Epithelial cell proliferation and gene mutation in the mucosa of gallbladder with pancreaticobiliary malunion and cancer

The significant association between pancreaticobiliary malunion (PBM), especially undilated-type PBM, and a high risk of gallbladder cancer is known. Reflux and stasis of pancreatic juice induce various epithelial changes in the gallbladder. Recently, epithelial hyperplasia of the gallbladder was shown to be significantly and frequently associated with undilated-type PBM, and it is suggested that the majority of epithelial hyperplasia may exist at birth or be acquired in early childhood, and thereafter present throughout the lives of PBM patients. Cell kinetic studies demonstrated a significant stepwise increase in cellular proliferative activity from normal gallbladder mucosa, through epithelial hyperplasia to cancer. Epithelial hyperplasia with increased proliferative activity may predispose the mucosa to mutational events, thereby increasing cancer risk in PBM patients. K-ras mutations were frequently detected in gallbladder cancer in PBM patients and in epithelial hyperplasia as well. Epithelial hyperplasia is demonstrated to be an important premalignant lesion of gallbladder cancer. A multistep process of carcinogenesis as a consequence of multiple genetic alterations of oncogenes and tumor suppressor genes has been demonstrated in various organs; however, there is limited information on the molecular mechanism in gallbladder carcinogenesis with PBM. Recent findings support the idea that epithelial hyperplasia plays an important role in gallbladder carcinogenesis with PBM and also support the concept that neoplastic development in gallbladder with PBM also evolves through a multistep process associated with hyperproliferation and genetic alterations.     

Lysis of colonic epithelial cells by allogeneic mononuclear and lymphokine activated killer cells derived from peripheral blood and intestinal mucosa: evidence against a pathogenic role in inflammatory bowel disease.

A sensitive 4 h 51Cr-release cytotoxicity assay has been developed using as targets colonic epithelial cells obtained by Dispase-collagenase digestion of resected mucosa or colonoscopic biopsies. Peripheral blood mononuclear cells (MNC) from most healthy donors showed low, but significant levels of cytotoxicity for normal epithelial cell target cells of 8.7 (4.4) % (mean (SD] and similar levels were found in 14 ulcerative colitis (6.5 (4.4) %) and 16 Crohn's disease (6.2 (5.2) %) patients. Neither drug therapy nor disease activity influenced the results. The sensitivity of colonic epithelial cells isolated from inflamed and histologically normal mucosa to lysis by peripheral blood MNC from a single donor was not affected by the underlying disease. Anti-epithelial cell activity did not correlate with anti-K562 activity and the cytotoxic cell was plastic non-adherent and Leu-11b-. None of 15 MNC populations isolated from mucosa of normal, tumour bearing, or chronically inflamed intestine exhibited significant lysis of colonic epithelial cells despite killing of K562 target cells in 10. Lymphokine activated killer (LAK) cells, generated by interleukin-2 stimulation in vitro of nine intestinal and seven peripheral blood MNC populations, exhibited high levels of lysis of K562 cells but, on every occasion, failed to lyse colonic epithelial cells. These data indicate that spontaneously cytotoxic or LAK cells are unlikely to play a role in the generation of colonic epithelial cell injury by direct cytotoxicity in inflammatory bowel disease.     

Alterations in protein kinase C system of colonic epithelium during fasting-refeeding

In the present study, we compared (1) incorporation of [³H]dThd into DNA, (2) total protein kinase C (PKC) activity, (3) the subcellular distribution of PKC, and (4) PKC isozyme (, and ) mass in colonic mucosal scrapings and isolated superficial and proliferative colonic epithelial cells from 48-hr fasted, 48-hr fasted-refed, and adlibitum-fed rats. Total colonic mucosal PKC activity and PKC mass were higher and thein vivo rate of [³H]dThd incorporation into mucosal DNA was markedly depressed in 48-hr fasted rats compared toad libitum-fed or fasted-refed rats. These alterations were localized predominantly to the proliferative pool of colonic epithelial cells. Despite an 11-fold increase in mucosal DNA synthesis, no alterations in total mucosal PKC activity were detected in fasted-refed rats compared to rats fedad libitum. Moreover, no differences in the subcellular distribution of PKC were noted among any of the dietary groups. Intrarectal instillation of deoxycholate activated PKC and increased DNA synthesis 1.5-to 2-fold. Deoxycholate-induced increases in DNA synthesis, but not those induced by refeeding, were inhibited by treatment of rats with the PKC inhibitors H-7, sphingosine, or staurospaurine. The results do not support a role for PKC in the mediation of increased proliferative activity of colonic mucosa induced by refeeding.     

