Dose response and proliferative characteristics of aberrant crypt foci: putative preneoplastic lesions in rat colon

Foci of aberrant crypts (ACF) have been identified in the unsectioned methylene blue stained rodent colons and hypothesized to represent precursor lesions of colon cancer. In the present study, induction and growth characteristics of ACF were investigated in response to a single injection of varying dosages of 1,2-dimethylhydrazine-2HCl (DMH), a colon carcinogen. Female Sprague-Dawley rats were given a single injection of DMH (5-150 mg/kg). Two and 19 weeks after the injection, animals were killed and their distal 10 cm of colons were enumerated for the number and crypt multiplicity of ACF. Number of ACF increased with increasing dosages of DMH plateauing at 100 mg/kg. However, percentage of ACF exhibiting different crypt multiplicity (1 to greater than 4) were similar among different dose groups. Aberrant crypts and normal crypts were enumerated for total number of cells and number and distribution of S-phase cells along the crypt height 19 weeks after DMH injection after autoradiography. The labeling index (LI) (percentage of S-phase cells) and LI along the crypt height were determined. Compared to the surrounding normal crypts, aberrant crypts exhibited significantly higher (P less than 0.05) number of cells (1122 +/- 81 versus 411 +/- 28) and higher (P less than 0.05) LI (21 +/- 1 versus 12 +/- 1). For the eight ACF analysed in the present study, the distribution of S-phase cells in the aberrant crypts were similar to that of normal crypts in that S-phase cells were restricted to the lower two-thirds of the crypts rather than distributed throughout the height of the crypts as reported for adenomatous epithelium.     

Frequent mutation of Apc gene in rat colon tumors and mucin-depleted foci, preneoplastic lesions in experimental colon carcinogenesis

Mucin-depleted foci (MDF) are microscopic dysplastic lesions induced in the colon of rodents by specific colon carcinogens. Most MDF show Wnt pathway activation, whereas only a subset shows mutations in the Ctnnb1 gene, coding for beta-catenin. Because Apc is a member of the Wnt pathway and the most frequent mutated gene in human colon cancer, we tested whether MDF harbor Apc mutations. F344 rats were treated twice with 150 mg/kg of 1,2-dimethylhydrazine. After 15 or 28 weeks, MDF, aberrant crypt foci (ACF), and tumors were collected. We screened a segment of the Apc gene comprising the region homologous to the mutation cluster region (MCR) of human APC, which frequently shows mutations in experimental colon tumors. Mutations were identified by PCR amplification and sequencing in 6:24 MDF (25%), 7:23 tumors (30%), 0:24 ACF (0%). Most of the mutations (92%) in MDF and tumors were localized in a region upstream from the MCR. All mutations were single-base substitutions and mainly formed by G:C-->A:T and C:G-->T:A transitions. The pattern of nucleotide changes was similar in MDF and tumors, and, interestingly, the same mutation in codon 1047 was found in two MDF and in three tumors. Four out of the six mutations found in MDF were nonsense mutations, and two were missense. All mutations in tumors determined a protein truncation. These results show that Apc mutations are present in MDF with a frequency similar to that of tumors, strengthening the evidence that they are precancerous lesions in colon carcinogenesis.     

K-ras mutations in aberrant crypt foci, adenomas and adenocarcinomas during azoxymethane-induced colon carcinogenesis

Ras mutations are an important early event in a number of carcinogen-inducedrodent tumors. Colon carcinogenesis induced in rats by azoxymethaneis a useful model as it mimics the adenoma-carcinoma sequenceobserved in humans. In addition, aberrant crypt foci developin the rat and these lesions appear to be potentially importantprecursors to adenomas in colorectal cancer. Recent studieshave shown that specific K-ras codon 12 and 13 mutations arepresent in up to 66% of carcinogen-induced rat colon adenocarcinomas.We studied the frequency of these mutations during the aberrantcrypt focus-adenoma-carcinoma sequence in azoxymethane-inducedFisher F344 rats. K-ras codon 12 GAT and codon 13 GAC mutationswere detected with a sensitive assay based on the amplificationof DNA using the polymerase chain reaction. No mutations werepresent in normal mucosa. Of 27 aberrant crypt foci, K-ras mutationswere identified in 2 lesions containing 5 and 10 aberrant crypts,respectively. Mutations were present in 1 of 23 and 10 of 27adenomas and adenocarcinomas, respectively. These data suggestthat K-ras mutations play a role during the stages of carcinogenesisin azoxymethane-induced rat colon cancer. The demonstrationof a genetic mutation in aberrant crypt foci provides furtherevidence for the significance of these lesions as precursormarkers of maligant potential during colorectal tumorigenesis.     

