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[Cancer Research 60, 3323-3327, July 1, 2000]
© 2000 American Association for Cancer Research

Advances in Brief

Frequent ß-Catenin Gene Mutations and Accumulations of the Protein in the Putative Preneoplastic Lesions Lacking Macroscopic Aberrant Crypt Foci Appearance, in Rat Colon Carcinogenesis1

Yasuhiro Yamada2, Naoki Yoshimi, Yoshinobu Hirose, Kunihiro Kawabata, Kengo Matsunaga, Masahito Shimizu, Akira Hara and Hideki Mori

Department of Pathology, Gifu University School of Medicine, Gifu 500-8705, Japan


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Activating mutations in the ß-catenin gene is thought to be responsible for the excessive ß-catenin signaling involved in the majority of carcinogen-induced colonic carcinomas. To determine whether ß-catenin signaling is involved in the initial stage of colon carcinogenesis, mutational analysis of the gene and immunohistochemistry for ß-catenin protein were performed in the early appearing lesions, including aberrant crypt foci (ACF), of colonic mucosa in rats given azoxymethane. Male F344 rats received s.c. injections of azoxymethane at a dose of 15 mg/kg body weight, once weekly for 3 weeks, and they were sacrificed 10 weeks after the carcinogen treatment. The colonic mucosa was examined in en face preparations and in serial sections after the observation in whole mount preparations. Microscopical observations in the cross sections have shown two populations of histologically altered crypts. The first type had a macroscopic feature resembling ACF [histologically altered crypts with ACF appearance (HACAs)]. The second type of altered crypts did not have the ACF-like appearance and could not be clearly distinguished from adjacent normal crypts in whole mount preparations [histologically altered crypts with macroscopically normal-like appearance (HACNs)]. The ß-catenin gene mutations were recognized in 10 of 15 HACNs (67%) and 3 of 15 HACAs (20%). Frequent immunoreactivity of ß-catenin protein was seen in the cytoplasm of HACNs (13 of 15 cases), whereas apparent accumulation was not confirmed in any HACAs analyzed. These results suggest that (a) there are two types of putative preneoplastic lesions in cancer-predisposed colonic mucosa, and ß-catenin signaling may contribute to the initial stage of colon carcinogenesis; and (b) HACNs are more likely to be direct precursors of colon tumors than HACAs in the rat colon carcinogenesis.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Colon carcinogenesis is a multistep process with discrete pathological changes from normal mucosa to abnormal hyperproliferative epithelium, finally leading to the neoplastic transformation (1) . ACF3 were first described by Bird (2) in methylene blue-stained preparations of colon in animals treated with colon-specific carcinogens. Several studies in rodent models have supported the hypothesis that ACF are putative premalignant lesions (3) . ACF have been used to evaluate potential chemopreventive agents against colon carcinogenesis in laboratory animal models (4) . Although the molecular analysis of these lesions from experimental animals has characterized the early events in colon carcinogenesis (3 , 5) , the preneoplastic nature of colon carcinogenesis has not been fully elucidated.

ß-Catenin, which was originally discovered as a cadherin-binding protein, has recently been proved to function as a transcriptional activator when complexed with members of the Tcf family of DNA binding proteins (6 , 7) . It is known that hTCF is expressed in normal and neoplastic colorectal epithelium, and the ß-Catenin-Tcf complex effects gene expressions that may play important roles in cell proliferation and/or apoptosis (8, 9, 10) . Activation of the ß-Catenin-Tcf pathway results in the accumulation of ß-catenin in the cytosol and nucleus (7 , 11) . It is also known that ß-catenin levels are regulated by degradation of this protein through the ubiquitin-proteasome pathway (12) , and intact APC cooperates with a serine/threonine kinase glycogen synthase kinase-3ß (GSK-3ß) to regulate this degradation via multiple phosphorylation sites of ß-catenin protein encoded by exon 3 (8) . Mutations in the APC gene are known to repress the degradation and result in accumulations of ß-catenin (13) . It has been estimated that at least 80% of human colorectal tumors have a somatic mutation of the APC gene (14 , 15) , which is predicted to activate the ß-Catenin-Tcf pathway. The frequency of APC mutations is just as high in small benign tumors as in cancers (15) . In addition, mutations of APC have been also found in human ACF, including those as small as a few crypts (16) . These observations suggest that mutations of APC is an early and perhaps initiating event in sporadic colorectal tumorigenesis. In contrast, mutations in the ß-catenin gene that alter functionally significant phosphorylation sites on exon 3 also prevent ß-catenin from the degradation and result in activation of the ß-Catenin-Tcf pathway, as is the case with APC mutations (13) . It has been reported that such oncogenic mutations are concerned with various tumors, including colon tumors without APC mutations (17, 18, 19) . Recently, frequent mutations in the ß-catenin gene and accumulations of ß-catenin were recognized in rat colon tumors induced by the colon-specific carcinogen, AOM (20) . Together with the fact that APC mutations are not common events in such tumors (21) , ß-catenin mutations may mainly activate the pathway and contribute to the development of most colon tumors in this model.

