Proliferation rate of colonic mucosa in normal subjects and patients with colonic neoplasms: a refined immunohistochemical method.

An increased colonic epithelial proliferation rate and an increase of the cryptal proliferative zone are probable markers of increased susceptibility to colonic cancer. In this study an immunohistochemical method using 5-bromo-deoxyuridine (BrdUrd) to measure the proliferation rate of colonic mucosa in vitro was used. Fresh endoscopic colonic biopsy specimens were incubated with BrdUrd and then processed for immunohistochemistry using a monoclonal antibody. Essential procedures with respect to the equal distribution of nuclei stained with BrdUrd in the biopsy specimens proved to be the cutting of the specimens before incubation and the use of a microwave oven at the beginning of incubation. The use of the procedure of the running average showed that 12 length cut crypts are sufficient to determine reliably the proliferation rate, expressed as the labelling index (LI). This was determined in the biopsy specimens of 10 subjects without organic colonic disease, eight patients with adenomatous colonic polyps, and in six patients with (recent) colonic carcinoma. Mean LI in the controls was significantly lower than in patients with colonic polyps and in those with colon cancer. It is concluded that this method is promising for screening persons at risk for colon cancer and will be of great potential in performing dietary intervention studies in these subjects.     

Expression of Sonic hedgehog pathway genes is altered in colonic neoplasia

The Hedgehog (Hh) signalling pathway is crucial for normal development and patterning of numerous human organs including the gut. Hh proteins are also expressed during gastric gland development and gastric epithelial differentiation in adults. Recently, dysregulation of these developmentally important genes has been implicated in cancer, leading to the present study of the expression of Hh signalling proteins in colon cancer. In this study, normal colon and colonic lesions (hyperplastic polyp, adenoma, and colonic adenocarcinoma) were examined by immunohistochemistry using antibodies against Hh signalling molecules: the secreted protein Sonic hedgehog (SHH), its receptor Patched (PTCH), and the PTCH-associated transmembrane protein Smoothened (SMOH). The study shows that Hh signalling pathway members are expressed in normal colonic epithelium. SHH was expressed at the top of the crypts and in a few basally located cells, while PTCH was detected in the neuroendocrine cells and SMOH at the brush border of superficial epithelium. RT-PCR analysis of laser-microdissected crypts from normal human colon confirmed that mRNAs encoding these proteins were expressed in colonic epithelium. Expression of SHH, PTCH, and SMOH was up-regulated in hyperplastic polyps, adenomas, and adenocarcinomas of the colon, and SHH expression correlated with increased expression of the proliferation marker Ki-67 in all lesions examined. To address whether the Hh signalling pathway is functional in the gut, the effect of Shh on epithelial cells in vitro was explored by treating primary murine colonocytes with either Shh peptide or neutralizing anti-Shh antibody. The proportion of cells in the S-phase was assessed by bromodeoxyuridine (BrdU) incorporation. It was found that exogenous Shh promotes cell proliferation in colonocytes, while anti-Shh inhibits proliferation, suggesting that Shh is required during proliferation of epithelial cells in vitro. It is suggested that SHH is required during epithelial proliferation in the colon and that there is a possible role for Hh signalling in epithelial colon tumour progression in vivo.     

Immunohistochemical detection of abnormal cell proliferation in colonic mucosa of subjects with polyps.

Previous studies have shown the presence of increased proliferation in the large bowel epithelium of those at high risk of developing colon cancer. An in vitro technique for labelling large bowel mucosa with the thymidine analogue bromodeoxyuridine (Brdu) was therefore developed and its ability to distinguish differences in mucosal proliferation between subjects with colorectal adenomas and normal controls was assessed. Sigmoid biopsy specimens from 15 subjects with polyps and 15 age and sex matched controls were labelled and the incorporated Brdu visualised with an immunohistochemical technique. Mean labelling index (LI) was significantly higher in those with polyps than in controls. Differences in the pattern of labelling in colonic crypts were compared by the generation of cumulative labelling distributions. Analysis showed a significant expansion of the proliferative compartment in the colon crypts of those with polyps. It is concluded that in vitro labelling with Brdu provides a useful method for the assessment of mucosal proliferation in subjects at high risk of developing colon cancer.     

