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

Colon carcinogenesis is a multistep process with discrete pathological changes from normal mucosa to abnormal hyperproliferative epithelium, finally leading to the neoplastic transformation(1). ACF³were 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.

β-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 theβ-Catenin-Tcf complex effects gene expressions that may play important roles in cell proliferation and/or apoptosis(8, 9, 10). Activation of the β-Catenin-Tcf pathway results in the accumulation of β-catenin in the cytosol and nucleus (7, 11). It is also known that β-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-3β (GSK-3β) to regulate this degradation via multiple phosphorylation sites of β-catenin protein encoded by exon 3 (8). Mutations in the APCgene are known to repress the degradation and result in accumulations of β-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 β-Catenin-Tcf pathway. The frequency of APCmutations 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 β-catenin gene that alter functionally significant phosphorylation sites on exon 3 also prevent β-catenin from the degradation and result in activation of the β-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 β-catenin gene and accumulations of β-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), β-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 β-catenin signaling is involved in the initial stage of colon carcinogenesis, mutational analysis of the β-catenin gene and immunohistochemistry forβ-catenin protein were performed in the earliest lesions analyzed on the cross sections.

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 facepreparation 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).

Extracted DNAs were amplified with primers designed to produce a 211-bp product of rat β-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:20μ m of each primer, 200μ m of each deoxynucleotide triphosphate, 1 unit of Taq polymerase in 1× PCR buffer [10 mm Tris-HCl (pH 9.0), 50 mmKCl, 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).

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.

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 β-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.

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). 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). The number of HACAs and HACNs per area was 4.79 ± 2.15/cm² and 1.87 ± 0.71/cm², respectively.

Genomic DNA was isolated from tissue that was microdissected by use of laser capture microdissection system and analyzed for exon 3 of the β-catenin gene. In SSCP analysis, frequent band shifts were recognized in HACNs, whereas they were rare in HACAs (Fig. 2,A). 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 1.

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

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. β-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 β-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 β-catenin, which had been recognized in rat colon tumors, was also apparent in the early occurring lesions,suggesting that β-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 β-catenin gene mutations more frequently than HACAs, thus, may indicate a potential of HACNs to progress into neoplastic lesions.

Frequent accumulation of cytoplasmic β-catenin protein was only seen in HACNs (13 of 15 cases), whereas they were not recognized in any HACAs. Excessive β-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 β-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 theβ-Catenin-Tcf pathway is an oncogenic pathway. Because β-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 theβ-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 β-catenin mutations, suggesting that β-catenin mutations are not sufficient for the accumulation of the protein. In contrast, HACNs without β-catenin mutations revealed cytoplasmic accumulation of β-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 β-catenin gene and an immunohistochemical analysis for the β-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 β-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.

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