Intestinal Polyp Development Research Articles

Inhibition of Intestinal Polyp Formation by Pitavastatin, a HMG-CoA Reductase Inhibitor

It has been suggested that hyperlipidemia is positively associated with colon carcinogenesis. Statins, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors, reduce serum lipid levels. In this study, we clarified the effects of a novel chemically synthesized statin, pitavastatin, on intestinal polyp formation in Min mice, and further examined serum lipid and adipocytokine levels, and proinflammatory and adipocytokine gene levels in intestinal mucosa of Min mice. Treatment with pitavastatin at doses of 20 and 40 ppm decreased the total number of polyps dose-dependently to 85.2% and 65.8% (P < 0.05) of the untreated value, respectively. Serum levels of total cholesterol and triglyceride were slightly reduced and those of IL-6, leptin, and MCP-1 were decreased by 40-ppm pitavastatin treatment. mRNA expression levels of cyclooxygenase-2, IL-6, inducible nitric oxide (iNOS), MCP-1, and Pai-1 were significantly reduced in intestinal nonpolyp parts by pitavastatin treatment. Among them, iNOS mRNA levels were also reduced in the intestinal polyps. Moreover, oxidative stress represented by 8-nitroguanosine in the small intestinal epithelial cells was reduced by pitavastatin treatment. Related to these proinflammatory genes, PPARγ activity was activated in the intestinal nonpolyp parts and in the liver of Min mice with pitavastatin treatment. These results indicated that pitavastatin has potential benefit for the suppression of intestinal polyp development.

SS6-4 IL-17A and IL-17F are important for the development of intestinal polyps in APCmin mice by accelerating blood vessel formation

SS6-4 IL-17A and IL-17F are important for the development of intestinal polyps in APCmin mice by accelerating blood vessel formation

Dietary-induced ERβ upregulation counteracts intestinal neoplasia development in intact male Apc Min/+ mice

Most sporadic colorectal cancers (CRCs) develop through the adenoma-carcinoma sequence pathway and are initiated by adenomatous polyposis coli (APC) gene mutations. Estrogen receptor beta (ERbeta) is recognized to progressively reduce its expression in adenomatous and carcinomatous tissues in humans. Moreover, ERbeta deficiency enhances small intestinal tumorigenesis in rodents. In the Apc(Min/+) mouse model, we evaluated intestinal polyp development and ERbeta expression plus other biological parameters influencing tumor growth (epithelial cell proliferation, apoptosis and migration) following the addition of a combination of the ERbeta-selective agonist silymarin (SIL) and/or lignin (LIG) to a high-fat/low-fiber diet. Forty-five Apc(Min/+) mice were divided in four groups: animals fed on the tumorigenic high-fat/low-fiber diet, the tumorigenic diet supplemented with SIL (0.02%) or purified LIG (6.24%) or SIL (0.005%) + LIG (6.24%). In these animals, we assessed polyp number and volume and their degree of dysplasia together with ERbeta messenger RNA (mRNA) and protein levels and epithelial cell proliferation, migration and apoptosis. The latter group of parameters was evaluated in normal and adenomatous mucosa and the results compared with those found in wild-type (WT) mice fed on the control diet. The addition of SIL or LIG to the diet and even more the specific combination of the two significantly counteracted intestinal tumorigenesis and increased ERbeta mRNA and protein levels. Cell proliferation and apoptosis were rebalanced and cell migration accelerated, restoring values similar to those observed in WT animals. Our results further support a protective effect of ERbeta in CRC suggesting the use of the combination of SIL-LIG as a potential approach against CRC development.

The Interaction of a High-Fat Diet and Regular Moderate Intensity Exercise on Intestinal Polyp Development in ApcMin/+ Mice