Repair of the surface epithelium after saponin-induced colonic mucosal injury in the rat

The superficial colonic epithelia of rats were exposed to 1.0% saponin solution for 3 min and fixed at various periods thereafter. The repair or restitution process was observed by light as well as by transmission and scanning electron microscopy. The exposure of the luminal surface to saponin resulted in uniform and extensive damage to the superficial epithelial cells without affecting the cells in the crypts. At 3 min after saponin treatment, the damaged epithelial cells exfoliated from the mucosa and the basal lamina was exposed. Within 15 min, most of the exposed basal lamina was covered by squamous to low-cuboidal epithelial cells, probably migrating from the crypts. These epithelial cells extended large lamellipodia over the denuded basal lamina. After 15 min the damaged surface was completely covered with epithelial cells, which became columnar at 1 h. Tight junction protein ZO-1 became positive along the restituted epithelium. Proliferating-cell nuclear antigen (PCNA) staining showed that proliferation of epithelial cells occurred after the restitution. These results suggest that saponin treatment serves as a good model system to study colonic restitution, which is carried out by rapid migration from the remaining crypt cells, followed by cellular proliferation. Rapid formation of tight junctions spanning the damaged regions allows rapid restoration of the barrier function of the colonic epithelium.     

Dietary calcium supplementation increases apoptosis in the distal murine colonic epithelium

BACKGROUND: Increased dietary calcium might reduce colorectal cancer risk, possibly by reduction of colonic epithelial hyperproliferation, but not all studies have demonstrated this. Little is known about the effects of calcium on colonic apoptosis. AIM: To quantify the effects of increasing calcium on apoptosis and cell proliferation in normal murine colonic crypt epithelium. METHODS: Twenty one day old male C57B1/6 mice were fed either control AIN-76 diet (0.5% calcium wt/wt; n = 10) or the same supplemented with calcium carbonate (1.0% calcium; n = 10) for 12 weeks. Apoptotic cells in proximal and distal segments were counted and expressed as an apoptotic index (AI: frequency of apoptosis/100 longitudinal crypts). The bromodeoxyuridine (BrdU) labelling index was also determined. Differences were analysed by the student's t test. RESULTS: In control animals, the AI was significantly higher in the caecum/proximal colon (mean, 28.6; SEM, 2.0) compared with the distal colon (mean, 19.9; SEM, 1.8; p = 0.004). In the calcium treated group, the AI in the caecum/proximal colon (mean, 30.6; SEM, 1.7) was similar to controls (p = 0.71) but the AI in the distal colon was significantly greater (mean, 32.6; SEM, 1.8; p = 0.001) than in control mice and was raised to values similar to those in the proximal colon. Calcium was also associated with reduced crypt cellularity and, in the proximal colon, a downward shift in the crypt position at which apoptosis occurred. There were no significant differences in the BrdU labelling index between groups or between proximal and distal colonic segments in each group. CONCLUSIONS: Increased dietary calcium is associated with the induction of apoptosis in normal mouse distal colonic epithelium without affecting cell proliferation. This might contribute to its putative chemopreventive role in colorectal carcinogenesis. Whether this effect is direct or indirect requires further study.     