In the present study, to determine if ß-catenin signaling is involved in the initial stage of colon carcinogenesis, mutational analysis of the ß-catenin gene and immunohistochemistry for ß-catenin protein were performed in the earliest lesions analyzed on the cross sections.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Animal Treatment.
Male F344 rats, 6 weeks old, were obtained from Shizuoka SLC, Co. (Shizuoka, Japan), received s.c. injections of AOM (15 mg/kg body weight; Sigma Chemical Co.) once a week for 3 weeks, and were killed 10 weeks after the first injection. Immediately after the sacrifice, colons were removed, cut open along its longitudinal axis, and fixed flat in 10% buffered formalin for 24 h at room temperature. We photographed and/or checked the aberration of the surface of colon mucosa, and we marked the position of mucosal aberrations, including ACF. Colonic mucosa was embedded in paraffin for histological analysis. Colonic mucosal sections were examined by using an en face preparation and 3- to 5-µm-thick serial sections. For each case, 20–40 serial sections of whole crypts were used to investigate whole crypts from the mucosal surface to the crypt bottom. For DNA sampling, sections were microdissected using the laser capture microdissection system (LM100, Arcturus Engineering, Inc., Mountain View, CA).

PCR-SSCP Analysis.
Extracted DNAs were amplified with primers designed to produce a 211-bp product of rat ß-catenin gene (Ctnnb1) corresponding to functionally important phosphorylation sites in CTNNB1. The primers used here were the same as those in previous studies (22, 23, 24) and designed to PCR-amplify the regions corresponding to exon 2 and 3 (codons 1–57) of Ctnnb1, including intron 2. IF4 (forward; 5'-GCTGACGTCGTACTCAGGCA-3') and R3 (reverse; 5'-TCCACATCCTCTTCCTCAGG-3') were included in the following PCR reaction mixture containing in a total volume of 50 µl:20 µM of each primer, 200 µM of each deoxynucleotide triphosphate, 1 unit of Taq polymerase in 1x PCR buffer [10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2; Pharmacia Biotech, Tokyo, Japan], and 50 ng of template DNA. The mixture was heated at 94°C for 5 min and subjected to 30 cycles of denaturation (94°C, 45 s), annealing (57°C, 45 s), and extension (72°C, 2 min) using a thermal cycler (Perkin-Elmer Cetus). Five µl of the PCR products were mixed with the same volume of SSCP buffer (0.1% SDS and 10 mM EDTA), and they were mixed with 2.5 µl of formamide dye (10 ml formamide, 10 mg xylene cyanol, 10 mg bromphenol blue, 10 mM EDTA). After denaturation at 90°C for 3 min, samples were applied to a 10% polyacrylamide gel with 1% or 10% glycerol. DNAs extracted from adjacent crypts were used as negative controls. For positive controls, DNAs that contain the G->A transition at the first position of codon 32 were used (24) .

Sequencing Analysis.
When the pattern of migration was abnormal, the corresponding PCR products were purified and amplified again by PCR using IF4 and R3 primers before sequencing. Sequencing was performed using an ALF Express DNA sequencer (Pharmacia Biotech), and the sequencing was repeated more than twice, including the use of forward and reverse primers. When the mutated products were underrepresented, bands were purified and cloned into the pCR2.1 TA vector (Invitrogen, San Diego, CA) before sequencing.