Sequential analyses of the growth and morphological characteristics of aberrant crypt foci: putative preneoplastic lesions

The main objective of the present study was to sequentially analyze growth and morphological characteristics of aberrant crypt foci (ACF) in the rat colon. Sprague-Dawley rats were given a single injection of a carcinogenic dose of 1,2-dimethylhydrazine-HCl and at varying time points ranging from 2 to 57 weeks, groups of 5 rats were terminated. The number and crypt multiplicity of ACF were determined in the distal 8 cm of the colon. In addition, ACF were processed for histology and then graded for the presence of nuclear atypia using a score of 0-4. The findings of the present study demonstrated that ACF exhibit the characteristics expected for precursor lesions. ACF were present at all time intervals in large numbers in the colons of rats treated with 1,2-dimethylhydrazine-HCl and were present when adenocarcinomas were observed. The number of ACF with 4 or more crypts and those exhibiting a higher grade (grade 4) of nuclear atypia increased significantly at or beyond 19 weeks. These features of ACF, particularly the presence of nuclear atypia indicative of dysplasia, provide strong support for the hypothesis that ACF are precursor lesions of chemically induced colon cancer.     

Tea can protect against aberrant crypt foci formation during azoxymethane induced rat colon carcinogenesis

Tea shows many health promoting activities including chemopreventive action during carcinogenesis due to the presence of antioxidative polyphenolic constituents. The present experiment evaluated the anticarcinogenic role of black tea infusion on azoxymethane induced colonic preneoplastic lesions, the aberrant crypt foci in Sprague-Dawley rats. Rats were injected with azoxymethane (15 mg/kg b.w.) and received oral administration of 1% and 2% (w/v) tea infusions from first day of carcinogen application. This treatment was continued for twelve weeks and assessed for aberrant crypt foci and compared with untreated carcinogen control group. Levels of lipid peroxidation were determined in liver as well as in colon tissue. During initiation phase of carcinogenesis, glutathione-S-transferase (GST) and glutathione peroxidase (GPx) activities were also evaluated. Significant reduction in the number of aberrant crypt foci and levels of lipid peroxidation among the tea-treated groups were observed. Induction of GST and GPx activities was noted during the initiation phase of carcinogenesis. Results of the present study indicate that the protective effect of black tea infusions may be due to an outcome of antioxidative influence of tea components on azoxymethane induced carcinogenesis.     

Whole almonds and almond fractions reduce aberrant crypt foci in a rat model of colon carcinogenesis

Almonds and other nuts appear to confer health benefits despite their high fat content. To assess the effect of almonds on colon cancer, whole almond-, almond meal- or almond oil-containing diet effects on aberrant crypt foci (ACF) in azoxymethane-treated F344 male rats were investigated. Six-week-old male F344 rats were fed the various almond and control diets and given subcutaneous injections of azoxymethane (15 mg/kg body weight) twice 1 week apart. After 26 weeks animals were injected with bromodeoxyuridine 1 h prior to sacrifice, after which colons were evaluated for ACF and cell turnover (labeling index, LI). Whole almond ACF and LI were both significantly lower than wheat bran and cellulose diet groups (-30 and -40%, respectively), while almond meal and almond oil ACF and almond meal LI declines were only significant vs. cellulose (P<0.05). These results suggest that almond consumption may reduce colon cancer risk and does so via at least one almond lipid-associated component.     

Abnormal expression of gastric mucin in human and rat aberrant crypt foci during colon carcinogenesis.

Colorectal cancer is a major cause of death in Europe and the USA, and much effort is therefore devoted to improve its early detection. In this article, we report the abnormal expression of gastric mucin in aberrant crypt foci (ACF) that appear in the colon mucosae removed from colorectal cancer patients and rats treated with methyl-N'-nitro-N-nitroso-guanidine (MNNG). We performed the immunoperoxidase test using monoclonal antibodies raised against gastric M1 mucin encoded by the MUC5AC gene and against rat gastric mucins (MAb 660), respectively. In both human and rat colon, these anti-gastric mucin MAbs stained specifically goblet cells within ACF. In humans, the M1/MUC5AC mucin was expressed in the upper part of the glands in hyperplastic ACF and in the typical ACF. In addition, the anti-gastric mucin MAbs stained some rare, scattered, histologically normal glands in the human and rat colon mucosae. These glands may be regarded as precursors of ACF. The abnormal expression of the MUC5AC gene constitutes a novel change in addition to genetic modifications already observed in ACF, and supports our previous findings demonstrating the potential of this gastric mucin as an early marker of human and rat colon carcinogenesis.     

