Epidermal growth factor attenuates tubular necrosis following mercuric chloride damage by regeneration of indigenous, not bone marrow-derived cells.

To assess effects of epidermal growth factor (EGF) and pegylated granulocyte colony-stimulating factor (P-GCSF; pegfilgrastim) administration on the cellular origin of renal tubular epithelium regenerating after acute kidney injury initiated by mercuric chloride (HgCl2 ). Female mice were irradiated and male whole bone marrow (BM) was transplanted into them. Six weeks later recipient mice were assigned to one of eight groups: control, P-GCSF+, EGF+, P-GCSF+EGF+, HgCl2 , HgCl2 +P-GCSF+, HgCl2 +EGF+ and HgCl2 +P-GCSF+EGF+. Following HgCl2 , injection tubular injury scores increased and serum urea nitrogen levels reached uraemia after 3 days, but EGF-treated groups were resistant to this acute kidney injury. A four-in-one analytical technique for identification of cellular origin, tubular phenotype, basement membrane and S-phase status revealed that BM contributed 1% of proximal tubular epithelium in undamaged kidneys and 3% after HgCl2 damage, with no effects of exogenous EGF or P-GCSF. Only 0.5% proximal tubular cells were seen in S-phase in the undamaged group kidneys; this increased to 7-8% after HgCl2 damage and to 15% after addition of EGF. Most of the regenerating tubular epithelium originated from the indigenous pool. BM contributed up to 6.6% of the proximal tubular cells in S-phase after HgCl2 damage, but only to 3.3% after additional EGF. EGF administration attenuated tubular necrosis following HgCl2 damage, and the major cause of this protective effect was division of indigenous cells, whereas BM-derived cells were less responsive. P-GCSF did not influence damage or regeneration.

Gut hypertrophy after gastric bypass is associated with increased glucagon-like peptide 2 and intestinal crypt cell proliferation.

OBJECTIVE: We aimed to determine changes in crypt cell proliferation and glucagon-like peptide-2 (GLP-2) in rodents and man after Roux-en-Y gastric bypass (RYGB). SUMMARY OF BACKGROUND DATA: Roux-en-Y gastric bypass results in sustained weight loss and reduced appetite with only mild gastrointestinal side effects. Glucagon-like peptide-2 released from intestinal l-cells after nutrient intake stimulates intestinal crypt cell proliferation and mitigates the effects of gut injury. METHODS: Wistar rats underwent either RYGB (n = 6) or sham procedure (n = 6) and plasma GLP-2, GLP-1, and gut hormone peptide YY (PYY) were measured after 23 days. Biopsies from the terminal ileum were stained using the antibody to Ki67, which detects cyclins and hence demonstrates cells in the S-phase of the cell cycle. The total number of cells, number of mitosis, and number of labeled cells per crypt were counted. Obese patients (n = 6) undergoing RYGB were evaluated following a 420 kcal meal preoperatively, and 1, 3, 6, 12, and 24 months later for responses in l-cell products such as GLP-2, GLP-1, total PYY, and PYY3-36. RESULTS: Rat GLP-2 levels after RYGB were elevated 91% above sham animals (P = 0.02). At necropsy, mitotic rate (P < 0.001) and cells positive for the antibody Ki67 (P < 0.001) were increased, indicating crypt cell proliferation. Human GLP-2 after RYGB reached a peak at 6 months of 168% (P < 0.01) above preoperative values. Area under the curve for GLP-1 (P < 0.0001), total PYY (P < 0.01), and PYY3-36 (P < 0.05) responses increased progressively over 24 months. CONCLUSIONS: RYGB leads to increased GLP-2 and mucosal crypt cell proliferation. Other gut hormones from l-cells remain elevated for at least 2 years in humans. These findings may account for the restoration of the absorptive surface area of the gut, which limits malabsorption and contributes to the long-term weight loss after RYGB.

The Apc 1322T mouse develops severe polyposis associated with submaximal nuclear beta-catenin expression.