Immunohistochemistry.
For immunohistochemical analysis, the labeled streptavidin biotin method was performed using a LSAB KIT (DAKO, Glostrup, Denmark) with microwave accentuation. The paraffin-embedded sections were heated for 30 min at 65°C, deparaffinized in xylene, and rehydrated through graded alcohols at room temperature. A 0.05 M Tris-HCl buffer (pH 7.6) was used to prepare solutions and for washes between various steps. Incubations were performed in a humidified chamber. Four-µm-thick sections were treated for 40 min at room temperature with 2% BSA and incubated overnight at 4°C with primary antibodies against ß-catenin (diluted 1:1000; Transduction Laboratories, Lexington, KY). For each case, negative controls were performed on serial sections. On the control section, incubation with the primary antibody was omitted. Horseradish peroxidase activity was visualized by treatment with H2O2 and diaminobenzidine for 5 min. At the last step, the sections were weakly counterstained with hematoxylin. These immunoreactivities were evaluated by two pathologists independently. In cases of disagreement, a third examination under a double-headed microscope was carried out.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Two Types of Altered Crypts.
At autopsy, there were no detectable AOM-induced tumors in all colons examined macroscopically. In the colonic mucosa of AOM-treated rats, two populations of altered crypts were histologically detected at the cross section, whereas these lesions were absent in the colonic mucosa of non-carcinogen-treated rats. The first type had a macroscopic feature resembling ACF (HACAs), which could be easily distinguished by their increased size, prominent epithelial cells, and increase of pericryptal space from surrounding normal crypts in colonic mucosa stained with methylene blue. Histological features of HACAs at cross sections were those where crypt size was enlarged. Hypertrophic cytoplasms with basophilic cytoplasms and hyperchromatic nuclei were present. Secretory activity of mucins was decreased, as shown in previous studies (25) . The second type of altered crypts did not have the ACF-like appearance in whole mount preparations. The crypts of this type never had prominent epithelial cells. Such altered crypts were not clearly distinguished from adjacent normal crypts in whole mount preparations (HACNs). Occasionally, HACNs could be detected as a depressed mucosal lesion. Even in such cases, they lacked the ACF-like appearance (Fig. 1)Citation . Aberrant crypts of HACNs resembled the crypts of HACAs in their basophilic cytoplasms and hyperchromatic nuclei. The secretory activity of mucins (in HACNs) was decreased more prominently than in HACAs. Epithelium of HACNs tended to show disruption of the parallel cell arrangement and loss of nuclear polarity as seen in epithelium of HACAs (Fig. 1)Citation . The number of HACAs and HACNs per area was 4.79 ± 2.15/cm2 and 1.87 ± 0.71/cm2, respectively.



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  		Fig. 1. Topographic view of methylene blue-stained mucosa of whole mount colons (A) and histological view of the corresponding area (B). A, ACF could be easily distinguished by their increased size, prominent epithelial cells, and increase of pericryptal space from surrounding normal crypts in colonic mucosa stained with methylene blue (arrows). B, two populations of altered crypts are histologically detected at the cross section. The first type has a macroscopic appearance resembling ACF (HACAs, arrows). The second type of altered crypts does not have the ACF-like appearance (HACNs). HACNs cannot be clearly distinguished from adjacent normal crypts in whole mount preparations. Occasionally, HACNs can be detected as a depressed mucosal lesion. Even in such cases, they lack the ACF-like appearance (open arrow). Bars, 250 µm.

 
ß-Catenin Mutation.
Genomic DNA was isolated from tissue that was microdissected by use of laser capture microdissection system and analyzed for exon 3 of the ß-catenin gene. In SSCP analysis, frequent band shifts were recognized in HACNs, whereas they were rare in HACAs (Fig. 2A)Citation . When the mutated products were underrepresented or the results of direct sequencing were indistinct, subcloning before sequencing was performed. The sequencing analysis lead to the identification of a genetic alteration in 10 of 15 HACNs (67%) and 3 of 15 HACAs (20%). All alterations identified were caused by single-nucleotide substitution and located within functionally significant phosphorylation sites on exon 3. G:C->A:T transitions were the most common mutation (57% of all mutations). In case N15, double mutations in the same allele were observed. Recognized mutations are summarized in Table 1Citation .