Abnormal cell proliferation and p52/p35-CSK expression in the colons of aging rats

In rodents and in humans, aging is associated with increased gastrointestinal epithelial cell proliferation and an expanded crypt proliferative compartment similar to that seen in the preneoplastic bowel. We have compared the distribution of a series of cytoskeletal antigens that are modified when colonic cancer cells differentiate in vitro in the colon of young (4–7 month) and aging (22–26 month) Fischer 344 rats. Two such proteins, p52 and p35, (that are increased in cultured senescent cells) differ in their position in the crypt axis and subcellular localization between young and aging rats. In young rats, immunoreactive p52 protein is present solely near the colonic crypt surface epithelium but in aging rats p52 expressing cells are found deeper in crypts. The intracellular localization of p35 also differs markedly in young and aging animals. The distribution of these proteins appears to be a reproducible biomarker of aging. Antigenic changes similar to those observed in aging colons also are seen in crypt cells of patients with ulcerative colitis and in the flat colonic mucosa of patients with adenomatous polyps and colon cancer. The combination of proliferative and differentiation changes suggest that the flat mucosa of the colon of aging rats has preneoplastic features.     

Increase of mitotic activity in the colonic mucosa of patients with colorectal cancer

A histologic and histochemical study of the colonic mucosa, including a study of the mitotic index, was performed in routinely processed specimens from control and tumor-bearing patients. A significant increase in the mitotic index (number of mitosis×1000 gland cells), without concomitant modifications in the distribution of mitotic figures along the crypt depth, in mucosal thickness, or in mucin secretion, was demonstrated in the colonic mucosa of patients with colonic or rectal cancer compared with controls. The results point to an accelerated cell renewal in the colonic mucosa of tumor-bearing patients compared with the controls, without concomitant dysplasia. Results are discussed in the light of the possibility that an increased cell proliferation may have preceded the onset of tumor and played a role in the second step of carcinogenesis,i.e., tumor promotion, independently of dysplasia.     

Increased expression of urokinase-type plasminogen activator in colorectal carcinoma and in adenomatous polyps

Human plasminogen activators (HPA) comprise the tissue type produced mainly by endothelial cells of 66 000 molecular weight (MW) which is principally involved in fibrinolysis (HPA66) and the urokinase type of 52 000 MW (HPA52) which is implicated in the invasion process of malignancy. The purpose of this study was to elucidate the pattern of HPA expression in histologically normal colonic mucosa, sporadic polyps, polyposis coli polyps, and in colon cancer tissue, to determine whether the expression of HPA52 is a correlate of neoplastic transformation of colonic epithelial cells. Homogenates of colonoscopic biopsies and resected colon tissue were subjected to polyacrylamide gel electrophoresis and the HPA activity was detected by a fibrin overlay gel. In histologically normal mucosal biopsies from non-cancer-bearing colons and in uninvolved mucosa from cancer-bearing colons, only HPA66 was detected. By contrast, all 19 colon cancer specimens expressed HPA52 and 16 of these also showed HPA66 activity. Two of three colon cancer cell lines showed HPA52 activity, but none expressed HPA66. HPA52 activity was observed in 17 of 20 adenomatous polyps, all of which displayed HPA66 activity. No correlation was found between polyp size, degree of epithelial dysplasia or the type of polyp architecture, and the semiquantitative estimates of HPA52 activity as judged by the areas of fibrinolysis generated.
This study of HPA52 in the colon epithelial neoplasms comprising adenomatous polyps, colon cancer tissue and colon cancer cell lines suggests that the transformation of the colon epithelial cell correlates with increased expression of HPA52, an enzyme that has been implicated in the invasive process of malignancy.     