BACKGROUND & AIMS: We previously demonstrated that the 2 APC mutations in human colorectal tumors are coselected, because tumorigenesis requires an optimal level of Wnt signaling. We and others subsequently showed that the truncated APC proteins in colorectal tumors usually retain a total of 1-2 beta-catenin binding/degradation repeats (20AARs); very few intestinal tumors have proteins with no 20AARs. The coselection of the "2 hits" at APC makes it difficult to undertake further mechanistic studies in this area in humans. In mice, however, second hits appear to vary with the strain or genetic background used. This suggested the possibility of creating suboptimal Apc genotypes in the mouse. METHODS: We have constructed a mouse, Apc(1322T), with a mutant protein retaining one 20AAR. After repeated backcrossing to the C57BL/6J background, we compared the 1322T animals with the widely used Min mouse in which the mutant Apc protein has zero 20AARs. RESULTS: In both mice, intestinal adenomas showed copy-neutral loss of heterozygosity, making them homozygous for the mutant Apc allele. 1322T animals had markedly more severe polyposis, with earlier-onset, larger, more numerous, and more severely dysplastic adenomas. 1322T tumors also had more marked Paneth cell differentiation and higher frequencies of crypt fission. Somewhat surprisingly, nuclear beta-catenin expression was lower in 1322T than Min tumors. CONCLUSIONS: We propose that the Apc(1322T) mutation produces submaximal beta-catenin levels that promote early tumor growth more effectively than the Apc(Min) mutation.

Involvement of KLF4 in sulforaphane- and iberin-mediated induction of p21(waf1/cip1).

Sulforaphane (SF; 4-methylsulfinylbutyl isothiocyanate), a dietary compound derived from broccoli, may exhibit chemopreventive properties by inducing cell cycle arrest via induction of cyclin-dependent kinase inhibitor 1A (p21(waf1/cip1)), but the exact molecular mechanism has not been determined. Here we evaluate the role of the transcription factor Kruppel-like factor 4 (KLF4) in mediating the induction of p21(waf1/cip1) and cellular differentiation by SF and iberin (IB; 3-methylsulphinyl propyl isothiocyanate), also derived from broccoli. Exposure of Caco-2 and Caco-2/TC7 cells to SF and IB increased expression of both KLF4 and p21(waf1/cip1), whereas exposure of HT29 cells resulted only in induction of p21(waf1/cip1). In Caco-2 cells, small interfering RNA knock down of KLF4 expression attenuated induction of p21(waf1/cip1) in response to either SF or IB treatment. Contrary to expectation, prolonged exposure to SF reduced sucrase isomaltase activity, a marker of small intestinal differentiation in Caco-2 cells. Additional support for the SF-mediated induction of p21(waf1/cip1) by KLF4 was obtained from analyses of gastric tissue of Apc(Min/+) mice following acute intervention with SF but not from the analyses of other tissue of the intestinal tract. These results suggest that induction of p21(waf1/cip1) by SF or IB may be partly mediated by KLF4 in some colon cancer cells and tissues.

Dual inhibition of VEGFR and EGFR signaling reduces the incidence and size of intestinal adenomas in Apc(Min/+) mice.

