.

14. Ogawa, K., Yamada, Y., Kishibe, K., Ishizaki, K., and Tokusashi, Y.b-Cateninmutations are frequent in hepatocellular carcinomas but absent in adenomas inducedby diethylnitrosamine in B6C3F1 mice. Cancer Res.,59: 1830–1833, 1999.

15. Bonner, R. F., Emmert-Buck, M., Cole, K., Pohida, T., Chuaqui, R., Goldstein, S.,and Liotta, L. A. Laser capture microdissection: molecular analysis of tissue. Science(Washington DC),278: 1481–1483, 1997.

16. Tam, A. S., Foley, J. F., Devereux, T. R., Maronpot, R. R., and Massey, T. E. Highfrequency and heterogeneous distribution of p53 mutations in aflatoxin B1-inducedmouse lung tumors. Cancer Res.,59: 3634–3640, 1999.

17. Gumbiner, B. M. Carcinogenesis: a balance betweenb-catenin and APC. Curr. Biol.7: R443–R446, 1997.

18. Tsujiuchi, T., Tsutsumi, M., Sasaki, Y., Takahama, M., and Konishi, Y. Differentfrequencies and patterns ofb-catenin mutations in hepatocellular carcinomas inducedby N-nitrosodiethylamine and a choline-deficientL-amino acid-defined diet in rats.Cancer Res.,59: 3904–3907, 1999.

19. Diwan, B. A., Henneman, J. R., and Rice, J. M. Further evidence for promoter-dependentdevelopment of hepatoblastoma in the mouse. Cancer Lett.,89: 29–35, 1995.

20. Ward, J. M., Rice, J. M., Creasia, D., Lynch, P., and Riggs, C. Dissimilar patterns ofpromotion by di(2-ethylhexyl)phthalate and phenobarbital of hepatocellular neoplasiainitiated by diethylnitrosamine in B6C3F1 mice. Carcinogenesis (Lond.),4: 1021–1029, 1983.

21. Diwan, B. A., Ward, J. M., and Rice, J. M. Origin and pathology of hepatoblastomasin mice. In: A. E. Sirica (ed.), The Role of Cell Types in Hepatocarcinogenesis, pp.71–87. Boca Raton: CRC Press, 1992.

22. Hulsken, J., Birchmeier, W., and Behrens, J. E-cadherin and APC compete for theinteraction withb-catenin and the cytoskeleton. J. Cell Biol.,127: 2061–2069, 1994.

23. Christofori, G., and Semb, H. The role of the cell-adhesion molecule E-cadherin as atumor-suppressor gene. Trends Biochem. Sci.,24: 73–76, 1999.

24. Risinger, J. I., Berchuk, A., Kohler, M. F., and Boyd, J. Mutations of the E-cadheringene in human gynecological cancers. Nat. Genet.,7: 98–102, 1994.

25. Birchmeier, W., and Behrens, J. Cadherin expression in carcinomas: role in theformation of cell junctions and prevention of invasiveness. Biochim. Biophys. Acta,1198: 11–26, 1994.

26. Fukuchi, T., Sakamoto, M., Tsuda, H., Maruyama, K., Nozawa, S., and Hirohashi, S.b-Catenin mutation in carcinoma of the uterine endometrium. Cancer Res.,58:3526–3528, 1998.

27. Blaker, H., Hofmann, W. J., Rieker, R. J., Penzel, R., Graf, M., and Otto, H. F.b-Catenin accumulation and mutation of the CTNNB1 gene in hepatoblastoma.Genes Chromosomes Cancer,25: 399–402, 1999.

2868

b-CATENIN MUTATIONS IN CHEMICALLY INDUCED HEPATOBLASTOMAS

Research. on June 11, 2020. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from

2000;60:2864-2868. Cancer Res   Colleen H. Anna, Robert C. Sills, Julie F. Foley, et al.   Anthraquinone or OxazepamHepatoblastomas Examined from B6C3F1 Mice Treated with

Mutations and Protein Accumulation in AllCatenin-β

  Updated version

  http://cancerres.aacrjournals.org/content/60/11/2864

Access the most recent version of this article at:

   

   

  Cited articles

  http://cancerres.aacrjournals.org/content/60/11/2864.full#ref-list-1

This article cites 23 articles, 12 of which you can access for free at:

  Citing articles

  http://cancerres.aacrjournals.org/content/60/11/2864.full#related-urls

This article has been cited by 5 HighWire-hosted articles. Access the articles at:

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/60/11/2864To request permission to re-use all or part of this article, use this link

Research. on June 11, 2020. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from