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[Cancer Research 61, 1874-1878, March 1, 2001]
© 2001 American Association for Cancer Research

Carcinogenesis

Sequential Analysis of Morphological and Biological Properties of ß-Catenin-accumulated Crypts, Provable Premalignant Lesions Independent of Aberrant Crypt Foci in Rat Colon Carcinogenesis1

Yasuhiro Yamada2, Naoki Yoshimi, Yoshinobu Hirose, Kengo Matsunaga, Masaki Katayama, Keiko Sakata, Masahito Shimizu, Toshiya Kuno 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
 
Our previous study (Cancer Res., 60: 3323–3327, 2000) showed that frequent ß-catenin gene mutations are present in ß-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 ß-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 ß-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 ß-catenin-accumulated crypts was significantly higher than for ACF at every time point (P < 0.001). The number of AgNOR/nucleus in ß-catenin-accumulated crypts was significantly higher than in ACF (P < 0.001). ß-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 ß-catenin-accumulated crypts, which are independent of ACF, are truly premalignant lesions for colon cancer.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Colon carcinogenesis is a representative multistep tumorigenesis that includes events of genetic alterations (1) . ACF3 were first described by Bird (2) with the use of methylene blue-stained preparations. ACF have been defined as crypts that: (a) have altered luminal openings; (b) exhibit thickened epithelia; and (c) are larger than adjacent normal crypts (3) . During the last decade, numerous studies including molecular analysis have focused on the significance of ACF as early events in colon carcinogenesis, and ACF are now regarded as putative premalignant lesions for colon cancers (4 , 5) . It is reported that the number of crypts/focus increases with time after the carcinogen treatment, and that ACF have an increased proliferative activity (6 , 7) . Nevertheless, there is evidence that documents the lack of correlation between tumor development and the expression of ACF (8 , 9) . Furthermore, some compounds with the potency to prevent the occurrence of ACF, e.g., 2-(carboxyphenyl) retinamide or genistain, have been found to enhance the development of colon cancers (10 , 11) . Yet, the premalignant nature of the colon carcinogenesis remains inconclusive thus far.

ß-Catenin, which was originally discovered as a cadherin-binding protein, has been proved to function as a transcriptional activator when complexed with members of the Tcf family of DNA binding proteins (12 , 13) . Recently, target genes of the ß-catenin-Tcf pathway were determined to be growth-promoting genes, such as c-myc and cyclin D1 (14 , 15) , which suggests that ß-catenin-Tcf is an oncogenic pathway. It is also known that ß-catenin levels are regulated by the degradation of the protein through the ubiquitin-proteasome pathway (16) , and intact APC cooperates with AXIN and GSK-3ß to regulate the degradation of ß-catenin protein (17 , 18) . Mutations in the APC or ß-catenin genes, which are associated with the majority of human and rat colon tumors (19 , 20) , were proved to repress the degradation of the protein and to generate ß-catenin accumulations (21) , which leads to activation of the oncogenic ß-catenin-Tcf pathway. Excessive ß-catenin protein has been shown in many colon cancers of rats and humans (20) . A recent report indicates that expression of nuclear ß-catenin is correlated with tumor size in colon carcinogenesis (22) .

Very recently, we demonstrated that ß-catenin gene mutations and accumulations of the protein are involved in the initial stage of colon carcinogenesis induced in rats by AOM (23) . Interestingly, accumulation of ß-catenin was more prominent in the crypts that did not present a ACF-like appearance, as recognized in the whole-mount preparations. We thus hypothesize that ß-catenin-accumulated crypts are independent lesions of ACF, and crypts with ß-catenin accumulation are direct precursors for colon cancer. To address this hypothesis, we performed a sequential analysis of the morphological and biological properties of ß-catenin-accumulated crypts.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Treatment.
Male F344 rats 6 weeks of age were obtained from the Shizuoka SLC Co. (Shizuoka, Japan). Animals were given s.c. injections of AOM (15 mg/kg body weight; Sigma Chemical Co.) once a week for 3 weeks and killed at 5, 10, and 20 weeks after the first injection. Immediately after death, the animals’ colons were removed, cut open along the longitudinal axis, and fixed flat in 2% paraformaldehyde in 0.1 ml of PBS (pH 7.4) for 2 h at 4°C. Then, whole-mount colons were stained with methylene blue or examined for hexosaminidase activity.