Diet and exercise are two environmental factors that can alter colon cancer risk. The purpose of this study was to determine if regular moderate-intensity treadmill exercise training could attenuate polyp formation in Apc(Min/+) mice fed the Western-style diet. Four-week-old male Apc(Min/+) mice (n = 12 per group) were assigned to AIN-76A Control, AIN-76A Exercise, Western Control, or Western Exercise treatment groups. Mice were weaned to these diets and either subjected to regular moderate-intensity treadmill exercise (18 m/min, 60 min/d, 6 d/wk) or remained sedentary for 6 weeks. Mice fed the Western-style diet consumed approximately 14% more calories and had 42% more epididymal fat compared with mice fed the AIN-76A diet. Exercise had no effect on fat pad mass with either diet treatment. Exercise reduced total intestinal polyp number by 50% and the number of large polyps (>1 mm diameter) by 67% in AIN-76A-fed mice. The Western-style diet increased polyp number by 75% when compared with AIN-76A-fed mice, but exercise did not decrease polyp number or alter polyp size in mice fed the Western-style diet. Markers of systemic inflammation and immune system function were improved with exercise in mice fed the AIN-76A diet. Mice fed the Western-style diet showed more inflammation and immunosuppression, which were not completely ameliorated by exercise. These data suggest that the induction of adiposity, inflammation, and immunosuppression by the Western-style diet may compromise the beneficial effect of moderate-intensity exercise on the intestinal polyp burden in Apc(Min/+) mice.

Nonsteroidal Anti-inflammatory Drug-activated Gene (NAG-1/GDF15) Expression Is Increased by the Histone Deacetylase Inhibitor Trichostatin A

Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) is a putative tumor suppressor whose expression can be increased by drug treatment. Glioblastoma is the most common central nervous system tumor, is associated with high morbidity and mortality, and responds poorly to surgical, chemical, and radiation therapy. The histone deacetylase inhibitors are under current consideration as therapeutic agents in treating glioblastoma. We investigated whether trichostatin A (TSA) would alter the expression of NAG-1 in glioblastoma cells. The DNA demethylating agent 5-aza-dC did not increase NAG-1 expression, but TSA up-regulated NAG-1 expression and acted synergistically with 5-aza-dC to induce NAG-1 expression. TSA indirectly increases NAG-1 promoter activity and increases NAG-1 mRNA and protein expression in the T98G human glioblastoma cell line. TSA also increases the expression of transcription factors Sp-1 and Egr-1. Small interfering RNA experiments link NAG-1 expression to apoptosis induced by TSA. Reporter gene assays, specific inhibition by small interfering RNA transfections, and chromatin immunoprecipitation assays indicate that Egr-1 and Sp-1 mediate TSA-induced NAG-1 expression. TSA also increases the stability of NAG-1 mRNA. TSA-induced NAG-1 expression involves multiple mechanisms at the transcriptional and post-transcriptional levels.

Targeting Cyclooxygenase-2 and the Epidermal Growth Factor Receptor for the Prevention and Treatment of Intestinal Cancer

Clinical and animal studies indicate a role for cyclooxygenase-2 (COX-2) and the epidermal growth factor receptor (EGFR) in the development and progression of intestinal polyps and cancers. Although this combination of enzyme inhibition has shown synergy in intestinal polyp and tumor models, the exact mechanism for these effects remains undefined. Therefore, we sought to define the molecular mechanisms through which this process occurs. We observed a significant reduction in the number and size of small intestinal polyps in APC(min+/-) mice treated with either celecoxib (a selective COX-2 inhibitor) or erlotinib (Tarceva, an EGFR inhibitor). However, in combination, there was an overall prevention in the formation of polyps by over 96%. Furthermore, we observed a 70% reduction of colorectal xenograft tumors in mice treated with the combination and microarray analysis revealed genes involved in cell cycle progression were negatively regulated. Although we did not observe significant changes in mRNAs of genes with known apoptotic function, there was a significant increase of apoptosis in tumors from animals treated with the combination. The inhibition of EGFR also induced the down-regulation of COX-2 and further inhibited prostaglandin E2 formation. We observed similar effects on the prevention of intestinal adenomas and reduction of xenograft tumor volume when nonselective COX inhibitors were used in combination with erlotinib. Together, these findings suggest that the inhibition of both COX-2 and EGFR may provide a better therapeutic strategy than either single agent through a combination of decreased cellular proliferation and prostaglandin signaling as well as increased apoptosis.

Carrageenan Reduces Bone Morphogenetic Protein-4 (BMP4) and Activates the Wnt/β-Catenin Pathway in Normal Human Colonocytes