Both the epidermal growth factor (EGF) and the vascular endothelial growth factor (VEGF) pathways are associated with intestinal cancer, and therapeutic approaches targeting either EGF receptor (EGFR) or VEGF receptor (VEGFR) signaling have recently been approved for patients with advanced colorectal cancer. The Apc(Min/+) mouse is a well-characterized in vivo model of intestinal tumorigenesis, and animals with this genetic mutation develop macroscopically detectable adenomas from approximately 6 weeks of age. Previous work in the Apc(Min/+) mouse has shown that therapeutic approaches targeting either VEGFR or EGFR signaling affect predominantly the size or number of adenomas, respectively. In this study, we have assessed the effect of inhibiting both these key pathways simultaneously using ZD6474 (Vandetanib, ZACTIMA), a selective inhibitor of VEGFR and EGFR tyrosine kinases. To assess the effects of ZD6474 on early- and later-stage disease, treatment was initiated in 6- and 10-week-old Apc(Min/+) mice for 28 days. ZD6474 markedly reduced both the number and the size of polyps when administered at either an early or a later stage of polyp development. This reduction in both adenoma number and size resulted in a total reduction in tumor burden in the small intestine of nearly 75% in both studies (P < 0.01). The current data build on the concept that EGFR-dependent tumor cell proliferation and VEGF/VEGFR2-dependent angiogenesis and survival are distinct key mechanisms in polyp development. Pharmacologic inhibition of both signaling pathways has significant antitumor effects at both early and late stages of polyp development. Therefore, targeting both VEGFR- and EGFR-dependent signaling may be a beneficial strategy in early intestinal cancer.

Increased colorectal epithelial cell proliferation and crypt fission associated with obesity and roux-en-Y gastric bypass.

BACKGROUND AND AIMS: The relationship between obesity, weight reduction, and future risk of colorectal cancer is not well understood. Therefore, we compared mucosal biomarkers in normal weight individuals [body mass index (BMI), 18.5-24.9 kg/m(2)] with those in morbidly obese patients (BMI >40 kg/m(2)) before and 6 months after Roux-en-Y gastric bypass (RYGB). METHODS: Rectal epithelial cell mitosis, crypt area, and crypt branching were measured following whole crypt microdissection. Apoptosis was measured by immunohistochemistry for neo-cytokeratin 18 on fixed tissue sections. Serum levels of C-reactive protein and cytokines were assayed in combination with quantification of mucosal proinflammatory gene expression by real-time RT-PCR. RESULTS: Twenty-six morbidly obese patients (mean BMI, 54.4 kg/m(2)) had significantly increased mitosis, crypt area, and crypt branching (all P < 0.01) compared with 21 age- and sex-matched normal weight individuals (mean BMI, 22.5 kg/m(2)). Morbidly obese patients underwent a mean excess weight loss of 41.7% at a mean of 26 weeks after RYGB. Surprisingly, this was associated with a further increase in mitosis and decreased apoptosis of epithelial cells. At the same time, lower levels of serum C-reactive protein and interleukin-6 following RYGB were accompanied by a reduction in mucosal IL-6 protein content but elevated mucosal expression of other proinflammatory genes such as cyclooxygenase-1 and cyclooxygenase-2. CONCLUSIONS: Mucosal biomarkers, accepted as indicators of future colorectal cancer risk, are increased in morbidly obese patients compared with normal weight controls. The hyperproliferative state that exists 6 months after RYGB may have important implications for long-term colorectal cancer risk in bariatric surgery patients.

Resistant carbohydrates stimulate cell proliferation and crypt fission in wild-type mice and in the Apc(Min/+) mouse model of intestinal cancer, association with enhanced polyp development.

Fermentation of carbohydrates in the colon can stimulate cell proliferation and could thus be a cancer risk. The effects of resistant carbohydrates, i.e. those not digested and absorbed in the small intestine, on cell proliferation, crypt fission and polyp development were investigated in wild-type and adenomatous polyposis coli multiple intestinal neoplasia (Apc(Min/+)) mice. Fifteen 4-week-old female wild-type and fifteen Apc(Min/+) mice were used for each group and fed a chow diet, a semi-synthetic diet or the semi-synthetic supplemented with wheat bran or an apple pomace preparation, both high in resistant carbohydrates, for 8 weeks. Tissue from all mice was used to measure cell proliferation and crypt fission and tissue from the Apc(Min/+) mice was scored for polyp number and tumour burden. There were slight reductions in intestinal mass in the mice fed the semi-synthetic diets and this was increased by the inclusion of resistant carbohydrates. The Apc(Min/+) mice had elevated cell proliferation and crypt fission in the distal small intestine and colon and these were increased by the resistant carbohydrates. Bran or apple pomace significantly increased polyp number in the proximal third of the small intestine. Apple pulp more than doubled polyp number throughout the small bowel (99.2 (SEM 11.1) v. 40.0 (SEM 8.2), P<0.004). Bran and apple pomace increased polyp diameter and hence burden in the colon by 243 and 150 %, respectively (P<0.05). In conclusion, both types of resistant carbohydrates increased polyp number and tumour burden and this was associated with elevated epithelial cell proliferation and crypt fission.