Scanning electron microscopy of aberrant crypt foci in rat colon

The surface of the colon mucosa of 1,2-dimethylhydrazinetreatedF344 rats was examined with the scanning electron microscope.A detailed examination of the mucosal topography revealed fociwith one to several aberrant crypts. These were seen as structureselevated from the background mucosa. The shape of the luminalopenings of the aberrant crypts varied from elongated or tortuousto circular. However, we found no ultrastructural variationsbetween the different aberrant crypt foci (ACF) or between theACF and the background mucosa. There was no direct relationshipbetween the size of ACF and the number of aberrant crypts perfocus, which may be explained by the mechanism of crypt fission;in two aberrant crypts we discovered the formation of a transverseepithelial septum, dividing the large crypt into two smallercrypts. The gross morphology of the ACF observed by scanningelectron microscopy and light microscopy was in principle thesame.     

Promotion of aberrant crypt foci and cancer in rat colon by thermolyzed protein.

BACKGROUND: We have previously shown that thermolyzed protein (casein) cooked with fat in the diet of the rat promotes the growth of aberrant crypt foci (putative precursors of colon cancer) assessed at 100 days. PURPOSE: To determine how thermolysis affects this promotion, we examined thermolysis conditions, quantity of thermolyzed protein in the diet, and duration of thermolysis. To determine whether the previous finding of promotion of aberrant crypt foci corresponds to promotion of cancers assessed much later, we carried out promotion studies until colon cancers appeared. METHODS: F344 rats were given an initiating dose of azoxymethane and were then randomly allocated to groups receiving diets differing in their quantity and quality of casein. The groups were examined for aberrant crypt foci and tumors in the colon. RESULTS: Aberrant crypt foci were promoted by diets containing thermolyzed casein (180 degrees C, 2 hours). Promotion increased with increasing level of thermolyzed casein in the diet (to 20%) and with increasing thermolysis time (to 4 hours). The number of animals with polyps and cancers was higher in the animals receiving thermolyzed protein (2 hours), 16/23 versus 9/26 (P less than .05) and 10/26 versus 3/27 (P less than .05), respectively. The number of aberrant crypts per focus and the number of large aberrant crypt foci were higher in the tumor-bearing animals. CONCLUSIONS: Thermolyzed casein promotes early colonic precursor lesions in a dose-dependent and thermolysis time-dependent manner; thermolyzed casein also promotes colon cancer. IMPLICATIONS: The promoter formed on thermolysis could be involved in colon cancers associated with diets cooked at elevated temperatures, such as can occur with high-fat diets.     

Sequential analysis of morphological and biological properties of beta-catenin-accumulated crypts, provable premalignant lesions independent of aberrant crypt foci in rat colon carcinogenesis

Our previous study (Cancer Res., 60: 3323-3327, 2000) showed that frequent beta-catenin gene mutations are present in beta-catenin-accumulated crypts, which occur early in rodent colonic carcinogenesis, with a lack of the appearance of aberrant crypt foci (ACF). To clarify the nature of such lesions, we performed a sequential analysis of the morphological and biological properties of beta-catenin-accumulated crypts. Azoxymethane was administered s.c. to male F344 rats (15 mg/kg body weight) once a week for 3 weeks, and the animals were sacrificed at 5, 10, and 20 weeks after the carcinogen treatment. Both the number of crypts/lesion and the diameter of beta-catenin-accumulated crypts were significantly increased with time courses of 5, 10, and 20 weeks from carcinogen exposure (P < 0.01). Likewise, the histological abnormality in those crypts, assessed by semiquantitative analyses, was also increased with time (P < 0.01). Conversely, ACF did not show any increase in histological abnormality during the time course and maintained a monotonous histology throughout the experiment. The histological abnormality score for beta-catenin-accumulated crypts was significantly higher than for ACF at every time point (P < 0.001). The number of AgNOR/nucleus in beta-catenin-accumulated crypts was significantly higher than in ACF (P < 0.001). Beta-catenin-accumulated crypts were accompanied frequently by Paneth cells and had decreased hexosaminidase activity. Such data, together with the results in our previous report, strongly suggest that beta-catenin-accumulated crypts, which are independent of ACF, are truly premalignant lesions for colon cancer.     