Carrageenans are highly sulfated polysaccharides that are widely used as food additives in the Western diet, in order to improve the texture of processed foods. Although native and degraded carrageenans induce colonic ulcerations, polyps, and colorectal tumors in animal models, very little is known about the effects of carrageenan on human colonocytes. We evaluated effects of lambda-carrageenan (lambdaCGN) on the normal human colonocyte cell line NCM460, using a concentration of 1 mug/ml, about less than one tenth the average daily exposure to carrageenan in the Western diet. We measured secreted bone morphogenetic protein-4 (BMP4) in spent media and quantified its expression by quantitative RT-PCR. Wnt-related genes were measured by an oligonucleotide array. Cellular beta-catenin was quantified by ELISA. We found a marked decline in secreted BMP4 (P < 0.001) following exposure of NCM460 cells to lambdaCGN for 24 hr. Quantitative RT-PCR for BMP4 transcripts revealed 24% and 45% inhibition of expression on days 2 and 4. cDNA gene expression array of Wnt signaling pathway target genes demonstrated significant changes, including 4.5-fold induction of Wnt 9A and suppression of Dickkopf 3 and RHOU genes. Measurement of beta-catenin by ELISA revealed concomitant accumulation with increases of 67.8%, 61.6%, and 73.9% on days 1, 2, and 4, compared to untreated controls. We conclude that treatment of normal human colonocytes with lambdaCGN activated the Wnt/beta-Catenin cascade and suppressed the expression and secretion of BMP4, inducing significant changes in cellular pathways that are associated with both sporadic and juvenile polyps. CGN may influence development of intestinal polyps in vivo by these mechanisms.

Negative energy balance induced by voluntary wheel running inhibits polyp development in APCMin mice

Treadmill running of approximately 0.9 km/day has had inconsistent effects on spontaneous intestinal polyp development in C57BL/6J-Apc(Min)/J (Min) mice; the amount of energy expenditure and/or a lack of hormonal changes could account for this variability. The purpose of this study was to examine the effects of a negative energy balance induced by voluntary wheel running on polyps, insulin-like growth factor-1 (IGF-1) and corticosterone in Min mice. Seven-week-old male Min mice were randomly assigned to control (CON, n = 23) or wheel running (EX, n = 24) conditions for a 10-week study period. All mice had water and AIN-76A diet ad libitum for the first approximately 3 weeks on study, after which the EX group was pair-fed to the CON group to maintain a negative energy balance due to the exercise. EX mice voluntarily ran 3.8 km/day (2.7-6.0 km/day) (median, interquartile range) and weighed less than CON mice throughout the study. More CON mice died before the end of the study versus EX mice (26 versus 0%, P < 0.01). CON mice had significantly more polyps versus EX mice (21.6 +/- 1.5 versus 16.9 +/- 2.0, P < 0.01; mean +/- SE), and daily running distance in EX was inversely correlated with total polyp number (r = -0.70, P < 0.01). Urinary corticosterone output (P < 0.01) and serum IGF-1 were significantly higher in EX than CON (P < 0.001); however, total polyp number was unrelated to corticosterone (r = 0.05, P = 0.84) and IGF-1 (r = -0.01, P = 0.93). In this study, a negative energy balance produced by wheel running exercise and restricted feeding decreased polyp burden in male Min mice and appeared to have a dose-response effect on polyp number. Although EX affected IGF-1 and corticosterone, neither marker was related to total polyp number.

Decreased intestinal polyp multiplicity is related to exercise mode and gender in ApcMin/+ mice

Moderate-intensity treadmill running can alter male Apc(Min/+) mouse polyp formation. This purpose of this study was to examine whether exercise mode differentially affects Apc(Min/+) mouse intestinal polyp development in male and female mice. Male and female Apc(Min/+) mice were randomly assigned to control, treadmill (18 m/min; 60 min/day; 6 days/wk), or voluntary wheel running (24-h access) groups. Nine weeks of training decreased total intestinal polyps by 29% in male treadmill runners (66 +/- 9; P = 0.038) compared with male controls (93 +/- 7). The number of large polyps (>/=1-mm diameter) were also reduced by 38% in male treadmill runners (49 +/- 6; P = 0.005) compared with male controls (79 +/- 6). Treadmill running in female Apc(Min/+) mice and wheel running in both genders did not affect polyp number or size. Spleen weight decreased in male treadmill runners (91 +/- 9 mg; P = 0.011) and wheel runners (75 +/- 6 mg; P = 0.004) compared with controls (141 +/- 13 mg). Plasma IL-6 was reduced by 96% in male treadmill runners (1.2 +/- 0.6 pg/ml) and 78% in male wheel runners (6.6 +/- 3.3 pg/ml) compared with control mice (27.9 +/- 2.8 pg/ml; P < 0.05). Female mice responded similarly with an 86% decrease in plasma IL-6 with treadmill running (3.2 +/- 1.2 pg/ml) and 90% decrease with wheel running (2.9 +/- 2.0 pg/ml) compared with control mice (21.1 +/- 5.3 pg/ml; P < 0.05). The crypt depth-to-villus height ratio in the intestine, an indirect marker of intestinal inflammation, decreased by 21 (P = 0.024) and 24% (P = 0.029), respectively, in male and female treadmill runners but not wheel runners. Physical activity-induced attenuation of intestinal polyp number and size is dependent on exercise mode and differs between genders. The modulation of systemic and intestinal inflammation may also depend on exercise mode.