Specific protein supplementation using soya, casein or whey differentially affects regional gut growth and luminal growth factor bioactivity in rats; implications for the treatment of gut injury and stimulating repair.

Modulation of regional growth within specific segments of the bowel may have clinical value for several gastrointestinal conditions. We therefore examined the effects of different dietary protein sources on regional gut growth and luminal growth factor bioactivity as potential therapies. Rats were fed for 14 days on isonitrogenous and isocaloric diets comprising elemental diet (ED) alone (which is known to cause gut atrophy), ED supplemented with casein or whey or a soya protein-rich feed. Effects on regional gut growth and intraluminal growth factor activity were then determined. Despite calorie intake being similar in all groups, soya rich feed caused 20% extra total body weight gain. Stomach weight was highest on soya and casein diets. Soya enhanced diet caused greatest increase in small intestinal weight and preserved luminal growth factor activity at levels sufficient to increase proliferation in vitro. Regional small intestinal proliferation was highest in proximal segment in ED fed animals whereas distal small intestine proliferation was greater in soya fed animals. Colonic weight and proliferation throughout the colon was higher in animals receiving soya or whey supplemented feeds. We conclude that specific protein supplementation with either soya, casein or whey may be beneficial to rest or increase growth in different regions of the bowel through mechanisms that include differentially affecting luminal growth factor bioactivity. These results have implications for targeting specific regions of the bowel for conditions such as Crohn's disease and chemotherapy.

Quantification of epithelial cell proliferation, cell dynamics, and cell kinetics in vivo.

The measurement of cell proliferation in vivo is usually carried out by the examination of static measures. These comprise the mitotic index or labeling indices using incorporation of DNA synthesis markers such as bromodeoxyuridine or tritiated thymidine, or intrinsic markers, such as Ki67 and proliferative cell nuclear antigen (PCNA). But static measures only provide a 'snapshot' of cell proliferation. Rate measures, including double labeling methods and the metaphase arrest method, can actually measure cell production rates but they are far less utilized at present. Transit times and migration rates can also be measured using pulse and chase labeling or by following the transit of labeled cells through the tissue. Simple indices of cell division can easily be confounded by concomitant changes in the compartment size and many alleged markers of proliferation have serious shortcomings, as the markers may be involved in multiple aspects of cell regulation. The complexities of studying proliferation in vivo are illustrated here with a focus on the gastrointestinal tract. Some of these methods can help elucidate the role of the stem cells and their relationship to label retaining cells. WIREs Dev Biol 2017, 6:e274. doi: 10.1002/wdev.274 For further resources related to this article, please visit the WIREs website.

Bottom-up histogenesis of colorectal adenomas: origin in the monocryptal adenoma and initial expansion by crypt fission.