Dietary polyamines promote the growth of azoxymethane-induced aberrant crypt foci in rat colon

We have examined whether dietary polyamines influence the formation and initial growth of azoxymethane (AOM)-induced aberrant crypt foci (ACF) in rat colon. Effects of a combination of dietary polyamines at three dose levels (putrescine: 50, 280, 740 nmol/g; spermidine: 10, 261, 763 nmol/g; spermine: 1, 31, 91 nmol/g) in the polyamine-poor AIN-76A diet were studied in animals in two different experimental situations: animals treated with AOM alone and animals treated with AOM + difluoromethylornithine (DFMO), a specific inhibitor of endogenous polyamine synthesis. In both experimental situations, dietary polyamines enhanced the growth of ACF, expressed as the number of large ACF (foci with three or more aberrant crypts, ACF > or = 3), whereas the formation of ACF, expressed as the number of ACF, was apparently not altered. In animals treated with AOM alone, maximal growth enhancing effect on ACF was nearly obtained with the median level of dietary polyamine. In rats fed a low polyamine diet, basic AIN-76A, DFMO reduced the growth of AOM-induced ACF by 83%. This inhibitory effect of DFMO was counteracted by dietary polyamines in a dose- dependent manner, and it was abolished at the highest level of polyamines. In conclusion, it was demonstrated that dietary polyamines are able to enhance the growth of AOM-induced ACF. Further, dietary polyamines reversed the DFMO-caused inhibition of ACF growth, probably by compensating for the DFMO-reduced endogenous polyamine synthesis.      

Newly defined aberrant crypt foci as a marker for dysplasia in the rat colon

Dysplasia represents a preneoplastic status in multistep colon carcinogenesis. Whereas laborious preparation of thin sections is required for its diagnosis, we here show that newly defined aberrant crypt foci (ACF) simply mark the majority of the dysplasia on the whole colon. Specifically, decoloring of the azoxymethane‐treated rat colon after scoring classical ACF (cACF) resulted in visualization of a subset of aberrant crypts that remained densely stained. They were morphologically classified into three subtypes, of which two with compressed luminal openings proved highly correlated with dysplasia. Accordingly, we designated those foci harboring either of the two crypt subtypes as dysplasia‐associated ACF (dACF). By serially applying different detection methods for known preneoplastic lesions to the same colon, we showed that most dACF had already been identified as cACF, and a few newly identified dACF contained an entire population of more advanced lesions, such as flat ACF and mucin‐depleted foci. Consequently, integrative scoring of cACF and dACF enabled capture of all early lesions of the colon. Furthermore, 94% of the dACF showed dysplasia and 90% of the dysplastic lesions proved to be dACF. Thus, dACF is a promising marker for dysplasia, likely facilitating precise identification of the early stages of colon carcinogenesis.     

Essential similarities between spontaneous and MeIQx-promoted aberrant crypt foci in the F344 rat colon

Aberrant crypt foci (ACFs) in the Fischer 344 (F344) rat colon, of control or 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-treated groups, were compared morphologically, immunohistochemically, and at the molecular biological level in order to elucidate their biological characteristics. Male 3-week-old rats were fed a diet supplemented with or without MeIQx at doses of 100 ppm or less for 16 weeks. The incidence of ACFs was the highest (90%) in animals given 100 ppm MeIQx but that in untreated rats was also surprisingly high (57%). Nine ACFs from nine MeIQx-treated rats and ten ACFs from ten untreated control rats were selected for detailed examination for their large size. There were no morphological differences in macroscopic and microscopic features between MeIQx-promoted and spontaneous ACFs. There were also no differences in immunohistochemical labeling for proliferating cell nuclear antigen (PCNA) and p53 protein between these ACFs although in both cases labeling was higher than in normal crypts. Dot blot hybridization revealed no c-K-ras mutations in codon 12 except in one ACF (11.1%) developing in a rat treated with 100 ppm MeIQx, in which a GGT-->GAT single base substitution was detected. Our results thus suggest that in terms of morphology, cell proliferation, P53 expression and c-K-ras mutation, most ACFs found in rats given 100 ppm MeIQx are essentially identical to their spontaneous counterparts.     

Classification of aberrant crypt foci and microadenomas in human colon.