Systemic And Intestinal Inflammation Changes With Physical Activity In The APCMin+/− Mouse

Colon cancer is the second leading cause of all cancer deaths in the US. Behavioral modifications, such as physical activity (PA) and diet, can effect its prevention and progression. The adenomous polyposis coli (APC) gene is one heritable genetic mutation that can trigger tumor development. To determine if Male and female APCMin+/− mice were randomly assigned to Control, Treadmill, and Activity Wheel groups. Treadmill runners ran on a treadmill at 18 m/min for 60 min, 6 d/wk. Activity wheel runners were housed with 24-h access to voluntary activity wheels. Splenic inflammatory cytokine mRNA levels were analyzed via RT-PCR. We have previously demonstrated that treadmill running can decrease intestinal polyp number and size in male APCMin+/− mice. Splenic IL-6 mRNA concentration decreased 29% in male treadmill runners compared to controls (p=0.036), but not in wheel runners. There was no effect of exercise on female spleen weight or splenic IL-6 mRNA abundance. The crypt depth:villus height ratio in the intestine (CVR), an indirect marker of intestinal inflammation, decreased 21% (p=0.024) and 24% (p=0.029), respectively, in male and female treadmill runners, but not wheel runners. Physical activity-induced attenuation of intestinal polyp number may be related to the systemic and intestinal inflammatory state. Further studies need to examine the interaction between nutraceutical treatments and physical activity on intestinal polyp development.

Physical Activity Decreases Intestinal Polyp Development in the APCmin+/ ??? Mouse

Physical Activity Decreases Intestinal Polyp Development in the APCmin+/ ??? Mouse

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.

Exercise and intestinal polyp development in APCMin mice.

Epidemiological evidence suggests that physical activity protects against colon cancer. We previously used a mouse predisposed to intestinal polyps (APCMin) to evaluate this association and found the suggestion of fewer polyps in exercised males but not females. The present study was designed to further explore the potential exercise x sex interaction on polyp development and to begin to look at potential mechanisms. Six-week-old APCMin mice (N = 60 males; 60 females) were randomly assigned to one of two groups by sex: treadmill running at 20 m.min-1, 5% grade, 45 min.d-1, 5 d.wk-1 (EX) or nonrunning controls (CON) (N = 30 per group). EX mice ran in running wheels while in quarantine (weeks 0-3), followed by treadmill running weeks 3-8. Body weights were measured weekly. Urine was collected at 5 wk and fasting blood at 7.5 wk. Body composition was measured, serum was frozen, and polyp number and size were measured at sacrifice. EX resulted in lower body weights (P < 0.01) and reduced fat mass (P < 0.01). Fasting glucose was lower in EX (P < 0.01), and leptin was lower in EX (P = 0.05) compared with CON. EX did not affect serum insulin-like growth factor-1 or urinary corticosterone. Total polyp number and size were not statistically different between groups; however, there were fewer jejunal polyps in EX (3.6 +/- 0.7, mean +/- SE) versus CON males (5.2 +/- 0.8; P = 0.04) and an even larger difference when only the consistent runners were kept in the analysis (2.7 +/- 0.5 in EX; P = 0.01). Despite favorable changes in body composition, blood glucose, and leptin, 8 wk of running resulted in only minor changes related to polyp development in male but not female APCMin mice.

Exercise and tumor development in a mouse predisposed to multiple intestinal adenomas.