The adenoma:carcinoma sequence is well established. Understanding the molecular pathology of the adenoma is therefore important. There is great controversy within the field. The Vogelstein group champions the "top-down" theory (colorectal adenomas arise and grow across the mucosal surface and down into the crypts), whereas other studies, including our own, propose "bottom-up" spread. Serial sections of 40 small (<3 mm) sporadic colorectal adenomas were stained with H&E, MIB-1, and for beta-catenin. 10 early adenomas were Feulgen-stained and microdissected. We also examined the flat mucosa of three patients who had undergone colectomies for familial adenomatous polyposis (FAP) and specimens from a XO/XY individual with FAP, the latter using in situ hybridization for the Y chromosome. In the earliest sporadic adenomas, there were crypts entirely filled with adenomatous epithelium, which showed proliferative activity and nuclear localization of beta-catenin. There was a sharp cutoff between crypt epithelial cells showing nuclear beta-catenin and surface cells with membrane staining. In slightly larger lesions, adenomatous spread from above was seen. Microdissected adenomas showed multiple fission events, with proliferation distributed equally throughout. In FAP tissue, numerous isolated monocryptal adenomas, which were clonal in origin, were seen. Examination of adenomas in the XO/XY individual showed no instances of XY or XO adenomatous epithelium growing down into crypts of the other genotype. Both sporadic and FAP adenomas start as a unicryptal adenomas and grow initially by crypt fission--a bottom-up pattern. Later, in sporadic adenomas, there is evidence of growth down into adjacent crypts (top-down).

Myh deficiency enhances intestinal tumorigenesis in multiple intestinal neoplasia (ApcMin/+) mice.

Monoallelic APC and biallelic MYH (homolog of Escherichia coli mutY) germ-line mutations are independently associated with a strong predisposition to colorectal adenomas and carcinoma in humans. Whereas mice heterozygous for mutant Apc develop intestinal tumors, mice homozygous for mutant Myh do not show increased tumor susceptibility. We analyzed the phenotype of Apc(Min/+)/Myh(-/-) mice and found that they developed significantly more adenomas in the small intestine than did Apc(Min/+)/Myh(+/+) or Apc(Min/+)/Myh(+/-) mice (median 231 versus 151 versus 152). In the large bowel, Apc(Min/+)/Myh(-/-) mice showed significant increases in the number of aberrant crypt foci. In addition, Apc(Min/+)/Myh(-/-) mice developed an increased number of mammary tumors. Molecular analyses suggested that at least 19% of intestinal tumors from Apc(Min/+)/Myh(-/-) mice had acquired intragenic Apc mutations rather than allelic loss. Consistent with a defect in base excision repair, three intragenic Apc mutations in polyps without allelic loss from Apc(Min/+)/Myh(-/-) mice were shown to be G:C to T:A transversions which resulted in termination codons; no such mutations were found in polyps from Apc(Min/+)/Myh(+/+) or Apc(Min/+)/Myh(+/-) mice. Tumors from Apc(Min/+)/Myh(+/-) mice harbored neither somatic mutations nor allelic loss at Myh. Thus, homozygous, but not heterozygous, Myh deficiency enhanced intestinal tumorigenesis in Apc(Min/+) mice. The excess small-bowel adenomas in Apc(Min/+)/Myh(-/-) mice, therefore, appear to be a model of MYH-associated polyposis in humans.

Intestinal growth in parenterally-fed rats induced by the combined effects of glucagon-like peptide 2 and epidermal growth factor.

BACKGROUND: Parenteral nutrition and the absence of luminal feeding result in impaired intestinal growth and differentiation of enterocytes. Glucagon-like peptide 2 (GLP-2) and epidermal growth factor (EGF) have each been shown to have trophic effects on the intestine, and thus have the potential to benefit patients fed parenterally, such as those with intestinal failure from short bowel syndrome. We report studies aimed to determine whether there may be synergistic effects of these 2 peptides. METHODS: Rats were established on parenteral nutrition (PN) and infused for 6 days with GLP-2 (20 microg/d), EGF (20 microg/d), or GLP-2 + EGF (20 microg/d of each). These groups were compared with untreated PN-fed and orally-fed controls. Tissue was obtained from small intestine and colon to determine growth, proliferation, and representative gene expression. RESULTS: Small intestinal weight was increased by 75%, 43%, and 116% in the GLP-2, EGF, and GLP-2 + EGF groups, respectively, compared with PN controls (all p < .001). Cell proliferation increased with GLP-2, EGF, and GLP-2 + EGF in proximal small intestine by factors of 2.3, 1.7, and 3.4 respectively (p < .001). A synergistic effect on villous and crypt area was observed in the proximal small intestine when GLP-2 and EGF were combined (p < .05). GLP-2 had no effect in the colon, unlike EGF. Further studies showed GLP-2 + EGF significantly increased expression in distal small intestine of transcripts for the bile acid transport protein IBABP (p < .05) and showed a significant correlation between the expression of IBABP and the transcription factor HNF-4. CONCLUSIONS: Both GLP-2 and EGF upregulate growth of the small intestine, and this is augmented when GLP-2 and EGF are combined. These findings may lead to improved treatment of patients receiving PN.