Effects of aging on expression of genes involved in regulation of proliferation and apoptosis in the colonic epithelium

The purpose of this study was to characterize the effects of aging on colonic messenger ribonucleic acid (mRNA) and protein levels of genes involved in the regulation of cell proliferation and apoptosis, and epithelial morphology in male Fischer 344 rats. Our study shows that, with aging, colonic expression of insulin-like growth factor-1 (IGF-1) and IGF-binding protein-3 (IGFBP-3) is significantly increased and decreased, respectively. Colonic Bax protein levels are increased significantly with aging. Immunohistochemical localization of Bax protein shows a greatly increased expression in colonic crypts, especially in the upper portion of crypts. p53 expression is unchanged with aging. No significant change in proliferation of colonic crypt cells is observed by bromodeoxyuridine (BrdU) labeling, although the increased colonic expression of IGF-1 and the decreased expression of IGFBP-3 with aging may result in an increased colonic IGF-1 bioactivity. The age-related changes in Bax and IGFBP-3 appear to be independent of p53. The finding of an unchanged colonic epithelium with aging in the face of a greatly increased Bax protein levels may suggest that the elevated Bax protein levels function to render colonic epithelial cells more sensitive to apoptotic stimuli.     

Ets2 regulates colonic stem cells and sensitivity to tumorigenesis

Ets2 has both tumor repressive and supportive functions for different types of cancer. We have investigated the role of Ets2 within intestinal epithelial cells in postnatal mouse colon development and tumorigenesis. Conditional inactivation of Ets2 within intestinal epithelial cells results in over representation of Ets2-deficient colon crypts within young and adult animals. This preferential representation is associated with an increased number of proliferative cells within the stem cell region and an increased rate of crypt fission in young mice that result in larger patches of Ets2-deficient crypts. These effects are consistent with a selective advantage of Ets2-deficient intestinal stem cells in colonizing colonic crypts and driving crypt fission. Ets2-deficient colon crypts have an increased mucosal thickness, an increased number of goblet cells, and an increased density. Mice with Ets2-deficient intestinal cells develop more colon tumors in response to treatment with azoxymethane and dextran sulfate sodium. The selective population of colon crypts, the altered differentiation state and increased sensitivity to carcinogen-induced tumors all indicate that Ets2 deficiency alters colon stem cell number or behavior. Ets2-dependent, epithelial cell-autonomous repression of intestinal tumors may contribute to protection from colon cancer of persons with increased dosage of chromosome 21.     

Xenografting of normal colonic mucosa in athymic mice

Colonic mucosa was implanted subcutaneously into nude mice in order to investigate the potential use of xenografts as an in vivo model system for the study of colonic epithelial proliferation and differentiation. The xenografts were followed for 5 weeks. Proliferation was studied by bromodeoxyuridine (BrdU) incorporation followed by visualization of the DNA-synthesizing cells with an anti BrdU monoclonal antibody. Differentiated cells were visualized by histochemical staining of goblet- and enterochromaffin cells and of columnar cells by immunoperoxidase staining for carcinoembryonic antigen (CEA), secretory component, and serotonin. In different strains of mice different observations were made. Less immunocompetent strains (Balb c nu/nu, NMRI nu/nu) developed abscesses at the site of the xenograft as well as wasting disease. These phenomena almost never occurred in CD-1 nu/nu mice. The success rate was highest in CD-1 nu/nu mice. After about 1 week, only crypt base cells remained vital and started to repopulate crypts with epithelial cells showing normal colonic differentiation features such as CEA, secretory component, and serotonin immunoreactivity and mainly sulphomucin production. After longer periods of time, crypts started to form cyst-like structures due to accumulation of secretion products and dead cells. DNA-synthesizing cells were seen in the basal areas of recognizable crypts and in the cyst-like structures in a random distribution. These results indicate that normal colon mucosa xenografted into nude mice maintains its proliferative and differentiating capacity for at least 5 weeks.     