Immunohistochemistry.
Colonic mucosal sections were examined using an en face preparation and 3- to 5-µm thick serial sections. For each case, 20–40 serial sections of crypts were used to investigate whole crypts from the mucosal surface to the crypt bottom. For immunohistochemical analysis, the labeled streptavidin biotin method was performed using primary antibodies against ß-catenin (diluted 1:1000; Transduction Laboratories, Lexington, KY), as described previously (23) . These immunoreactivities were evaluated by two pathologists independently. At least two sections/preparation were examined for ß-catenin immunohistochemistry, and samples were regarded as positive when apparent immunoreactivities in cytoplasms or nucleus were recognized in more than one section.

Histological Examination.
Histological evaluation was performed by routine procedures with H&E staining. The stained sections were examined for grade of histological abnormality. The grade of histological abnormality was semiquantitatively scored using the following four parameters, which are distinctive features of colonic dysplasia and neoplasia (24) : (a) increase of nuclear/cytoplasmic ratio (0, 0–25%; 1, 25–50%; 2, >50%); (b) nuclear stratification (0, none; 1, mild; 2, severe); (c) loss of nuclear polarity (0, none; 1, mild; 2, severe); and (d) structural abnormality (0, none; 1, mild; 2, severe). At least four sections/preparation were examined for grading. The total score of each parameter was regarded as the score of histological abnormality. When we used this score of histological abnormality, almost all colon cancers were scored as 8 points (full score), and normal colonic crypts were scored as 0 points. The Mann-Whitney nonparametric U test was used to analyze the histological abnormality scores. Statistical significance was established on the basis of 95% confidence intervals.

AgNOR.
AgNOR staining, which is suggested to be a useful biomarker of cell proliferation, was carried out according to the method described previously (25) , with minor modifications. Colonic mucosa from rats at the 10-week time point was used for the analyses. AgNOR counts/nucleus were scored on AgNOR-stained sections by microscopy at a magnification of x400.

Hexosaminidase Activity.
Decreased hexosaminidase activity has been reported to be involved in the early stage of colon carcinogenesis in rats (26) . The activity in colonic mucosa from rats at the 10-week time point was examined using whole-mount staining methods. Substrate solution was prepared according to the procedure described by Pretlow et al. (26) . Fixed colons were rinsed in PBS and incubated with the substrate solution for 2 h at 37°C. Finally, they were stained with 0.5% methyl green for 10 s.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ß-Catenin Immunohistochemistry.
ß-Catenin-accumulated crypts were recognized only in the colons of rats treated with carcinogen. The protein in those crypts was localized in the cytoplasms and nucleus, as was the case with colon tumors. Basically, ß-catenin-accumulated crypts did not present an ACF-like appearance in the whole-mount preparations, suggesting that those crypts are different lesions from ACF (Fig. 1)Citation . The crypts often consisted of small crypts rather than enlarged crypts when compared with adjacent normal crypts. Those crypts were detected in all segments of the colon and were often detected as small lesions or as a single crypt. In contrast, ACF did not generally reveal the accumulation of ß-catenin protein, and the localization of ß-catenin immunoreactivity of ACF was only detected as restricted to the membrane in the cell-to-cell borders as in the case of adjacent normal crypts.



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  		Fig. 1. Topographic view of whole-mount preparation stained with methylene blue (A). ß-Catenin immunohistochemistry of the corresponding area (B). A, mucosal topography of cancer-predisposed colon. Arrows indicate ACF that have altered luminal openings and exhibit thickened epithelia. Note that crypts in ACF are larger than adjacent normal crypts. In contrast, area indicated by the open arrow does not have ACF-like appearance. B, accumulation of ß-catenin protein is seen in altered crypts without ACF-like appearance, whereas the accumulation is not recognized in ACF. Bars, 200 µm.