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine.

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and 'roll' mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card, tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.

Inhibition of Aurora-B kinase activity confers antitumor efficacy in preclinical mouse models of early and advanced gastrointestinal neoplasia.

The Aurora family of kinases, play a fundamental role in cell division and are overexpressed in several cancers including colon. The activity of barasertib-hQPA, a selective inhibitor of Aurora-B kinase (ABK) was investigated in a range of preclinical models of gastrointestinal cancer. Treatment with barasertib-hQPA produced anti-proliferative and cytotoxic effects across a panel of human colorectal cancer (CRC) cell lines in vitro. Prodrug, barasertib [48-h subcutaneous (s.c.) infusion; 150 mg/kg/day] inhibited the growth of SW620, Colo205, HCT116 human colorectal tumor xenografts in nude mice significantly (Student's t-test, P<0.05, n=10-12 per group). Flow cytometric analysis of single cells from disaggregated barasertib-treated SW620 tumors revealed a decrease in phosphorylated histone H3 (phH3) and an increase in tumor cells with ≥4N DNA content P<0.05). The activity of barasertib was then examined in ApcMin/+ mice, a spontaneous model of early intestinal neoplasia. Macroscopic evaluation of the small intestine revealed that barasertib treatment [25 mg/kg intra-peritoneal (i.p.) Q1Dx4 each week for 3 weeks] of 8-week old ApcMin/+ mice produced a 39% reduction in macroadenoma number (P=0.02) and a 43% reduction in overall adenoma burden (P=0.02) compared with vehicle-treated controls. Quantification of microscopic adenomas revealed a >64% reduction in the number of adenomas spanning more than one villus. Histological analysis of these adenomas revealed a number of distinct changes in barasertib-treated ApcMin/+ mice, including a 94% reduction in the proportion of phospho-histone H3-positive cells (P<0.001) and a 53% reduction in the number of cells per adenoma (P=0.001). These results provide a scientific rationale for investigating ABK inhibitors as a treatment for intestinal cancer.

Effects of vandetanib on adenoma formation in a dextran sodium sulphate enhanced Apc(MIN/+) mouse model.

The Apc(MIN/+) mouse is a well-characterised model of intestinal tumourigenesis in which animals develop macroscopically detectable adenomas. However, most of the adenomas are formed in the small intestine and resolution of events in the colon, the most relevant site for human disease, is limited. Inducing colitis with dextran sodium sulphate (DSS) can selectively enhance the development of lesions in the colon. We demonstrated that a DSS pre-treatment is well tolerated and effective at inducing colon adenomas in an Apc(MIN/+) mouse model. We then investigated the effect of inhibiting vascular endothelial growth factor (VEGFR)- and epidermal growth factor receptor (EGFR)-dependent signalling pathways on the development of adenomas induced in DSS-pretreated (DSS/Apc(MIN/+)) or non-DSS-pretreated (Apc(MIN/+)) mice using vandetanib (ZD6474), a potent and selective inhibitor of VEGFR and EGFR tyrosine kinase activity. Eight-week old Apc(MIN/+) mice were given either drinking water or 1.8% DSS and then vandetanib (ZD6474) (50 mg/kg/day) or vehicle by oral gavage for 28 days and sacrificed 24 h after the last dose and assessed for adenoma formation in the intestines. DSS pre-treatment was well tolerated and significantly enhanced formation of adenomas in the colon of control Apc(MIN/+) mice. Vandetanib treatment significantly reduced adenoma formation in the small intestine by 68% (P=0.001) and the colon by 77% (from 13.8 to 3.1, P=0.01) of DSS-pretreated Apc(MIN/+) mice. In the Apc(MIN/+) group, vandetanib also reduced the mean number of adenomas in the small intestine by 76% (P<0.001) and in the colon by 60% (from 3.9 to 1.5, P=0.1). DSS-pre-treatment increased the resolution of the model, allowing us to confirm statistically significant effects of vandetanib on the development and growth of colon adenomas in the Apc(MIN/+) mouse. Moreover these preclinical data provide a rationale for studying the effects of vandetanib in early stages of intestinal cancer in the clinic.