Impaired growth of small intestinal epithelium by adrenalectomy in weaning rats

Functional maturation of the small intestine occurs during the weaning period in rats. It is known that this development is facilitated by glucocorticoid. However, the effect of glucocorticoid on morphological development of small intestine has yet to be clarified. The present study evaluated the morphological development and cell proliferation of the small intestine in adrenalectomized (ADX) rat pups. To further understand the mechanism of glucocorticoid effects on intestinal development, we examined the localization of the glucocorticoid receptor in the small intestine. Microscopic analysis showed that growth of villi and crypts is age-dependent, and is significantly attenuated in ADX rats compared with sham-operated rats. BrdU-positive cells, i.e. proliferating cells, were primarily observed in crypt compartments and rapidly increased in number during the early weaning period. The increase in BrdU-positive cells could be attenuated by adrenalectomy. The morphological development of small intestine may be associated with increased proliferation of epithelial cells. On the other hand, glucocorticoid receptors were found in epithelial cells of the mid- and lower villi and not in crypts where BrdU-positive cells were localized. These results indicate that the growth of small intestine is attenuated by adrenalectomy, and that glucocorticoid indirectly acts on proliferation of epithelial cells during the weaning period.     

Targeted inactivation of the mouse guanylin gene results in altered dynamics of colonic epithelial proliferation

Heat-stable enterotoxin (STa), elaborated by enterotoxigenic Echerichia coli, is a worldwide cause of secretory diarrhea in infants and travelers. Both STa and guanylin, a peptide structurally similar to STa, increase intracellular cGMP levels after binding to the same intestinal receptor, guanylate cyclase C (GC-C). Distinct from its role as an intestinal secretagogue, guanylin may also have a role in intestinal proliferation, as guanylin expression is lost in intestinal adenomas. To determine the function of guanylin in intestinal epithelia, guanylin null mice were generated using a Cre/loxP-based targeting vector. Guanylin null mice grew normally, were fertile and showed no signs of malabsorption. However, the levels of cGMP in colonic mucosa of guanylin null mice were significantly reduced. The colonic epithelial cell migration rate was increased and increased numbers of colonocytes expressing proliferating cell nuclear antigen (PCNA) were present in crypts of guanylin null mice as well. The apoptotic index was similar in guanylin null mice and littermate controls. We conclude from these studies that loss of guanylin results in increased proliferation of colonic epithelia. We speculate that the increase in colonocyte number is related to decreased levels of cGMP and that this increase in proliferation plays a role in susceptibility to intestinal adenoma formation and/or progression.     

Morphology of isolated colonic crypts

The traditional paradigm of colonic fluid and electrolyte transport includes a spatial separation of absorptive and secretory processes to surface and crypt cells, respectively. Recent studies of isolated microperfused colonic crypts revealed constitutive Na-dependent fluid absorption while secretion is regulated by one or more neurohumoral agonists. One obvious reason for the difference found in microdissected crypts is their separation from the lamina propria milieu. While it has been shown that isolated crypts are devoid of obvious lamina propria elements, including pericryptal fibroblasts, detailed morphologic information of the content of isolated crypts has been lacking. To characterize the morphology of the isolated crypt, we performed transmission electron microscopy (TEM) and immunofluorescence on microdissected and Ca2+ chelated crypts. Crypt cell type analysis was carried out separately on intact rat colon using light microscopy. TEM revealed a complete lack of either lamina propria cells or extracellular material in crypts isolated by either technique. TEM also revealed a subtle difference between the two isolation methods, with intact basal membranes in microdissected crypts but focal disruption of basal membranes in Ca2+- chelated crypts. Immunofluorescent stains for two basement membrane components (laminin and collagen type IV) revealed the presence of adherent basement membrane only on microdissected crypts; evidence that the plane of separation differs in these two preparations. Crypt cell type analysis on intact rat colon revealed an equal proportion of goblet cells in the right and left colon (approximately 50%) when measuring the middle 70% of the crypts - the area studied during crypt microperfusion. This morphologic analysis will increase our understanding of the observed physiology of isolated colonic crypts.     