 
Histological Features of ß-Catenin-accumulated Crypts.
Fig. 2Citation shows the histological features of ß-catenin-accumulated crypts and ACF in H&E staining. Interestingly, ß-catenin-accumulated crypts were frequently accompanied by Paneth cells with eosinophilic apical granules. In general, the epithelium of ß-catenin-accumulated crypts bore basophilic cytoplasms and hyperchromatic nuclei, and had an increase in the nuclear/cytoplasmic ratio. Mucin production was usually absent in those crypts. ß-Catenin-accumulated crypts tended to possess nuclear stratifications and disruptions of nuclear polarity and to have structural abnormality (A and B). Infrequently, ß-catenin-accumulated crypts with large diameters revealed adenomatous crypts (B). In contrast, ACF usually kept cellular and nuclear polarity (C and D) and did not exhibit adenomatous crypts (D).



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  		Fig. 2. The histological features of two types of altered crypts. A and B, ß-catenin-accumulated crypts. Epithelium had basophilic cytoplasms and hyperchromatic nuclei and tended to show disruption of nuclear polarity. Mucin production is almost absent. ß-Catenin-accumulated crypts are detected in small lesions even in a single crypt (A). Some large lesions have adenomatous crypts with extensive branchings (B). ß-Catenin-accumulated crypts are accompanied by Paneth cells (A and B). Arrows indicate instances of Paneth cells. C and D, ACF having hyperchromatic nuclei and decreased activity for mucin production. However, cellular and nuclear polarity is retained even in large ACF (D). Histological characteristics of ACF are monotonous regardless of size of lesion (C and D). The histological abnormality scores assessed by semiquantitative analyses were 3 points in A (nuclear/cytoplasmic ratio = 1; nuclear stratification = 1; loss of nuclear polarity = 1; and structural abnormality = 0); 6 points in B (nuclear/cytoplasmic ratio = 1; nuclear stratification = 2; loss of nuclear polarity = 1; and structural abnormality = 2); 2 points in C (nuclear/cytoplasmic ratio = 1; nuclear stratification = 0; loss of nuclear polarity = 1; and structural abnormality = 0); and 1 point in D (nuclear/cytoplasmic ratio = 1; nuclear stratification = 0; loss of nuclear polarity = 0; and structural abnormality = 0). Bars, 100 µm.

 
Sequential Analysis of ß-Catenin-accumulated Crypts.
ß-Catenin-accumulated crypts were already detectable in the carcinogen-treated rats at the first time point (week 5). Both the mean diameter of lesion and the mean number of crypts/lesion steadily increased with time, being significant at 5, 10, and 20 weeks after the first carcinogen treatment (P < 0.01; Student’s t test; Fig. 3Citation ). To evaluate the grade of histological abnormality, we performed semiquantitative analyses using four parameters that are characteristic features of colonic dysplasia and colon cancer (24) . The nuclear/cytoplasmic ratio and structural abnormality were found to be significantly increased at the 20th week when compared with the 5th week (P < 0.01 and P < 0.001, respectively; Mann-Whitney U test). Nuclear stratification was present more frequently at the 20th week than at the 10th week (P < 0.005; Mann-Whitney U test). The total score of histological abnormality was significantly greater at the 20th week than that at the 5th and the 10th weeks (P < 0.003 and P < 0.01, respectively; Mann-Whitney U test). In contrast, ACF generally exhibited a monotonous histological appearance during the experiment, and the ACF score did not increase sequentially. The grade of histological abnormality was significantly higher in ß-catenin-accumulated crypts than in ACF at all time points (P < 0.001; Mann-Whitney U test). The results of the grading of histological abnormality are summarized in Table 1Citation and in Fig. 4Citation . The number of ß-catenin-accumulated crypts/area was 2.90 ± 0.81/cm2 at week 5; 3.54 ± 1.14/cm2 at week 10; and 3.46 ± 1.06/ cm2 at week 20.



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  		Fig. 3. Time course of the mean number of crypts (A) and the mean diameter (B) of ß-catenin-accumulated crypts. Both the multiplicity and the diameter of the lesion increased with time. Values presented are means ± SD (bars). *, significantly different (P < 0.001; Student’s t test) from week 5. **, significantly different (P < 0.01; Student’s t test) from week 10.