Leptin reduces the development of the initial precancerous lesions induced by azoxymethane in the rat colonic mucosa

BACKGROUND & AIMS: Recent studies suggest that leptin, a hormone involved in food intake regulation, released into the circulation and gastrointestinal juice, may be a growth factor for intestine and may be involved in carcinogenesis; however, data are contradictory. This study investigates in rat colonic mucosa (1) the effects of hyperleptinemia on epithelial cell proliferation and development of aberrant crypts, earliest preneoplastic lesions, and (2) whether luminal leptin affects cell proliferation. METHODS: Leptin (1 mg/kg/d) or vehicle was administered systemically by miniosmotic pump in Fischer 344 rats either for 7 days (BrdU-labeling indices study) or 23 days (azoxymethane-induced colonic lesions study). The effects of injections or continuous infusion of leptin into the colon were also studied. RESULTS: In systemic leptin-treated rats, plasma leptin levels were 4- to 5-fold increased (P < 0.008 to P < 0.001); labeling indices were higher in proximal colon than in pair-fed control rats (P = 0.006) but unaffected in distal colon. Unexpectedly, in azoxymethane-treated rats, leptin significantly inhibited aberrant crypt foci formation in the middle and distal colon compared with controls (P = 0.006). Under these conditions, plasma insulin levels were reduced by 41%-58%, but gastrin levels were unchanged. In controls, luminal immunoreactive leptin reached the colon. A 3.6-fold increase in intraluminal leptin had no effect on epithelial cell proliferation. CONCLUSIONS: This study provides the first evidence that leptin reduces the development of chemically induced precancerous lesions in colon, perhaps through decreased insulinemia, and thus does not support an important role for leptin in carcinogenesis promotion. Moreover, the study indicates that leptin is not a potent growth factor for normal intestine.     

Increased proliferation and apoptosis of colonic epithelial cells in dextran sulfate sodium-induced colitis in rats

We have evaluated morphologic alterations and epithelial cell apoptosis and proliferation of colonic mucosa in the acute and chronic phases of DSS-induced colitis. Colitis was induced in Sprague-Dawley rats by 7 days of 4% DSS oral administration followed by 7 days of tap water for one, two, and three cycles. Control rats receved tap water only. Morphological changes in colonic mucosa were evaluated and scored by light and scanning electron microscopy. Apoptosis was studied by TUNEL assay and cell proliferation by Ki-67 immunoreaction. The expression of both proapoptotic (Fas, FasL, Bax, p53) and antiapoptotic (Bcl2) cellular proteins was determined by immunohistochemistry. Morphologic assessment showed the most severe colonic epithelial lesions and inflammation in the distal colon with a trend to increasing severity from the first to the third DSS cycle. In DSS rats, the epithelial apoptotic index increased 20-fold after the first cycle and 120-fold after the second and third cycles compared with the controls; in the same way, the expression index of proapoptotic proteins (Fas, FasL, Bax, p53) dramatically increased. The proliferative index increased about 40 to 60-fold compared to controls, with no difference among the three DSS cycles. In conclusion, DSS-induced colitis in rats, which has many structural and ultrastructural features similar to those seen in human ulcerative colitis, is a suitable model for studying increased epithelial apoptosis and proliferation. Further studies employing this model will permitt two hypotheses to be tested. (1) Increased apoptosis may lead to a breakdown of the epithelial barrier function and facilitate the mucosal invasion of intraluminal microorganisms and/or antigens. (2) Abnormal and persistent epithelial hyperproliferation could be causally related to the development of colorectal cancers in the setting of chronic colonic inflammation.     