Apoptosis, anoikis and their relevance to the pathobiology of colon cancer

The maintenance of a constant number of cells in an adult organism is a tightly regulated process. This is particularly important in organs where cells are in a constant rate of renewal during the entire lifespan. In these organs, cell number homeostasis is the direct consequence of a balance between cell proliferation and apoptosis. The colonic epithelium is an example of such a site and the high prevalence of colon cancer makes the understanding of cell number homeostasis more important to define. Normal colonic epithelium is organized in crypts where cell proliferation, migration, differentiation and apoptosis are topographically organized in a linear fashion along the crypt axis. Normal colonic crypts are composed of stem cells at the base, a proliferation and a differentiation zone in the lower third of the crypt, a migration zone in the upper two-thirds, and the surface epithelium where senescent cells are eliminated by apoptosis. Globally, apoptosis can be defined as a normal process of cell suicide, critical for development and tissue homeostasis. Colonic epithelial cells migrate from the base of the crypt to the surface epithelium in 6-7 days. The normal architecture of the crypt is maintained by a balance between cell proliferation at the base and apoptosis at the top of the crypt and surface epithelium.     

Immunohistochemical analysis of cell kinetic parameters in colonic adenocarcinomas, adenomas, and normal mucosa

The monoclonal antibody Ki-67 identifies a nuclear antigen that is expressed in proliferating cells in G1, G2, S, and M phases of the cell cycle. An immunoperoxidase method and this antibody were used to identify proliferating cells in sections of colorectal tissues--normal colon (n = 10), colorectal polyps (n = 20), and adenocarcinoma (n = 28). Colorectal adenomas showed a uniform distribution of positive nuclear staining throughout the sections, including the cells of the adenoma surface, while staining in the normal mucosa was confined to the middle third and lower third of the crypts. Areas of polyps with numerous Ki-67-positive epithelial cells invariably showed immature or dysplastic histology and, conversely, glands that lacked such histologic features had low Ki-67 staining frequency or were negative. In adenomas, nuclei located toward the luminal surface of glands were more likely to be Ki-67-positive than those located basally in the cells. The mean Ki-67 score (a measure of positive staining nuclei) for adenomas was 45.5 compared to a mean score of 66.3 for adenocarcinomas in the carcinomas studied (P less than .001). Ki-67 score did not correlate with histologic grade or Duke's stage. Ki-67 staining can be used to characterize the proliferative characteristics of normal colonic mucosa, adenomas, and carcinomas.     

CD44 negatively regulates apoptosis in murine colonic epithelium via the mitochondrial pathway

Regulation of epithelial cell proliferation and apoptosis are important determinants of colonic crypt homeostasis, and their dysregulations are key features of colon cancer. In this study, we investigated whether CD44, an adhesion protein overexpressed in colon cancer, plays a role in colonocyte proliferation and apoptosis, and the molecular mechanisms involved in these processes. Using a CD44 knockout mouse model devoid of a gross phenotype, we found that CD44 null colonocytes have alterations at the ultrastructural and molecular levels. Mitochondria in CD44 null colonocytes at the top of the crypt have disrupted cristae. The ratio of anti-apoptotic Bcl-xl to pro-apoptotic Bak was shifted toward apoptosis in CD44 null colon due to decreased Bcl-xl expression. Caspase 9 was upregulated and active in CD44 null colon. Its expression shifted from a location restricted to the top of the control crypts to the whole crypt axis in CD44 null colon. Caspase 3 was also activated in CD44 null colon suggesting that CD44 null colonocytes are apoptotic via the intrinsic pathway. Cell cycle regulators, cyclin A, p21, and pRb protein were abrogated in CD44 null mice. Overall, CD44 negatively regulates apoptosis via the mitochondrial pathway in the colonic epithelium through the regulators/effectors of cell cycle and apoptosis.     

Localization of CEA messenger RNA by in situ hybridization in normal colonic mucosa and colorectal adenocarcinomas.