 

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  		Table 1 Semiquantitive analyses of grades of histological abnormality

 


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  		Fig. 4. Sequential analysis of histological abnormality in ß-catenin-accumulated crypts ({blacktriangleup}) and ACF ({blacksquare}). In ß-catenin-accumulated crypts, the total score of histological abnormality, assessed by semiquantitative analyses, increased with time after the carcinogen administrations ({blacktriangleup}; P < 0.01; Mann-Whitney U test). Note that ACF never showed an increase in score ({blacksquare}). The score was significantly higher in ß-catenin-accumulated crypts than in ACF at all time points (P < 0.001; Mann-Whitney U test).

 
AgNOR Count/Nucleus.
The data for numbers of AgNOR count/nucleus of the epithelium in ß-catenin-accumulated crypts, ACF, and adjacent normal crypts are shown in Fig. 5Citation . The mean number of AgNOR count/nucleus was greatest in ß-catenin-accumulated crypts, and the number in ß-catenin-accumulated crypts was significantly greater than that in ACF and normal crypts (P < 0.001; Student’s t test). ACF had a larger AgNOR count/nucleus than the normal crypts (P < 0.03; Student’s t test).



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  		Fig. 5. Number of AgNOR/nucleus in ß-catenin-accumulated crypts. Mean number of AgNOR/nucleus in ß-catenin-accumulated crypts was significantly larger than that in ACF and accompanying normal crypts (*, P < 0.001; Student’s t test). The number in ACF was also increased when compared with adjacent normal crypts (**, P < 0.03; Student’s t test).

 
Hexosaminidase Activity.
The decreased hexosaminidase activity has been reported in the two types of early-appearing lesions in rodents’ colonic mucosa by Pretlow et al. (26) . They described one of the lesions as having a normal-like appearance. We investigated the hexosaminidase activity to determine whether ß-catenin-accumulated crypts coincide with such enzyme-altered foci with normal-like crypts. In the present study, a decrease of the enzyme activity was present in both the ACF-like crypts and the normal-like crypts of cancer-predisposed colonic mucosa, indicating that there are two types of enzyme-altered foci (Fig. 6A)Citation . However, accumulation of ß-catenin protein was only recognized in the enzyme-altered foci with the normal-like crypts, whereas enzyme-altered foci with ACF-like crypts did not have the accumulation. Translocation of the protein into cytoplasms and/or nucleus was only detected in the normal-like crypts (Fig. 6C)Citation .



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  		Fig. 6. Whole-mount staining for hexosaminidase activity (A). A corresponding area of histological section (B) and ß-catenin immunohistochemistry (C). A, mucosal topography of cancer-predisposed colon after staining for hexosaminidase activity (red stain). Hexosaminidase activity is observed predominantly in the epithelial cells of the normal crypts. Note that there are two types of lesion with decreased hexosaminidase activity (arrow and open arrow). One type has aberrant crypts that are much larger than normal crypts and have thickened epithelium (ACF; arrow). The other type does not have ACF-like appearance (normal-like crypts; open arrow). Crypts presented here have small crypts rather than large crypts. B, both types of enzyme-altered foci are distinguishable from adjacent normal crypts in histological sections stained with H&E staining. C, accumulation of ß-catenin protein is detected only in enzyme-altered foci with the normal-like appearance, whereas it is not recognized in any ACF. Localization of the excessive protein is apparent in the cytoplasms and nucleus of the normal-like crypts, although it is restricted to membranes of the epithelial cells in ACF and adjacent normal crypts. Bars, 100 µm.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The advent of epithelial dysplasia has been indicated to reflect the frequency of genetic alteration in the process of colon carcinogenesis, and numerous reports suggest that colonic dysplasia is a hallmark of malignant potential (1) . In this study, we demonstrated that the cell population of ß-catenin-accumulated crypts exhibits histological abnormalities, including disruption of cellular morphology. Also, the grade of histological abnormality of such lesions, which is assessed by four parameters, was found to be increased sequentially during the experiment. Because the parameters used to score the histological abnormality are regarded as distinctive features of colonic dysplasia and neoplasia (24) , such scores are considered to well indicate the degree of dysplasia. Thus the results of this study indicate that the grade of dysplasia of ß-catenin-accumulated crypts was increased with time after the carcinogen treatment. Such results, together with the data showing increased proliferative activity in the crypts, suggest that the ß-catenin-accumulated crypts are premalignant lesions for colon cancer. In this study, crypts with accumulation of ß-catenin were infrequently recognized as adenomatous crypts having extensive branchings. Previously, we reported that ß-catenin-accumulated crypts in the large bowel harbor frequent mutations in the ß-catenin gene, providing evidence that those crypts are premalignant lesions. Histopathological results presented here may also support that ß-catenin-accumulated crypts are truly precursors of colon cancer.