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  		Fig. 2. Detection of ß-Catenin mutations in HACAs and HACNs. A, SSCP analysis: Representative results of PCR-SSCP analysis of the rat ß-catenin gene are shown. PCR products were electrophoresed on a 10% polyacrylamide gel containing 1% glycerol at 15°C. Lanes N2–9, samples of HACNs; Lanes A6–A13, samples of HACAs. Note that band shifts (closed arrows) are frequently recognized in HACNs, whereas they are rare in HACAs. B, DNA sequencing: The types of mutations are shown (a–c). All alterations identified are caused by single-nucleotide substitution and located within functionally significant phosphorylation sites on exon 3. Arrowheads, the nucleotide substitutions in histologically altered crypts. In case N15, double mutations in the same allele are observed (c). We detected one mutation (case N4) without the alteration in SSCP analyses (A, b, open arrow).

 

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  		Table 1 ß-Catenin status and ß-catenin accumulation

 
Immunohistochemistry.
In this study, samples that have been determined for the status of the ß-catenin gene were used for immunohistochemistry. In adjacent normal crypts, ß-catenin expression was immunohistochemically detected restrictedly to the membrane of the cell-to-cell borders. In contrast, cytoplasmic overexpression of ß-catenin was detected in 13 of 15 HACNs (Fig. 3, B and D)Citation . Furthermore, almost all HACNs revealed an altered localization of ß-catenin, and the expression was also confirmed in the nuclei. Conversely, all HACAs, regardless of the status of the ß-catenin gene, had no cytoplasmic accumulation or translocation of ß-catenin into nucleus (Fig. 3C)Citation . The immunohistochemical results accompanied by the status of the ß-catenin gene are summarized in Table 1Citation . No positive reaction was recognized in the slides of negative control.



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  		Fig. 3. Immunohistochemical analysis of ß-catenin expression in histologically altered crypts. A, H&E (H.E.) staining. Two populations of histologically altered crypts (HACAs and HACNs) are present in cross sections. B, ß-Catenin staining is immunohistochemically detected restrictedly to the membrane of the cell-to-cell borders in normal crypts. The overexpression of ß-catenin is recognized in epithelium of HACNs, whereas it is hardly detected in HACAs. C, at higher magnifications, the stain in HACAs is selectively localized to the membrane of the cell-to-cell borders as seen in normal crypts. Note that HACAs had no cytoplasmic or nuclear immunostaining. D, ß-Catenin overexpression and altered localization are present in HACNs. Strong immunopositivity is recognized in both cytoplasm and nuclei. In this case, the staining intensity was regarded as ++ in cytoplasms and + in the nucleus. Bars, 100 µm.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
In this study, we used en face preparations that demonstrate sheets of mucosa with the crypts in cross sections to indicate the early morphological changes of cancer-predisposed colonic mucosa. Because the orientation to mucosal topography is analogous to macroscopical examination, an en face preparation technique is considered to provide a useful tool for the mapping and quantitation of mucosal aberration, including ACF (26) . We were able to demonstrate two populations of altered crypts (HACAs and HACNs) by microscopical observations in the cross sections of the colonic mucosa. ß-Catenin gene mutations were present in 10 of 15 HACNs (67%) and in 3 of 15 HACAs (20%). Although the functional significance of the mutations in rat tumorigenesis is not fully elucidated, most mutants were located within the functionally significant phosphorylation sites on exon 3 and presumably interfere with the ubiquitination and degradation of ß-catenin (12) , with the exception of case N7. Because case N7 creates a stop codon that would truncate the protein at codon 34, the mutations may not relate to tumorigenesis. However, most of mutations detected in this study were in the same codons as observed in rat colon tumors (20 , 22 , 23) . Additionally, the most common mutation (G:C->A:T transitions), which was found in the early lesions, is also recognized as the representative mutation in rat colon tumors (20) . Such genetic evidences seem to support that both HACAs and HACNs are the preneoplastic lesions. Mutation of ß-catenin, which had been recognized in rat colon tumors, was also apparent in the early occurring lesions, suggesting that ß-catenin gene mutations are importantly associated with the initial stage of colon carcinogenesis in rats. Thus, these mutations seem to play a gate keeper role in the development of colon tumors. The results showing that HACNs had ß-catenin gene mutations more frequently than HACAs, thus, may indicate a potential of HACNs to progress into neoplastic lesions.