Physiological and pathological role of local and immigrating colonic stem cells

The latest avenue of research is revealing the existence of and role for the colonic stem cells in the physiological renewal of the mucosa and in pathological circumstances where they have both positive and negative effects. In the case of human colon, different levels of stem cell compartments exist. First, the crypt epithelial stem cells, which have a role in the normal crypt epithelial cell dynamics and in colorectal carcinogenesis. Close to the crypts, the second layer of stem cells can be found; the local subepithelial stem cell niche, including the pericryptic subepithelial myofibroblasts that regulate the epithelial cell differentiation and have a crucial role in cancer progression and chronic inflammation-related fibrosis. The third level of stem cell compartment is the immigrating bone-marrow-derived stem cells, which have an important role in wound healing after severe mucosal inflammation, but are also involved in cancer invasion. This paper focuses on stem cell biology in the context of physiological and pathological processes in the human colon.     

Transforming growth factor-alpha in vivo stimulates epithelial cell proliferation in digestive tissues of suckling rats.

BACKGROUND: The role that exogenous transforming growth factor-alpha (TGF-alpha) may exert on cell proliferation in vivo is poorly understood. AIM: To investigate the effect of rat TGF-alpha on epithelial cell proliferation in all suckling rat digestive tissues and to compare it with that of rat epidermal growth factor (EGF). ANIMAL AND METHODS: TGF-alpha and EGF were given three times daily either subcutaneously (10 or 20 micrograms/kg) or intraperitoneally (100 micrograms/kg) to rats from the ninth postnatal day. Cell proliferation was assessed through 5-bromo- 2-deoxyuridine incorporation and estimation of labelling indices. RESULTS: For both growth factors, the highest dose given for only two days significantly increased stomach and intestinal weights compared with controls (p < 0.05 to p < 0.001). The proliferative responded depended on the dose given, colonic mucosa being the most sensitive whereas oxyntic mucosa remained unresponsive. TGF-alpha was as potent as EGF in stimulating epithelial cell proliferation in antral, duodenal, and colonic mucosae. However, EGF was more active on oesophageal and jejunal cell proliferation whereas TGF-alpha was more active on pancreatic exocrine cell proliferation and the differences between the two growth factor treated groups were significant. CONCLUSIONS: These results prove for the first time the stimulating effect in vivo of exogenous rat TGF-alpha on epithelial cell proliferation in rat digestive tissues during the developmental period and support a functional role for TGF-alpha at that time.     

Epithelial cell turnover and apoptosis

The homeostasis of gastric epithelial cells is maintained by the balance between cell proliferation and apoptosis. Alterations of these physiological cellular events in chronic pathological conditions of the stomach. As far as the proliferative pattern is concerned, an increase in the total number of epithelial proliferating cells and an abnormal distribution of the latter are frequently observed in chronic gastritis, gastric atrophy, intestinal metaplasia, gastric dysplasia and gastric cancer. Conversely, apoptosis has been found to be impaired in intestinal metaplasia, gastric dysplasia and cancer. Helicobacter pylori infection is associated with changes in epithelial-cell turnover, though their significance in gastric carcinogenesis is still controversial. An increase in overall epithelial cell proliferation and the upward shift of replicating cells toward the superficial part of the gastric pits are patterns usually observed during Helicobacter pylori infection and these changes can be reversed by successful eradication of the infection. However, it seems that this reversibility will be lost during progression through the steps of gastric carcinogenesis, such as intestinal metaplasia, probably representing the phenotypic expression of the true initiating phase of the carcinogenetic process. The influence of Helicobacter pylori infection on gastric epithelial apoptosis in humans is still controversial, since different results having been obtained by different authors. It seems that cagA status influences the effect of Helicobacter pylori on epithelial apoptosis, so that a different cagA make-up of the studied groups could explain these conflicting results. However, further studies are needed to elucidate this issue in humans.     

Cell kinetics analysis of background colonic mucosa of patients with intestinal neoplasms by ex vivo autoradiography.

Studies of the cell kinetics of the colonic crypts could explain the morphogenesis of colonic adenomas and results suggest that a derangement of the proliferative zone of the colonic crypts takes place before adenoma development. This study was conducted to determine whether this is the case or not. The labelling distribution and labelling index (LI) of the colonic crypts was examined with ex vivo autoradiography. Eight patients with intestinal neoplasms, three with familial polyposis coli, four with ordinary colon cancer, and one with two colonic adenomas. Similar labelling distributions and absent upward shift of the active proliferative zone in crypts were observed in patients with familial polyposis and in those with non-polyposis cancer, or adenomas. The non-proliferative zone of the crypts was well preserved in all eight patients. The mean of the labelling index of the three patients with familial polyposis coli was 8.35% (3.17), values expressed as means (SD) and that of the five patients with non-polyposis cancer, or adenomas was 8.55% (3.20). None of the eight patients showed derangement of the proliferative zone of colonic crypts. This result is compatible with the hypothesis that adenomas are detected first as 'buds of adenomas', which sprout into the lamina propria mucosae in the middle part of normal crypts.     