It is noteworthy that such crypts with accumulation of ß-catenin did not present an ACF-like appearance. ACF have been widely accepted as putative preneoplastic lesions of colon cancer. However, in this study, ACF did not exhibit excessive ß-catenin protein, which is associated with the majority of colon cancers and is suggested to play a critical role in the initial stage of colon carcinogenesis (23) . ß-Catenin-accumulated crypts had higher histological abnormality scores than did ACF, with monotonous histology at every time point, and, importantly, the proliferative activity of crypts with an accumulation of ß-catenin was significantly higher than that in ACF. It is reasonable to conclude that ß-catenin-accumulated crypts are more likely to progress into malignant transformation than are ACF. However, it cannot be entirely ruled out that ACF are preneoplastic lesions, because an increase of cell proliferative activity and genetic alterations in the ß-catenin and/or k-ras gene have been reported in a part of ACF (23 , 27) . It may be true that such ACF with genetic alterations, although perhaps only a portion of the total ACFs, also have a neoplastic potential. The lesions have been used as biomarkers to evaluate various agents for their potential chemopreventive properties, and most outcomes of these studies have well indicated the chemopreventive efficacy against colon carcinogenesis (28) . Because ACF can be detected easily without tissue sectioning, such early-appearing lesions could still remain one of the useful biomarkers for the screening of compounds for their chemopreventive activities.

Previously, Pretlow et al. have shown putative preneoplastic lesions of rat colon cancer by the use of whole-mount staining for hexosaminidase activity (26) . The results of this study seem to be in agreement with the previous investigations, showing that decreased hexosaminidase activity was detected in two types of lesions in cancer-predisposed colonic mucosa (26) . Presently, accumulation of ß-catenin protein was detected in only the normal-like lesions with decreased hexosaminidase activity. This suggests that ß-catenin- accumulated crypts coincide with the enzyme-altered foci with normal-like appearance, and that those two types of enzyme-altered foci are biologically different lesions. Although our understanding of the functional significance of hexosaminidase activity may be evolving still, the results here imply that the enzyme-altered lesions with normal-like appearance are more likely to be direct precursors of colon cancer than those with the ACF-like appearance.

It is interesting to note that Paneth cells were associated frequently with ß-catenin-accumulated crypts. Despite extensive examinations, the biological role of Paneth cells has not yet been clearly defined. However, Paneth cells are considered to provide a number of cell-growth-related factors, such as tumor necrosis factor-{alpha} (29) , guanylin (30) , epidermal growth factor (31) , and matrilysin (32) . Interestingly, such factors are suggested to regulate epithelial proliferation and differentiation. In fact, mature Paneth cells sometimes are present in colonic tumors, whereas they rarely occur in normal colonic epithelium. The results may suggest a disdifferentiating potential of such dysplastic cryptal lesions.

In conclusion, the present study on early lesions strongly implicates that: (a) ß-catenin-accumulated crypts are independent lesions of ACF, and they are premalignant lesions of colon cancer; and (b) ß-catenin is a useful marker for premalignant lesions of AOM-induced colon cancer in rats. Additional investigations of the sequential analyses of cellular and molecular features of ß-catenin-accumulated crypts may provide an important clue to understanding the nature of the premalignant population.


   ACKNOWLEDGMENTS
 
We thank Dr. Takuji Tanaka for helpful discussions. We also thank Kyoko Takahashi, Chikako Usui, Tomoko Kajita, Ayumu Nagata, and Kazumasa Sato for their excellent technical assistance.


   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 Welfare, 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 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; Tcf, T cell factor; AOM, azoxymethane; APC, adenomatous polyposis coli; and AgNOR, silver-stained nucleolar organizer regions. Back

Received 7/10/00. Accepted 12/21/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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