Epidemiological evidence suggests that physical activity may be protective against the development of colon cancer. Potential mechanisms remain largely unexplored due to the paucity of appropriate experimental models. The purpose of this study was to examine the effect of exercise training on polyp development in an induced mutant mouse strain predisposed to multiple intestinal neoplasia (Min mouse). Three-week-old male and female heterozygotes were randomly assigned to control (CON; 10 males, 6 females) or exercise (EX; 11 males, 11 females) groups. In the first week, EX mice were acclimated to treadmill running at 10-18 m x min(-1) for 15-60 min x d(-1). From 4-10 wk of age, mice ran at 18-21 m x min(-1) for 60 min. CON mice sat in Plexiglas lanes suspended above the treadmill for the same time periods. At 10 wk of age, the mice were sacrificed and the intestines removed, opened, and counted for polyps. Skeletal muscle oxidative capacity increased with training as shown by a 64% increase in citrate synthase activity in the gastrocnemius/soleus muscle of EX compared with CON (P = 0.009). There were no significant effects of exercise in the males and females combined on small intestine, colon, or total intestinal polyps (P > 0.05). When analyzed separately, however, there were fewer colon and total polyps in the EX than in the CON males, although the difference was not statistically significant (P = 0.06). These results suggest that seven weeks of exercise training do not affect the development of intestinal polyps in the Min mouse. Further studies are required to determine if a true sex difference exists or if variations on the current training protocol may affect tumor outcomes.

Suppression by peroxisome proliferator activated receptora ligand of intestinal polyp development in APC Min mice

Suppression by peroxisome proliferator activated receptora ligand of intestinal polyp development in APC Min mice

Cell Type-specific Autoregulation of theCaudal-related Homeobox Gene Cdx-2/3

The caudal-related homeobox gene Cdx-2/3 is a critical "master" control gene in embryogenesis. Mice heterozygous for a null mutation in Cdx-2/3 exhibit multiple malfunctions including tail abnormalities, stunted growth, a homeotic shift in vertebrae, and the development of multiple intestinal adenomatous polyps, indicating that Cdx-2/3 is haplo-insufficient. In vitro studies have identified more than a half-dozen downstream target genes expressed in pancreatic and intestinal cells for this transcription factor. We have examined the transcriptional properties of the mouse Cdx-2/3 promoter. This promoter could be autoregulated in pancreatic and intestinal cells that express endogenous Cdx-2/3. In contrast, Cdx-2/3 transfection represses the Cdx-2/3 promoter in fibroblasts, which do not express endogenous Cdx-2/3. Since Cdx-2/3 activates proglucagon gene promoter in both pancreatic and intestinal cells and in fibroblasts, we suggest that some, yet to be identified, cell type-specific components are required for activating selected target gene promoters of Cdx-2/3, including the Cdx-2/3 promoter itself. Cdx-2/3 binds to the TATA box and another AT-rich motif, designated as DBS, within an evolutionarily conserved proximal element of the Cdx-2/3 promoter. The DBS motif is critical for the autoregulation, whereas the TATA box may act as an attenuating element for the autoregulatory loop. Finally, overexpression of Cdx-2/3 in a pancreatic cell line activated the expression of the endogenous Cdx-2/3. Taken together, our results indicate that the dose-dependent phenotype of Cdx-2/3 expression on its downstream targets in vivo could be regulated initially via a transcriptional network involving cell type-specific autoregulation of the Cdx-2/3 promoter.

Inhibitory effects of bovine lactoferrin on intestinal polyposis in the Apc Min mouse

Chemopreventive effects of bovine lactoferrin (bLF), previously shown to strongly inhibit intestinal carcinogenesis in rats (K. Sekine, E. Watanabe, J. Nakamura, N. Takasuka, D.J. Kim, M. Asamoto, V. Krutovskikh, T.H. Baba, T. Ota, M.A. Moore, M. Masuda, H. Sugimoto, H. Nishino, T. Kakizoe, H. Tsuda, Inhibition of azoxymethane-initiated colon tumor by bovine lactoferrin administration in F344 rats, Jpn. J. Cancer Res. 88 (1997) 523–526; K. Sekine, Y. Ushida, T. Kuhara, M. Iigo, H. Baba-Toriyama, M.A. Moore, M. Murakoshi, Y. Satomi, H. Nishino, T. Kakizoe, H. Tsuda, Inhibition of initiation and early stage development of aberrant crypt foci and enhanced natural killer activity in male rats administered bovine lactoferrin concomitantly with azoxymethane, Cancer Lett. 121 (1997) 211–216), on spontaneous intestinal polyp development were assessed in the Apc Min mouse, a model for both familial adenomatous polyposis and sporadic colon cancers. In the experiment, 54 mice at 6 weeks of age were given 2% bLF (15 mice), 0.2% bLF (15 mice) and AIN-93G (24 mice) as basal diet ad libitum for 8 weeks. An overall tendency for a reduction in the total number of polyps in the small intestine was evident in the bLF-treated animals, along with significant suppression in the jejunum at the 2% dose ( P<0.05, 68% of the control). In addition, body growth suppression, presumed to be due to anemia and/or intussusception as a consequence of numerous polyps in the intestine, was alleviated. No toxic effects were observed in the intestinal epithelium. Although not as obvious as observed for the rat case, the data suggest that bLF may be a chemopreventor of intestinal polyposis.

