Conjugated linoleic acids differentially alter polyp number and diameter in the Apc(min/+) mouse model of intestinal cancer.

OBJECTIVE: Dietary conjugated linoleic acids (CLA) have had many health benefits claimed for them, including antineoplastic actions. MATERIALS AND METHODS: The effects of the predominant forms of CLA, namely the c9t11 and t10c12 isomers, or a mixture of these on polyp development, were investigated in the Apc(Min/+) mouse. CLAs have also been linked to altered rates of cell renewal and cell proliferation so this was also studied, as was a further means of increasing tissue mass, namely crypt fission. RESULTS: The stomach and small intestine were significantly heavier in the t10c12, and in the mixture-treated groups (P < 0.001). Crypt fission was increased in the middle small intestine by the t10c12 diet while colonic weight was reduced by c9t11 provision and crypts were 20% shorter. The t10c12 and the mixture significantly reduced polyp number in the proximal small intestine but they increased polyp diameter in the middle and distal small intestine, to an extent that the polyp burden was significantly increased at these sites. All CLAs significantly reduced polyp number in the colon, but the mixture significantly increased polyp diameter in the colon. CONCLUSION: Increased polyp diameter associated with t10c12 diet and especially with the mixture is a cause of concern, as this is the commercially available form. The naturally occurring isomer, c9t11 decreased colonic polyp number and did not increase diameter, suggesting that this natural isomer is the most likely to be protective.

The development of duodenal microadenomas in FAP patients: the human correlate of the Min mouse.

UNLABELLED: The morphological changes associated with the adenoma-carcinoma sequence are well documented in the colorectum. Small intestinal carcinogenesis is thought to progress through a similar adenoma-to-carcinoma pathway, but there is a relative dearth of studies examining the associated morphological changes. The best-known mouse model of intestinal neoplasia, the multiple intestinal neoplasia (Min) mouse, has been criticized as a genetic model of intestinal neoplasia, as the majority of its tumours occur in the small intestine. We examined pancreatico-duodenal resection specimens from seven familial adenomatous polyposis (FAP) patients. Serial sections of these were stained with haematoxylin and eosin for beta-catenin and its downstream target CD44, for BMPR1a, lysozyme, carbonic anhydrase II, and with MIB-1. Individual dysplastic crypts were isolated and mutations in the FAP (APC) gene compared between the top and bottom of the crypt. We found that: (a) duodenal microadenomas are extremely common in FAP patients; (b) these grow in the core of duodenal villi, forming lesions similar to those described in the Min mouse; (c) many lesions arise as monocryptal adenomas and grow by a process of crypt fission and branching; (d) migrating adenomatous cells lose their dysplastic phenotype as they migrate up the crypt villous axis; and (e) Paneth cells lose positional information. IN CONCLUSION: (a) the morphological similarity of adenomas in the Min mouse and human suggest the Min mouse is a good model of FAP; (b) duodenal adenomas in FAP originate in monocryptal adenomas and follow the 'bottom-up' rather than the 'top-down' model of morphogenesis; (c) early microadenomas show evidence of cellular differentiation; (d) defects in the positioning of Paneth cells suggests disruption of the EphB2:EphB3 receptor system.

To best measure cell proliferation in samples from the intestine.

Arrangement of the intestinal cell lining, as it is, into distinct anatomically defined zones where proliferation is confined to the crypts, makes it an ideal tissue to study growth control mechanisms. While many methods have been used to quantify cell proliferation in the gut, several of them have severe limitations and others (although potentially better) have been misused and misinterpreted. Here, correct use and interpretation of labelling studies will be described as will a well established alternative method that provides equivalent results for one-sixth of the effort.

A simple device to rapidly prepare whole mounts of murine intestine.