Carcinoembryonic antigen (CEA) mRNA expression was studied in 14 cases of normal colorectal mucosa and colonic adenocarcinomas using in situ hybridization with a 32P-labelled cDNA probe to the unique 3'-untranslated region of CEA. This approach has the advantage that the target mRNA remains in the cell of origin, whereas there is considerable ambiguity in immunocytochemistry data for CEA because the protein is secreted. Furthermore, the specific cDNA probe overcomes potential problems of immunological cross-reactivity with other members of the CEA family. The results demonstrated that abundant, heterogeneously distributed CEA mRNA was present in colorectal adenocarcinomas, with the highest levels in cells lining glandular structures. Parallel immunohistochemistry with anti-CEA monoclonal antibody A5B7 showed that the regions of tumours with the highest levels of CEA mRNA also had the highest CEA protein levels, suggesting that the heterogeneous distribution reflects CEA expression rather than differential secretion of the protein. In the normal colonic mucosa, CEA mRNA expression was observed in surface epithelial cells and goblet cells of the upper crypts, with very low hybridization in the mid crypt and at the base. This crypt-surface distribution was identical to that observed for CEA protein. In situ hybridization therefore confirms that high levels of CEA mRNA are expressed in differentiated surface epithelial cells of the normal colon.     

Autofluorescence characterisation of isolated whole crypts and primary cultured human epithelial cells from normal, hyperplastic, and adenomatous colonic mucosa

BACKGROUND/AIMS: In vivo autofluorescence endoscopic imaging and spectroscopy have been used to detect and differentiate benign (hyperplastic) and preneoplastic (adenomatous) colonic lesions. This fluorescence is composed of contributions from the epithelium, lamina propria, and submucosa. Because epithelial autofluorescence in normal and diseased tissues is poorly understood, this was the focus of the present study. METHODS: Whole colonic crypts were isolated, and short term primary cultures of epithelial cells were established from biopsies of normal, hyperplastic, and adenomatous colon. Autofluorescence (488 nm excitation) was examined by confocal fluorescence microscopy. Fluorescently labelled organelle probes and transmission electron microscopy were used to identify subcellular sources of fluorescence. RESULTS: Mitochondria and lysosomes were identified as the main intracellular fluorescent components in all cell types. Normal and hyperplastic epithelial cells were weakly autofluorescent and had similar numbers of mitochondria and lysosomes, whereas adenomatous (dysplastic) epithelial cells showed much higher autofluorescence, and numerous highly autofluorescent lysosomal (lipofuscin) granules. CONCLUSIONS: Short term primary cell cultures from endoscopic biopsies provide a novel model to understand differences in colonic tissue autofluorescence at the glandular (crypt) and cellular levels. The differences between normal, hyperplastic, and adenomatous epithelial cells are attributed in part to differences in the intrinsic numbers of mitochondria and lysosomes. This suggests that the detection of colonic epithelial fluorescence alone, if possible, may be sufficient to differentiate benign (hyperplastic) from preneoplastic and neoplastic (adenomatous) colonic intramucosal lesions during in vivo fluorescence endoscopy. Furthermore, highly orange/red autofluorescent intracellular granules found only in dysplastic epithelial cells may serve as a potential biomarker.     

Morphological changes and cellular proliferation in mouse colon during fetal and postnatal development