Aberrant crypt foci (ACF) can be observed and quantified on the mucosal surface of formalin-fixed human colon resections after staining with methylene blue. To determine whether these ACF could be identified in fresh tissue, 10 colon resections were collected after surgery for colorectal cancer. Unfixed and fixed flat normal colonic mucosa from each colon were scored for ACF under a dissecting microscope after methylene blue staining. The number of ACF per cm2 and the average number of crypts per foci correlated highly in unfixed and fixed mucosa (r = 0.93 and 0.78, respectively). A significantly higher frequency of lesions was found in left-sided compared to right-sided colon resections. To determine whether the topographic features of the ACF gave an indication of the histological appearance, 68 specimens containing ACF or normal mucosa were examined histologically. The presence of slit-like lumen in the crypts of ACF on the mucosal surface correlated with the presence of dysplasia at histology, thus identifying microadenomas. These two observations suggest that the topographic classification of ACF in vivo could be used to distinguish microadenomas, a putative precursor lesion of colon cancer.     

Effect of dietary oligofructose and inulin on colonic preneoplastic aberrant crypt foci inhibition

Oligofructose and inulin, naturally-occurring fermentable chicory fructans, have been shown to stimulate the growth of bifidobacteria which are regarded as beneficial strains in the colon and inhibit colon carcinogenesis in the laboratory animal models. The present study was designed to determine the effect of oligofructose and inulin on the azoxymethane (AOM)-induced preneoplastic lesions such as aberrant crypt foci (ACF) formation in the colon of male F344 rats. At 5 weeks of age, groups of animals were fed the AIN-76A (control) and the experimental diets containing 10% oligofructose or inulin. At 7 weeks of age, all animals received s.c. injection of AOM dissolved in normal saline at a dose rate of 15 mg/kg body wt, once weekly for 2 weeks. The animals were necropsied 7 weeks after the last AOM injection, and the ACF were visualized under light microscopy in the formalin-fixed, unsectioned methylene blue-stained colons. They were distinguished by their increased size, more prominent epithelial cells and pericryptal space. The feeding of oligofructose or inulin significantly inhibited the ACF formation and the crypt multiplicity in the colon. The degree of ACF inhibition was more pronounced in animals fed inulin than in those fed oligofructose. The findings suggest that chicory fructan supplements inhibit ACF formation, an early preneoplastic marker of malignant potential in the process of colon carcinogenesis.      

Flat dysplastic aberrant crypt foci are related to tumorigenesis in the colon of azoxymethane-treated rat

We evaluated the role of aberrant crypt foci (ACF) as biomarkers of colon cancer by studying the sequential development (6-28 weeks) from early lesion to tumor in the colon of azoxymethane-exposed F344 rats (15 mg/kg bw x 2). Surface examination of unsectioned methylene blue-stained colon preparations, transilluminated in the inverse light microscope, revealed two types of early lesions: classic elevated ACF and small flat lesions, which we denoted flat ACF and which were characterized by bright blue staining, compressed crypt openings, and crypts not elevated above the surrounding mucosa. At a later stage, the crypts surrounding large flat ACF became enlarged, a change that slightly raised the structure; principally, large flat ACF and nascent tumors displayed the same surface morphology. Furthermore, flat ACF with 18.6 +/- 10.6 crypt/focus and tumors showed a uniform picture of severe dysplasia with frequent presence of Paneth cells, compressed crypts, cytoplasmic/nuclear overexpression of beta-catenin, and nuclear overexpression of cyclin D1. In contrast, classic elevated ACF with 5.3 +/- 2.5 crypts/focus did not display such changes: they showed mainly hyperplasia, mild or moderate dysplasia but never severe dysplasia. Along the time course, the number of flat ACF + tumors, including microscopic and macroscopic, was virtually constant, approximately 2.5 lesions/rat. The number of classic elevated ACF was initially approximately 180 lesions/rat and terminally approximately 80 lesions/rat. Flat ACF grew significantly faster than classic elevated ACF. In conclusion, our data indicate a continuous developmental growth from small flat dysplastic ACF to the stage of a tumor. In contrast, classic elevated ACF do not seem to be as closely related to tumorigenesis.     

Immunohistochemical demonstration of a mRNA-transport protein in rat liver putative preneoplastic foci

A 65K protein, known for promoting nucleocytoplasmic mRNA transport in a cell-free system, was previously found in fetal and tumor cells of the rat. The primary objective of this study was to show specificity of immunohistochemical staining for the 65K protein in the livers of rats subjected to a hepatocarcinogenesis protocol. Altered hepatic foci were induced by feeding male weanling Sprague-Dawley rats 2-acetylaminofluorene (AAF) followed by a phenobarbital (PB) diet. It was shown, using polyclonal antibodies produced in rabbits, that the 65K protein was present in the cells of rat liver putative preneoplastic foci, with little or none being detected in the surrounding cells.     