Many mouse models of neoplasia and pre-neoplasia require the examination of whole mounts of the gastrointestinal tract. A simple device has been produced to facilitate the rapid preparation of mouse intestines for subsequent quantification of tumours and pre-neoplastic lesions such as aberrant crypt foci. The device greatly speeds up the production of whole mounts and also provides far more consistent and better-quality preparations.

Inhibiting vascular endothelial growth factor receptor-2 signaling reduces tumor burden in the ApcMin/+ mouse model of early intestinal cancer.

The Apc(Min/+) mouse model is a clinically relevant model of early intestinal cancer. We used AZD2171, an oral, highly potent and selective vascular endothelial growth factor (VEGF) signaling inhibitor, to investigate the role of VEGF receptor-2 (VEGFR-2) signaling in adenoma development and growth in Apc(Min/+) mice. AZD2171 (5 mg/kg body wt/day) was administered once daily for 28 days to 6-week-old (early-intervention) or 10-week-old (late intervention) mice. In the early-intervention study, AZD2171 reduced the number of macroscopic polyps in the small bowel and colon. Macropolyp diameter was lower in the small bowel, but remained unchanged in the colon. In animals receiving AZD2171, microscopic evaluation of the small intestine showed a significant reduction in the number of larger lesions. In the late-intervention study, AZD2171 treatment reduced macropolyp diameter (but not number) in the small intestine. Microscopic analysis revealed that AZD2171 significantly reduced the number of larger micropolyps in the small bowel, with no large micropolyps present in the colon. AZD2171 treatment had no effect on microvessel density or localization of beta-catenin staining in adenomas or non-tumor intestinal tissue, but significantly reduced the number of cells expressing VEGFR-2 mRNA. In conclusion, the effects of AZD2171 in the small intestine of Apc(Min/+) mice are consistent with an antiangiogenic mechanism of action, limiting growth of adenomas to < or =1 mm. These data also suggest that an early step in adenoma development may depend on VEGFR-2 signaling. Together, these results indicate that VEGFR-2 signaling may play key roles in the development and progression of intestinal adenomas.

Leptin, cell proliferation and crypt fission in the gastrointestinal tract of intravenously fed rats.

Many peptides, hormones and growth factors have been implicated in the control of cell renewal in the gastrointestinal epithelium. Leptin is present in the stomach and salivary glands and leptin receptors are seen throughout the gut. Leptin can stimulate mitogen-activated protein kinase activity in vitro and short-term infusion has been reported to have a proliferative action on the colon in vivo, suggesting a biological link between obesity, physical activity and colon cancer. Food intake is one of the most important determinants of intestinal mucosal cell renewal, thus any direct effects of leptin on the gut may be hidden. This problem has been avoided experimentally by maintaining animals on total parenteral nutrition (TPN). Male Wistar rats were anaesthetized and cannulae were inserted into the jugular vein to deliver the TPN diet to which had been added 0, 0.5, 2.5, or 10 mg/kg of recombinant murine leptin. Orally fed rats were also studied. After 6 days of treatment, all animals were injected with vincristine and killed 2 h later. Tissue weight was recorded and crypt cell proliferation (arrested metaphases) and crypt fission were scored in 'microdissected' crypts. Leptin infusion led to a small decrease in body weight and in the weight of the caecum. Intestinal cell proliferation was significantly reduced by TPN when compared to the orally fed rats, but the addition of leptin had no effect on the small intestine or colon. Crypt fission was also significantly lowered in the TPN group. Fission was slightly but significantly increased in the proximal and mid-colon of the leptin-treated rats, but was decreased in the distal colon. Although leptin did not significantly alter cell proliferation, it had significant effects on the process of crypt fission in the colon, which varied according to the exact locality.

Both suboptimal and elevated vitamin intake increase intestinal neoplasia and alter crypt fission in the ApcMin/+ mouse.