Increased microvascular blood content is an early event in colon carcinogenesis

BACKGROUND: Increased premalignant epithelial microvascular blood content is a common theme in neoplastic transformation; however, demonstration of this phenomenon in colon carcinogenesis has been stymied by methodological limitations. Our group has recently developed a novel optics technology, four dimensional elastic light scattering fingerprinting (4D-ELF), which allows examination of the colonic mucosal architecture with unprecedented accuracy. In this study, we utilised 4D-ELF to probe the preneoplastic colonic microvasculature. METHODS: Colonic mucosal blood content was assessed by 4D-ELF at serial preneoplastic time points from azoxymethane (AOM) treated Fisher 344 rats and age matched control animals. We also examined the pretumorigenic intestinal mucosa of the MIN mouse, and compared with wild-type mice. Finally, in a pilot study, we examined superficial blood content from the endoscopically normal mid transverse colon in 37 patients undergoing screening colonoscopy. RESULTS: In the AOM treated rat model, augmentation of superficial mucosal and total mucosal/superficial submucosal blood supply preceded the appearance of aberrant crypt foci (ACF) and temporally and spatially correlated with future ACF occurrence. These findings were replicated in MIN mice. The 4D-ELF based results were corroborated with immunoblot analysis for haemoglobin on mucosal scrapings from AOM treated rats. Moreover, 4D-ELF analysis of normal human colonic mucosa indicated that there was a threefold increase in superficial blood in patients who harboured advanced adenomas. CONCLUSION: We report, for the first time, that blood content is increased in the colonic microvasculature at the earliest stages of colon carcinogenesis. These findings may provide novel insights into early biological events in colorectal carcinogenesis and have potential applicability for screening.     

Colonic mucosal atrophy induced by a liquid elemental diet in rats

The weight, the total crypt cell population, and the proliferation parameters of the colon were estimated in rats during a 4-week administration of a liquid elemental diet (Vivonex^® standard). Both mitotic and DNA synthesis activity were decreased (P<0.01) in the colonic mucosa during the administration of the diet. The weight of the colon was electively decreased (P<0.01) from the first week of the treatment. After four weeks, a 75% decrease in the cell population of mucosal glands was observed. This showed that considerable atrophy of the colonic mucosa occurred under the effect of feeding the elemental diet. This atrophy was probably mediated by a reduction in the proliferative activity of the stem cells in the mucosal glands.     

Frequent expression of mucin core protein MUC1 in non-neoplastic gallbladder mucosa from patients with pancreaticobiliary maljunction

Abstract: Background: Gallbladder carcinoma is known to develop frequently in patients with pancreaticobiliary maljunction, though the causal relationship remains speculative. Methods: Histopathologic changes, expression of mucin core protein MUC1 and MUC2, and cell proliferative activities in the gallbladder mucosa from 27 patients with panceaticobiliary maljunction and 21 control gallbladders were examined. Three cases of pancreaticobiliary maljunction were associated with gallbladder carcinoma. Results: The lining epithelia of the non-neoplastic gallbladder mucosa of pancreaticobiliary maljunction showed frequently papillary hyperplasia and higher proliferative activities, when compared to the control. In 3 cases with carcinoma, MUC1 was expressed on the luminal border and in the cytoplasm of carcinoma cells, particularly in de-differentiated and invasive areas. MUC1 was variably expressed on the luminal surface of the lining epithelia of non-neoplastic gallbladder mucosa in babies, children, youths and adults with pancreaticobiliary maljunction. However, such expression was focally seen in 2 of the 21 control cases (p<0.01). MUC2 was scattered in the hyperplastic and carcinomatous epithelial cells appearing as goblet cells in pancreaticobiliary maljunction and control groups. Conclusions: This study suggests that persistent MUC1 expression and increased cell proliferative activities of non-neoplastic gallbladder epithelium of the patients with pancreaticobiliary maljunction after birth reflect an altered phenotype of epithelial cells and these abnormalities may be related to carcinogenesis in such patients.     