Loss of heterozygosity in human aberrant crypt foci (ACF), a putative precursor of colon cancer

Aberrant crypt foci (ACF), the earliest neoplastic lesions of the colon, have genetic and epigenetic alterations. Loss of heterozygosity (LOH) of tumor suppressor gene loci is seen in most colon cancers, but it is not known how early in tumorigenesis this takes place. Nine microsatellite markers close to specific genes, that is, APC (5q21), PTPRJ (11p11), p53 (17p13) and DCC (18q21), were analyzed in 32 ACF and samples of normal crypts from the same 28 patients. Six losses of heterozygosity were found in 5 of 32 ACF: 4 losses of heterozygosity were at 11p11, the location of the gene for protein tyrosine phosphatase receptor type J (PTPRJ) and of a second independent region of deletion; the others were at 5q21 and 18q21. Microsatellite instability (MSI) with markers for a single locus was found in 4 of 32 ACF. All the observed allelic alterations (LOH and MSI) were in 8 of 32 ACF. The finding of LOH in ACF with normal expressions of adenomatous polyposis coli (APC) and beta-catenin proteins suggests that LOH can occur very early in colon neoplasia and perhaps even before APC mutations. The finding of 3 of 4 of the losses of heterozygosity at 11p11 for PTPRJ and half of all the losses of heterozygosity in this study at PTPRJ suggest that this gene plays a role early in colon neoplasia.     

Sequential and morphological analyses of aberrant crypt foci formation in mice of differing susceptibility to azoxymethane-induced colon carcinogenesis

Aberrant crypt foci (ACF), putative preneoplastic lesions, are early morphological changes induced by the colon carcinogen azoxymethane (AOM). Although inbred mice differ markedly in their susceptibility to AOM carcinogenesis, we have previously shown that ACF develop in both resistant and sensitive mouse strains after AOM treatment. The purpose of this study was to examine the sequential development and identify the morphological characteristics of ACF induced by AOM in the distal colon of sensitive and resistant mice. A/J (highly susceptible), SWR/J (relatively susceptible) and AKR/J (resistant) mice were treated with 10 mg/kg AOM or saline i.p. once a week for 6 weeks and were killed at 1, 2, 4, 6, 9 and 24 weeks after the last injection. The distal colons were stained with methylene blue and the numbers of ACF and tumors determined. Tumors were present as early as 4 weeks after AOM exposure in SWR/J and A/J mice and increased in frequency throughout the study in both strains. No tumors developed in the AKR/J mice. ACF, however, formed in all strains of mice. The greatest difference between susceptible and resistant strains was in the number of large ACF that developed at later time points. Furthermore, morphometric analysis revealed that A/J mice had the highest percentage of dysplastic ACF, followed by SWR/J mice. These data indicate that the difference in cancer risk from AOM may be due to the lack of progression of smaller ACF in the resistant mice and to the development of dysplasia in a higher percentage of ACF from susceptible strains.     

Frequent mutations of the beta-catenin gene in mouse colon tumors induced by azoxymethane

The beta-catenin gene is frequently mutated at codons 33, 41 and 45 of the glycogen synthase kinase-3beta phosphorylation motif in human colon cancers in patients without APC mutations. Frequent mutations at codons 32 and 34, as well as 33 and 41, have been detected in rat colon tumors induced by azoxymethane (AOM), with the second G of CTGGA sequences being considered as a mutational hot-spot. In the present study, exon 3 of the beta-catenin gene in mouse colon tumors induced by AOM was amplified by PCR and mutations were detected by the single strand conformation polymorphism method, restriction enzyme fragment length polymorphism and direct sequencing. All 10 colon tumors tested were found to have beta-catenin mutations, four in codon 34, three in codon 33, two in codon 41 and one in codon 37, nine being G:C-->A:T transitions. However, no mutations were found in codon 32 of the mouse beta-catenin gene. On immmunostaining, beta-catenin was observed in the cytoplasm and nucleus of the tumor cells. The cytoplasmic staining was homogeneous, while both homogeneous and heterogeneous patterns were noted for the nuclei. Highly frequent mutations of the beta-catenin gene in AOM-induced mouse colon tumors suggest that consequent alterations in the stability and localization of the protein may play an important role in this colon carcinogenesis model.