To document regional structural and cellular proliferation changes in the developing mouse colon, tissues from fetal, sukling, and weanling mice were analyzed by light microscopy (LM), transmission electron microscopy (TEM), scanning electron microscopy (SEM), [³H]-thymidine incorporation studies, and radioautography. The proximal and distal colon were studied independently at all ages. At 17–18 days of gestation, the mouse proximal colonic mucosa was projected into high and low longitudinal folds disposed in a V-shaped pattern. From birth up to 9 days, the mucosal folds observed by SEM can easily be misinterpreted as being a succession of high and low villus-like structures at LM level. TEM study confirmed the presence of highly specialized absorptive cells in the upper halves of the mucosal folds during this period. No recognizable crypts were noted at birth. Instead, LM and radioautography showed the presence of cell aggregates developing at the base of the epithelium at all levels of the mucosal folds. These cell aggregates evolved into rudimentary crypts giving fully differentiated crypts by day 16 with radiolabeled cells located in the midcrypt portion. As opposed to the proximal segment, a flat mucosa interspersed with well defined short crypts at birth was observed in the distal colon. During the following days, crypts further developed and by 16 days, the radiolabeled epithelial cells were still exclusively located at the base of the crypt. TEM observations illustrated that specialized cells as those found in the proximal segment did not differentiate in this segment. From birth up to 30 days, the labeling indices continuously decreased in the external muscle layer while increasing in the crypt epithelium at different time intervals in both colonic segments. The results show that true villus structures do not develop in proximal colonic mucosa and document regionally related morphological and cellular proliferation changes during mouse colonic maturation. © 1994 Wiley-Liss, Inc.     

The role of bile acids in colonic carcinogenesis

Summary Several lines of evidence suggest that bile acids may be implicated in the pathogenesis of colonic cancer. A high consumption of fat and animal protein and a low dietary intake of fiber have been shown to be related to the incidence of colonic cancer. From these epidemiologic observations the hypothesis was proposed that the correlation between diet and colon cancer might be explained by the involvement of bile acids. Populations at a high risk of developing cancer were shown to have an increased excretion both of total and bacterially modified bile acids in their feces. Animal studies demonstrated a cocarcinogenic effect of bile acids and experimental diets containing large amounts of fat did not only induce an increased bile acid excretion but also an enhanced tumor formation in the colon. Furthermore, microbial in vitro tests showed a comutagenic activity of secondary bile acids. However, case control studies comparing the fecal bile acid excretion pattern in colonic cancer patients and control subjects failed to show such a clear relationship, which might be explained by rather similar dietary habits within one population and individual differences in sensitivity to environmental factors contributing to the tumor development. Cholecystectomy, leading to an increased exposure of bile acids to the intestinal microflora, has been suggested as a predisposing factor for the development of colonic cancer, but the results of experimental and epidemiologic studies so far are rather inconsistent. More recent studies of epithelial cell kinetics showed that bile acids alter cell proliferative activity in the colonic mucosa by increasing the number of DNA synthesizing cells and expanding the proliferative compartment up to the middle third of the crypt. This enhanced cell proliferation seems to be related to the bile acid induced tumor formation due to an increased opportunity of malignant transformation. Bile acids may be implicated in the causation of human colonic cancer by stimulating the growth of a small-size benign adenoma to a large size with a correspondingly high risk of malignancy.     

Morphogenesis of colonic carcinoma. Ultrastructural studies of azoxymethane-induced early lesions in colon epithelium of Fischer 344 rats

Marked dilation and distortion of the crypts have been observed in the colonic epithelium of Fischer-344 rats after four to six weekly doses of azoxymethane (8 mg/kg). These changes precede malignant transformation in this model and, therefore, may be considered premalignant. Ultrastructural morphologic aspects of these crypts can arbitrarily be divided into three stages: an early stage of increased mucus secretion and "exhausted" appearance of mucous cells; an intermediate stage of severe crypt dilation, with extreme thinning of crypt epithelium and neutrophilic emigration to the mucous pool of the crypt lumen; and a late stage characterized by repopulation of the crypts, with epithelial cells showing an increased nuclear-cytoplasmic ratio and bizarre nuclei and nucleoli. The surface epithelium between the crypts shows increased cell loss and alterations of cell membrane. Similar structure of crypts has been described in the mucosa remote from carcinomas in rats and humans, and in various precancerous changes in humans (ie, ulcerative colitis and Crohn's disease). Therefore, I propose that these lesions are significant in human colon carcinogenesis as well.