The effects of vitamin deficiency on intestinal cancer are unclear, and even less is known about the consequences of excessive intake. We therefore investigated the actions of altered vitamin content on intestinal polyp development, cell proliferation and crypt fission in a mouse model of neoplasia. Ninety multiple intestinal neoplasia (ApcMin/+) mice and 90 wild-type littermates, 4 weeks old, were divided into six groups and fed either a control semi-synthetic diet, or the semi-synthetic diet with the vitamin content lowered to a third of the RDA or the semi-synthetic diet with the vitamin content increased 5-fold (except for retinol and folate, which were doubled). The number and size of polyps in the small and large intestines were scored after 8 weeks on the diets, as was cell proliferation (native mitoses per crypt) and crypt fission. The small intestines of the low and high vitamin groups were heavier than the controls. There were significantly more polyps and the tumour burden was higher in both the low and the high vitamin groups (P < 0.02). Proliferation was slightly reduced by the vitamin alteration and crypt fission was significantly increased in the ApcMin/+ mice when compared with the wild-type (P < 0.001). Fission indices were decreased by vitamin alteration in the small intestine, and increased in the colon, but only in the ApcMin/+ mice. The effects of vitamin alteration on polyp number were most pronounced in the proximal intestine, which is also the site of maximum crypt fission. Both vitamin deficiency and over-supplementation can markedly enhance polyp number and tumour burden.

Effect of epidermal growth factor administration on intestinal cell proliferation, crypt fission and polyp formation in multiple intestinal neoplasia (Min) mice.

Recombinant epidermal growth factor (EGF) may be useful to treat severe ulcerative gastrointestinal injury. There is concern, however, that systemic use of this potent mitogen might increase tumour development and/or progression in susceptible subjects. We therefore examined the effect of chronic administration of systemic EGF to multiple intestinal neoplasia (Min ) mice, who have a genetic defect in the adenomatous polyposis coli (APC) gene, leading to increased polyp development. Min mice (n =26) and wild-type littermates (n =26) received saline or EGF (223 microg of EGF/kg per day) for 4 weeks using subcutaneous osmotic mini-pumps. Cell proliferation and crypt fission were analysed using microdissection techniques and the number and size of polyps in the small and large intestines were determined. EGF increased wet weight and crypt cell proliferation rate by approx. 20% (all P <0.01 compared with the relevant control) in the small intestine and colon of both control and Min mice. In both groups, EGF reduced the colonic fission index by approx. 40% (P <0.01), but did not affect crypt fission in the small intestine. In Min mice, administration of EGF did not increase numbers of polyps or degree of dysplasia, but resulted in a 40% increase in the polyp size in the proximal intestine (P <0.02), but not in the remainder of the small intestine or colon. No polyps were found in control mice given EGF. EGF did not initiate polyp formation in control or Min mice. However, as polyp size is an important determinant for subsequent risk of malignant change in human colon cancer, further studies appear justified.

Dietary lectins can stimulate pancreatic growth in the rat.

Lectins are proteins or glycoproteins of nonimmune origin, which bind specifically to carbohydrate structures. They are widespread in the human diet, and many are resistant to digestion. High doses of lectins have been shown to stimulate intestinal and pancreatic growth. The aim of the present study was to investigate the long-term actions of low doses of lectins on the rat intestine and pancreas. A long-term carcinogenesis study was performed using low levels (40 micro g/rat/day) of peanut (PNA) or mushroom lectin (ABA) which bind to O-linked (mucin-type) oligosaccharides in the gut. While this was primarily designed as a colon carcinogenesis study, the pancreas was also investigated. No significant changes in colon carcinogenesis were seen, however, the colons were slightly heavier in the lectin treated groups. The weight of the pancreas was significantly greater (by 18 and 23%) in both lectin treated groups (P < 0.03/0.001). The weights of the acini and septal tissue were also increased by 39-46% in PNA and ABA fed animals, respectively (P < 0.002); there was no significant change in the endocrine pancreas. In conclusion, long-term feeding of low doses of lectin can influence pancreatic growth, and this trophic action may have potential adverse implications for the development of pancreatic cancer in humans.