Detection of metabolic alterations in non-tumor gastrointestinal tissue of the Apc(Min/+) mouse by (1)H MAS NMR spectroscopy.

In this study, we have used metabolic profiling (metabolomics/metabonomics) via high resolution magic angle spinning (HRMAS) and solution state (1)H NMR spectroscopy to characterize small bowel and colon tissue from the Apc(Min/+) mouse model of early gastrointestinal (GI) tumorigenesis. Multivariate analysis indicated the presence of metabolic differences between the morphologically normal/non-tumor tissue from approximately 10 week-old Apc(Min/+) mice and their wild-type litter mates. The metabolic profile of isolated lamina propria and epithelial cells from the same groups could also be discriminated on the basis of genotype. Accounting for systematic variation in individual metabolite levels across different anatomical regions of the lower GI tract, the metabolic phenotype of Apc(Min/+) lamina propria tissue was defined by significant increases in the phosphocholine/glycerophosphocholine ratio (PC/GPC, +21%) and decreases in GPC (-25%) and the gut-microbial cometabolite dimethylamine (DMA, -40%) relative to wild type. In the whole tissue, elevated lactate (+15%) and myo-inositol (+19%) levels were detected. As the metabolic changes occurred in non-tumor tissue from animals of very low tumor burden (<2 polyps/animal), they are likely to represent the specific consequence of reduced Apc function and very early events in tumorigenesis. The observed increase in PC/GPC ratio has been previously reported with immortalisation and malignant transformation of cells and is consistent with the role of Apc as a tumor suppressor. Phospholipase A2, which hydrolyses phosphatidylcholine to Acyl-GPC, is a known modifier gene of the model phenotype (Mom1), and altered expression of choline phospholipid enzymes has been reported in gut tissue from Apc(Min/+) mice. These results indicate the presence of a metabolic phenotype associated with "field cancerization", highlighting potential biomarkers for monitoring disease progression, for early evaluation of response to chemoprevention, and for predicting the severity of the polyposis phenotype in the Apc(Min/+) model.

Reduced susceptibility to azoxymethane-induced aberrant crypt foci formation and colon cancer in growth hormone deficient rats.

OBJECTIVES: To evaluate the role of GH in colon carcinogenesis, we examined the formation of aberrant crypt foci (ACFs) and tumor development in wild type (WT) and GH-deficient, spontaneous dwarf rats (SDRs) exposed to the carcinogen azoxymethane (AOM). DESIGN: ACF were quantified by stereomicroscopy and tumor number and weights were recorded for each animal. Cell proliferation was measured by vincristine metaphase arrest, flow cytometry, and bromodeoxyuridine (BrdU) incorporation. Apoptosis was measured by TUNEL staining and cleaved caspase-3 immunohistochemistry. IGF-I was measured by radioimmunoassay (RIA). Hexokinase activity was measured by spectrophotometric assay. PARP cleavage, and IGF-IR, and p27(kip/cip) expression were measured by Western blotting. RESULTS: ACFs detected by stereomicroscopy were markedly reduced ( approximately 85%) in SDRs vs. WT rats at 10, 25, and 28 weeks after AOM. Tumor incidence, number, and weight also were reduced in SDR vs. WT animals. AOM treatment increased cell proliferation in the distal colon (where tumors occur) of WT rats but not SDRs, and these changes corresponded to increased ACF and tumor formation. Apoptosis rates were similar in AOM-treated WT and SDRs. Alterations in serum IGF-I levels may contribute to differences in the proliferative response to AOM and decreased ACF formation in SDR vs. WT rats. CONCLUSIONS: We conclude that early neoplastic lesions (ACFs) were reduced in GH-deficient animals. This effect corresponds with differences in AOM-induced proliferation, but not apoptosis. These data indicate that GH is required for the full effect of AOM on colon ACF and tumor development, and that the SDR rat is a promising model for studies regarding the role of GH/IGF system in the initiation and promotion of colon cancer.