Frequent accumulation of cytoplasmic ß-catenin protein was only seen in HACNs (13 of 15 cases), whereas they were not recognized in any HACAs. Excessive ß-catenin protein attributable to gene mutations is expected to activate cyclin D, c-myc, and matrilysin gene transcription, which have been determined as target genes of the ß-Catenin-Tcf pathway (27, 28, 29) . Such genes are reported to be overexpressed in colon tumors and are modifying factors in colon carcinogenesis, implying that the ß-Catenin-Tcf pathway is an oncogenic pathway. Because ß-catenin protein seems to play an essential role in the pathway, the amount of the protein may be interrelated with activation of that pathway. Additionally, cytoplasmic accumulation and translocation of the protein into the nucleus has been shown in the majority of AOM-induced rat colon tumors (20) . Thus, it is suggested that the ß-Catenin-Tcf pathway is also activated in these large bowel tumors. Presently, our data indicate that activation of the pathway may be associated with the initial stage of colon carcinogenesis. It is also noteworthy that no HACAs revealed apparent cytoplasmic accumulation and translocation of the protein, although they were frequently recognized in HACNs. These findings strongly suggest that molecular characteristics, as well as morphological features of HACAs and HACNs, are quite different. We would like to emphasize that HACNs are more likely to be the precursor of colon tumors. Interestingly, in this study, no cytoplasmic immunoreactivity was recognized in HACAs with ß-catenin mutations, suggesting that ß-catenin mutations are not sufficient for the accumulation of the protein. In contrast, HACNs without ß-catenin mutations revealed cytoplasmic accumulation of ß-catenin. Because about 18% of AOM-induced rat colon tumors have been reported to harbor Apc mutations (21) , it may be true that early appearing lesions like HACNs have mutations of the Apc gene.

In summary, we have carried out a genetic analysis of the functionally critical exon 3 of the ß-catenin gene and an immunohistochemical analysis for the ß-catenin on the two types of early lesions in cancer-predisposed colonic mucosa, HACAs and HACNs. Frequent mutations and cytoplasmic accumulation as well as translocation of ß-catenin was detected in HACNs. Our results suggest that preneoplastic lesions lacking macroscopic ACF appearance are likely to be direct precursors of colon cancers in rats.


   ACKNOWLEDGMENTS
 
We thank Kyoko Takahashi, Chikako Usui, Tomoko Kajita, Kimiko Yamada, and Ayumu Nagata for their excellent technical assistance, and Kazumasa Sato for animal care.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by Grants-in-Aid from the Ministry of Health and Walfare, a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan, and the Program for Promotion of Fundamental Studies in Health Science from the Organization for Pharmaceutical Safety and Research, Japan. Back

2 To whom requests for reprints should be addressed, at the Department of Pathology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan. Phone: 81-58-267-2235; Fax: 81-58-265-9005; E-mail: y-yamada{at}cc.gifu-u.ac.jp Back

3 The abbreviations used are: ACF, aberrant crypt foci; AOM, azoxymethane; Tcf, T-cell factor; APC, adenomatous polyposis coli; HACA, histologically altered crypt with ACF appearance; HACN, histologically altered crypt with macroscopically normal-like appearance; SSCP, single-strand conformational polymorphism. Back

Received 1/11/00. Accepted 5/10/00.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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Correspondence re: Y. Yamada et al., Frequent {beta}-Catenin Gene Mutations and Accumulations of the Protein in the Putative Preneoplastic Lesions Lacking Macroscopic Aberrant Crypt Foci Appearance, In Rat Colon Carcinogenesis. Cancer Res., 60: 3323-3327, 2000; and Sequential Analysis of Morphological and Biological Properties of {beta}-Catenin-accumulated Crypts, Provable Premalignant Lesions Independent of Aberrant Crypt Foci in Rat Colon Carcinogenesis. Cancer Res., 61: 1874-1878, 2001.
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