Lectins can reverse the distal intestinal atrophy associated with elemental diets in mice.

BACKGROUND: Elemental diets cause intestinal atrophy and reduced intestinal integrity, which can lead to significant increases in intestinal permeability and bacterial translocation. Recently, several lectins have been shown to have trophic effects on the intestine. AIMS: We examined the effects of concanavalin-A and phytohaemagglutinin on cell proliferation and crypt fission throughout the intestine of mice fed on elemental diets. METHODS: Mice were randomized to chow fed, elemental diet, elemental diet plus concanavalin-A and elemental diet plus phytohaemagglutinin groups. Cell proliferation and crypt fission were estimated in microdissected crypts. Plasma gastrin and enteroglucagon levels were measured by radioimmunoassay. RESULTS: Elemental diet feeding significantly decreased cell proliferation and crypt fission of the middle and distal small intestine and throughout the colon. Phytohaemagglutinin significantly increased the weight of the intestine, but concanavalin-A had little effect. Cell proliferation in the small intestine was significantly increased by both lectins. However, in the stomach and colon, only phytohaemagglutinin increased proliferation. Crypt fission in the colon was dramatically increased by phytohaemagglutinin. Phytohaemagglutinin increased the plasma gastrin level, but not the enteroglucagon level. CONCLUSIONS: Lectins have significant trophic effects on the small intestine and colon of mice fed elemental diets, and these actions vary between different sites in the gastrointestinal tract.

Histogenesis of human colorectal adenomas and hyperplastic polyps: the role of cell proliferation and crypt fission.

BACKGROUND: The histogenesis of human colorectal hyperplastic polyps and colorectal adenomas is poorly understood even now. METHOD: Human colorectal adenomas, hyperplastic polyps, and normal colorectal mucosae (patients with familial adenomatous polyposis and hereditary non-polyposis colorectal carcinoma were excluded) were obtained during colonoscopy and microdissected into individual crypts. Morphology, cell proliferation characteristics, and fission indices of crypts isolated from these lesions were then studied. RESULTS: Crypts isolated from colorectal adenomas and colorectal hyperplastic polyps were significantly larger (p<0.001) than crypts from normal colorectal mucosae. Crypt fission was an uncommon event in normal colonic mucosae but common in crypts isolated from adenomas and hyperplastic polyps (p<0.001). Analysis of the distribution of mitoses suggested an upward expansion of the proliferation compartment in adenomas to the surface of the crypt with no reversal of proliferating cell distribution, as has previously been described. CONCLUSIONS: Sporadic human colorectal adenomas and hyperplastic polyps grow by the process of crypt fission. Expansion of the proliferative compartment was demonstrated in crypts from adenomas, consistent with deregulation of cell cycle control.

Glicentin, an active enteroglucagon, has a significant trophic role on the small intestine but not on the colon in the rat.

BACKGROUND: Many experiments have indicated that the gut glucagons (enteroglucagons) are associated with cell proliferation in the small intestine. However, recent studies have failed to show trophic effects of glicentin (enteroglucagon) on the intestine. AIMS: To examine the effects of glicentin on intestinal proliferation in vivo in the rat. METHODS: Rats were established on total parenteral nutrition for 6 days. Four experimental groups were given daily doses of 1, 4, 20 and 80 microg/rat of glicentin via the jugular vein. Rats fed by total parenteral nutrition and rats fed chow ad libitum were used as controls. Tissues taken from the duodenum, jejunum, ileum and colon were fixed in Carnoy's fluid and microdissected to determine the metaphase arrest scores and crypt fission ratios. RESULTS: The mean metaphase arrest scores per crypt of the small intestine were significantly increased in the rats given 4, 20 and 80 microg of glicentin. These responses were dose-dependent, and were most prominent in the ileum. Crypt fission of the ileum was significantly decreased in the 20 and 80 microg glicentin groups. Glicentin had no effects on proliferation or fission in the colon. CONCLUSIONS: Glicentin is trophic to the rat small intestine, but not the colon.