Suppression of intestinal polyp development by nimesulide, a selective cyclooxygenase-2 inhibitor, in Min mice.

Nonsteroidal anti‐inflammatory drugs (NSAIDs) suppress colon carcinogenesis in man and experimental animals. However, conventional NSAIDs inhibit both cyclooxygenase (COX) isoforms, COX‐1 and COX‐2, and cause gastrointestinal side‐effects. Nimesulide, a selective inhibitor of COX‐2, is much less ulcerogenic. We, therefore, examined its influence on the development of intestinal polyps in Min mice. Female Min mice at 4 weeks old were given 400 ppm nimesulide in their diet for 11 weeks. This treatment resulted in a significant reduction of the numbers of both small and large intestinal polyps, the total being 52% of that in untreated control Min mice. The size of the polyps in the nimesulide‐treated group was also significantly decreased. The results suggest that nimesulide is a good candidate as a chemopreventive agent for human colon cancer with low toxicity.

Suppression of intestinal polyp development by low-fat and high-fiber diet in Apc(delta716) knockout mice.

Most epidemiological and animal studies show a positive correlation of the dietary intake of fat with the incidence of colon cancer, whereas an inverse correlation of the dietary intake of fiber. In rats fed a diet low in fat and high in wheat bran fiber and calcium, a significant decrease was reported in the number of azoxymethane-induced aberrant crypt foci compared with those fed a high-fat, low-fiber and low-calcium diet. Mutations in the human APC gene play a key role, not only in familial adenomatous polyposis, but also in many sporadic cancers of the entire digestive tract. We previously constructed a mouse strain Apc(delta716), carrying a truncation mutation at codon 716 of the Apc gene, the homolog of human APC (10). The heterozygous mice developed numerous intestinal polyps, and all microadenomas dissected from the earliest polyps had already lost the wild-type allele, indicating the loss of heterozygosity. Using these Apc(delta716) knockout mice, we have investigated the effect of a low-fat and high-fiber diet (LRD for 'low-risk' diet) on intestinal polyposis, and compared it with that of a high-fat and low-fiber diet (HRD for 'high-risk' diet). The mice were fed either diet for 7 weeks, and the number and size of intestinal polyps were scored. The LRD-fed mice had fewer polyps than the HRD-fed mice, by 36% in the small intestine and by 64% in the colon. As for the polyp size distribution, there was no significant difference between the HRD- and LRD-fed mice. These results indicate that LRD can suppress intestinal polyposis compared with HRD which does not, and suggest that its suppression is at the initiation of polyp formation. This is likely to be due to a decreased frequency of loss of heterozygosity, rather than a retarded growth of the polyp adenomas.

Effects of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine on intestinal polyp development in ApcΔ716 knockout mice

Damage to the human adenomatous polyposis coli (APC) gene is responsible for not only familial adenomatous polyposis but also many sporadic cancers of the entire digestive tract. Using homologous recombination in embryonic stem cells, we recently constructed gene knockout mice with a truncation mutation in the Apc gene. These heterozygous mice developed intestinal polyps. We found that all microadenomas dissected from the earliest polyps had already lost the wild-type allele, indicating loss of heterozygosity (LOH) (Oshima et al., Proc. Natl. Acad. Sci. USA 92:4482-4486, 1995). Using these knockout mice, we investigated the effects of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP), one of the most abundant heterocyclic amines found in cooked meat and fish. When PhIP was fed to these mice at 400 ppm for 8 wk, the polyp distribution shifted to a larger size range, although the total polyp number did not change significantly. Similar, but weaker, effects were observed with the other heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline. On the other hand, intraperitoneal injections of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhlP) at a higher dose (50 mg/kg) for five consecutive days increased the polyp number significantly. This increment was not associated with mutations in the Apc gene; however, most polyps showed loss of the full-length Apc allele (LOH). These results suggest that PhIP affects intestinal polyp development by accelerating the growth rate of microadenomas. It is also possible that high doses of N-OH-PhIP increase the frequency of Apc gene LOH.