Ectopic expression of P-cadherin correlates with promoter hypomethylation early in colorectal carcinogenesis and enhanced intestinal crypt fission in vivo.

P-cadherin is normally expressed in the basal layer of squamous epithelia and absent from the healthy intestine and colon. We have previously shown it to be expressed in all inflamed, hyperplastic, and dysplastic intestinal and colonic mucosa. This study aimed to better understand the mechanisms controlling the expression of P-cadherin and the biological effects of its ectopic presence in the intestine and colon. We investigated the CpG methylation status of the P-cadherin (CDH3) promoter and P-cadherin mRNA and protein expression in cases of familial and sporadic colorectal cancer (CRC). The CDH3 promoter was hypomethylated in colonic aberrant crypt foci, in CRC, and, occasionally, in the normal epithelium adjacent to cancer, demonstrating a potential "field effect" of cancerization. The hypomethylation was also associated with induction of P-cadherin expression in the neoplastic colon (P < 0.0001). We then created transgenic mice that overexpressed P-cadherin specifically in the intestinal and colonic epithelium under the liver fatty acid binding protein promoter. Forced ectopic expression of P-cadherin accompanied by indomethacin-induced inflammation resulted in a 3-fold higher crypt fission rate within the small and large intestines in the homozygous mice compared with the wild-type animals (P < 0.02). We conclude that epigenetic demethylation of the P-cadherin promoter in the human intestine permits its ectopic expression very early in the colorectal adenoma-carcinoma sequence and persists during invasive cancer. Induced P-cadherin expression, especially in mucosal damage, leads to an increased rate of crypt fission, a common feature of clonal expansion in gastrointestinal dysplasia.

Human pancreatic secretory trypsin inhibitor stabilizes intestinal mucosa against noxious agents.

Pancreatic secretory trypsin inhibitor (PSTI) is a serine protease inhibitor, expressed in gut mucosa, whose function is unclear. We, therefore, examined the effects of PSTI on gut stability and repair. Transgenic mice overexpressing human PSTI within the jejunum (FABPi(-1178 to +28) hPSTI construct) showed no change in baseline morphology or morphometry but reduced indomethacin-induced injury in overexpressing hPSTI region by 42% (P < 0.01). Systemic recombinant hPSTI did not affect baseline morphology or morphometry but truncated injurious effects in prevention and recovery rat models of dextran-sodium-sulfate-induced colitis. In vitro studies showed PSTI stimulated cell migration but not proliferation of human colonic carcinoma HT29 or immortalized mouse colonic YAMC cells. PSTI also induced changes in vectorial ion transport (short-circuit current) when added to basolateral but not apical surfaces of polarized monolayers of Colony-29 cells. Restitution and vectorial ion transport effects of PSTI were dependent on the presence of a functioning epidermal growth factor (EGF) receptor because cells with a disrupted (EGFR(-/-) immortalized cells) or neutralized (EGFR blocking antibodies or tyrosine kinase inhibitor) receptor prevented these effects. PSTI also reduced the cytokine release of lipopolysaccharide-stimulated dendritic cells. We conclude that administration of PSTI may provide a novel method of stabilizing intestinal mucosa against noxious agents and stimulating repair after injury.