Cell surface-expressed Thomsen-Friedenreich antigen in colon cancer is predominantly carried on high molecular weight splice variants of CD44.

Increased mucosal expression of TF, the Thomsen-Friedenreich oncofetal blood group antigen (galactose beta1-3 N-acetylgalactosamine alpha-) occurs in colon cancer and colitis. This allows binding of TF-specific lectins, such as peanut agglutinin (PNA), which is mitogenic to the colorectal epithelium. To identify the cell surface TF-expressing glycoprotein(s), HT29 and Caco2 colon cancer cells were surface-labeled with Na[(125)I] and subjected to PNA-agarose affinity purification and electrophoresis. Proteins, approximately 110-180 kDa, present in HT29 but not Caco2 were identified by Western blotting as high molecular weight splice variants of CD44 (CD44v). Selective removal of TF antigen by Streptococcus pneumoniae endo-alpha-N-acetylgalactosaminidase substantially reduced PNA binding to CD44v. Immunoprecipitated CD44v from HT29 cell extracts also expressed sialyl-Tn (sialyl 2-6 N-acetylgalactosaminealpha-). Incubation of PNA 15 microg/ml with HT29 cells caused no additional proliferative effect in the presence of anti-CD44v6 mAb. In colon cancer tissue extracts (N = 3) PNA bound to CD44v but not to standard CD44. These data show that CD44v is a major PNA-binding glycoprotein in colon cancer cells. Because CD44 high molecular weight splice variants are present in colon cancer and inflammatory bowel disease tissue but are absent from normal mucosa, these results may also explain the increased PNA reactivity in colon cancer and inflammatory bowel disease. The coexpression of oncofetal carbohydrate antigens TF and sialyl-Tn on CD44 splice variants provides a link between cancer-associated changes in glycosylation and CD44 splicing, both of which correlate with increased metastatic potential.

Proglucagon-derived peptides in intestinal epithelial proliferation: glucagon-like peptide-2 is a major mediator of intestinal epithelial proliferation in rats.

Proglucagon-derived peptides have been implicated in the control of intestinal mucosal cell division. To investigate the actions of these peptides on intestinal cell proliferation, different doses of enteroglucagon, oxyntomodulin, glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2) were tested in male Wistar rats maintained on total parenteral nutrition. Crypt cell proliferation was assessed by the analysis of arrested metaphases in microdissected crypts. Enteroglucagon and oxyntomodulin had no effect on intestinal weight or cell proliferation. GLP-1 had a slight effect on stomach and small intestinal weights and on epithelial cell proliferation in the small and large intestines. GLP-2 infusion dose-dependently increased the weights of the stomach, small intestine, colon, and cecum and increased crypt cell proliferation in the small and large intestines of parenterally fed rats. In orally fed animals, GLP-2 increased intestinal weight but had little effect on proliferation. Therefore, of the proglucagon-derived peptides, GLP-2 appears to be a major mediator of intestinal epithelial proliferation.

Comparison of the effects of concanavalin-A and epidermal growth factor on epithelial cell proliferation in the rat intestine.

BACKGROUND: Concanavalin-A, the lectin present in Jack beans, binds to mannose- and glucose-containing residues and can interact with the epidermal growth factor receptor and moderate cell proliferation in vitro. AIM: To compare the actions of concanavalin-A and epidermal growth factor on the gastrointestinal tract in vivo. METHODS: Rats maintained on total parenteral nutrition were given intragastric concanavalin-A, intravenous epidermal growth factor or concanavalin-A and epidermal growth factor. Cell proliferation and crypt fission were assayed in 'micro-dissected' crypts. RESULTS: Concanavalin-A and epidermal growth factor both significantly elevated proliferation in the small intestine and colon. No significant interaction between the effects of these two agents was seen, except in the mid small intestine where there was a synergistic interaction. Concanavalin-A had no effect on crypt branching. Epidermal growth factor significantly reduced branching in the distal small intestine and mid colon. CONCLUSION: The effects of the two agents appeared to be separate, except in the mid small intestine where they were additive. This is in marked contrast with the actions reported in vitro, where concanavalin-A is a powerful inhibitor of epidermal growth factor-induced cell proliferation. Concanavalin-A thus has potential for enhancing the functions of the small intestine.