Effect of long-term intraluminal perfusion of pentagastrin on rat small-intestinal mucosa

Mucosal changes occurring during long-term intraluminal perfusion of pentagastrin in the duodenum of conscious adult rats (100 micrograms/24 h/kg, 6 days) were studied. Significant increases of labelling and of mitotic indices were noted in the whole small intestine, with an enlargement of the crypt epithelial proliferative compartment. Differential kinetic variations were observed between upper and lower parts of the small intestine when labelling indices were measured in accordance with the cell position in the crypts. Kinetic variations were correlated to the increases of villous and microvillous area. Alkaline phosphatase and leucine-aminopeptidase-specific activities were significantly increased, suggesting modifications of synthesis and/or maturation of these enzymes. The ileal Paneth cell number was also significantly increased in the ileum. Pharmacologic doses of pentagastrin intraluminally perfused have a trophic effect at all levels of the small-intestinal mucosa.     

Crypt alterations and collagen deposition in hyperplastic polyps of colorectum

In order to investigate the nature of hyperplastic polyps in the colorectum, 44 longitudinally sectioned crypts from biopsied polyps were analyzed morphometrically and compared with 81 control crypts. Although the crypts in hyperplastic polyps were longer and wider, containing more cells, their cell density was less, particularly in the serrated epithelium. In these crypts, both the tall and short epithelial cells contained cytoplasmic vacuoles, even in the surface epithelium. These cells exhibited increased expression of carcinoembryonic antigen. The subepithelial collagen table was of similar thickness in the polyp and control colonic mucosa, but it extended down along the cryptal wall to a greater depth in the polyp. These and other data indicate an aberrant differentiation of cryptal epithelial cells in the polyp. On upward migration to the surface, these cells appeared to undergo an arrested maturational process. Hence, the hyperplastic polyp may be considered a disease of epithelial cell differentiation.     

Colonic cell proliferation in two different ethnic groups with contrasting incidence of colon cancer: is there a difference in carcinogenesis?

Most studies on colorectal carcinogenesis suggest a field defect, preceding overt development of cancer. The low incidence of adenomatous polyps in the African population, however, suggests that there may be an alternative route for cancer development. The aim of the study was to discover if the difference in incidence of colorectal cancer in Africans compared with the white population is reflected in a different pattern of cell proliferation. Histological normal mucosa from 30 patients (15 white South African (W), 15 South African Africans (A)) with confirmed colon cancer were examined. Proliferating cells were detected using the Ki-67 antigen. In addition, cell proliferation data were obtained, from 30 age matched controls (15 Africans, 15 white South Africans), without colorectal disease. The African controls were significantly younger (mean (SD) (A: 42 (20), W: 66 (13), p < 0.05)) than the white controls. The second control group had a significantly higher mean (SD) total labelling index (W: 11 (3), A: 6 (4), p < 0.05). In addition the proliferative pattern of the white group without evidence of colorectal cancer showed a comparatively large amount of dividing cells in compartment 2, compared with African controls (mean (SD) (W: 21 (8), A: 9 (8), p < 0.05)). Mucosa from Africans with cancer showed a proliferative pattern with the same increased total labelling index (A: 15 (5), W: 16 (6), p = NS, phase II proliferative lesion) and an even more pronounced upward expansion (phase I proliferative lesion) compared with white cancer patients. This suggests that the mechanism of colorectal carcinogenesis is similar in Africans and the white population. The lack of clinical evidence of the adenoma-carcinoma sequence, and the incidence of cancer at a comparatively young age in Africans may be explained by the fact that colorectal cancer in this ethnic group behaves more aggressively and that adenomatous polyps are rapidly converted into overt cancer before detection.