Gastrointestinal cell proliferation and crypt fission are separate but complementary means of increasing tissue mass following infusion of epidermal growth factor in rats.

BACKGROUND AND AIMS: Epidermal growth factor (EGF) is a potent mitogen for the gastrointestinal tract and also influences the number of new crypts formed by crypt fission. The time course of these events and possible linkage between these two complementary mechanisms is however poorly understood. We therefore examined the temporal relationship of proliferation and fission in rats treated with EGF. METHODS: Osmotic minipumps were implanted subcutaneously into male Wistar rats to infuse EGF continuously (60 microg/rat/day) for periods of 1-14 days. Proliferation and crypt branching were quantified following vincristine induced metaphase arrest and morphometric assessment of microdissected tissue. RESULTS: In the small intestine, EGF significantly increased epithelial cell proliferation and crypt and villus area after 24 hours of EGF, although maximal effects were only reached following six days of infusion. EGF also resulted in an approximate 30% reduction in crypt fission in the small bowel. In the colon, EGF caused a twofold increase in epithelial cell proliferation one day after infusion, from 15.3 (2.3) to 29.6 (3.5) metaphases per crypt (p<0.01). Maximal effects were seen in rats receiving EGF for seven days. For all time points, colonic crypt size increased in response to EGF. The amount of branching increased following one day of infusion with EGF (from 15.3 (1.9) to 32.4 (5.5)%; p<0.001) but was significantly lower (approximately 25% of control values) following longer periods of infusion. Crypt fission did not correlate with crypt area. CONCLUSION: EGF has profound effects on cell proliferation and also altered crypt fission, with its actions on crypt fission most pronounced in the colon where it first increased and then decreased fission. EGF can thus be a potent stimulus for crypt fission during short term infusion and may reduce the number of branched crypts present in a resting or quiescent stage. Growth factors can alter cell mass by two separate but linked mechanisms, namely altered cell production and crypt fission.

Effect of ectopic expression of rat trefoil factor family 3 (intestinal trefoil factor) in the jejunum of transgenic mice.

To further examine the function of the trefoil factor family (TFF), the expression of which is up-regulated at sites of injury, we have produced transgenic mice that chronically express rat TFF3 within the jejunum (using a rat fatty acid-binding protein promoter). The expression of rat TFF3 was limited to the villi of the jejunum and had no effect on base-line morphology. Rat TFF3 expression did result, however, in a reduced sensitivity to indomethacin (85 mg/kg subcutaneously), which only caused a 29% reduction in villus height in transgenics versus 51% reduction in controls (p < 0.01). Indomethacin increased initial intestinal epithelial cell proliferation and migration, but the presence of rat TFF3 caused no additional change in proliferation (bromodeoxyuridine), cell migration ([(3)H]thymidine and bromodeoxyuridine), apoptosis (terminal deoxyuridine nucleotidyl nick end labeling), or E-cadherin immunostaining. In vitro studies following changes in resistance of intestinal strips in Ussing chambers (voltage-clamp technique) showed increased base-line resistance in the rat TFF3-expressing region (326 +/- 60 versus 195 +/- 48 ohm.cm(2) in controls, p < 0.05) and reduced the fall in resistance following HCl exposure by about 40% (p < 0.01). Overexpression of TFF3 stabilizes the mucosa against noxious agents, supporting its role in mucosal protection/repair. It may therefore provide a novel approach to the prevention and/or treatment of intestinal ulceration.