Activation of NF-kappaB is required for mediating proliferative and antiapoptotic effects of progastrin on proximal colonic crypts of mice, in vivo

Mice overexpressing progastrin (PG) in intestinal mucosa (fatty acid-binding protein (Fabp)-PG mice) are at an increased risk of proximal colon carcinogenesis in response to azoxymethane. Here, we report a significant increase in the length of proximal colonic crypts in Fabp-PG mice, associated with potent antiapoptotic effects of PG, which likely contributed to the previously reported increase in colon carcinogenesis in Fabp-PG mice. Phosphorylation of kinase of IkappaBalpha (IKKalpha/beta), inhibitor of kappaB (IkappaB)alpha and p65NF-kappaB was significantly elevated in proximal colonic crypts of Fabp-PG versus wild-type mice, which was associated with degradation of IkappaBalpha and nuclear translocation/activation of p65. Surprisingly, distal colonic crypt cells were not as responsive to elevated levels of PG in Fabp-PG mice. Annexin II, recently described as a high-affinity receptor for PG, strongly co-localized with PG intracellularly and on basolateral membranes of proximal crypt cells, providing evidence that annexin-II binds PG in situ in colonic crypt cells. Proliferative and antiapoptotic effects of PG on proximal crypts of Fabp-PG mice were attenuated to wild-type levels, on treatment with NEMO peptide (an inhibitor of nuclear factor-kappaB (NF-kappaB) activation), demonstrating for the first time a critical role of NF-kappaB in mediating hyperproliferative affects of PG on colonic crypts of Fabp-PG mice, in vivo. Thus, downregulation of NF-kappaB may significantly reduce the increased risk of colon carcinogenesis in response to PG.     

Intestinal expression of mutant and wild-type progastrin significantly increases colon carcinogenesis in response to azoxymethane in transgenic mice

BACKGROUND: The authors recently reported that transgenic mice (hGAS) expressing pharmacologic levels of progastrin (PG) (> 10 nM to 100 nM) exhibited increased susceptibility to colon carcinogenesis in response to azoxymethane (AOM). It is not known whether PG functions as a cocarcinogen at the concentrations observed in patients with hypergastrinemia (approximately 1.0 nM). METHODS: The authors generated transgenic mice that overexpressed either wild-type (wtPG) or mutant (mtPG) human PG in the intestinal mucosa using the murine fatty acid binding protein (Fabp) promoter. Fabp-PG mice and their wild-type littermates were treated with AOM, and their colons were examined for preneoplastic (aberrant crypt foci [ACF]) and neoplastic (adenomas [Ads] and adenocarcinomas [AdCas]) lesions after 2 weeks and 6 months of treatment. RESULTS: ACF and tumors were significantly more common (by a factor of approximately 2) in colon specimens from both Fabp-wtPG mice and Fabp-mtPG mice relative to wild-type mice. It is noteworthy that the multiplicity of ACF and the total number of small and large Ads and AdCas were significantly greater in colon specimens from Fabp-PG mice compared with colon specimens from wild-type mice, irrespective of gender. CONCLUSIONS: The results of the current study suggest that at concentrations (approximately 1.0 nM) far lower than the ones observed in hGAS mice, PG functions as an equally potent cocarcinogen and significantly increases the risk of colon carcinogenesis in response to AOM. Thus, PG may represent a clinically relevant target molecule in patients with hypergastrinemia or colon carcinoma.     

Development of aberrant crypt foci in the colons of ob/ob and db/db mice: evidence that leptin is not a promoter

Leptin is elevated in obesity and has been suggested to increase the risk of colorectal cancer (CRC), although the evidence is conflicting. The objective of this study was to compare the susceptibility to colon carcinogenesis of db/db mice that have highly elevated circulating leptin and leptin-deficient ob/ob mice, both of which are obese. Seven-week-old male ob/ob, db/db, and WT mice received 4 weekly i.p. injections of 5 mg/kg azoxymethane (AOM) and were killed 14 wk later for the analysis of putative preneoplastic aberrant crypt foci (ACF). There were no differences in ACF number or multiplicity between ob/ob and db/db mice. Leptin has been shown to induce CYP2E1, the main enzyme that activates AOM, but we observed no differences in hepatic CYP2E1 activity or colonic CYP2E1 protein levels between ob/ob and db/db mice. We also induced ACF with 2 oral doses 3 d apart of 30 mg/kg methylnitrosourea (MNU), a direct-acting carcinogen. There were no differences in ACF number or multiplicity between the two groups of obese animals 5 wk following the last dose of MNU. The colonic mucosa of db/db mice expressed significantly lower mRNA levels of ObRa, the predominant short form of the leptin receptor, compared to ob/ob mice, and following i.p. injection with 1 mg/kg recombinant mouse leptin, exhibited significantly reduced p44/42 pMAPK compared to saline-treated controls. These results show that ObRa is functionally active in the colons of db/db mice. We conclude that leptin does not play a significant role in ACF development.     

Cancer, aging and the optimal tissue design

Division patterns or mammalian tissues, like every other feature of life, have been subject to evolutionary pressures throughout the natural history. A particular and very important design principle that we discuss in this paper is the protective role of tissue architecture against cancer. We present a stochastic dynamical model of cell renewal of epithelial tissue (colonic crypts) which explicitly includes asymmetric indefinite divisions of stem cells and symmetric, finite divisions of daughter cells. We find that the hierarchical structure of crypts plays a protective role against accumulation of double-mutants. We argue that daughter cells, and not only stem cells, can play a role in carcinogenesis. Our model also predicts the optimum number of stem cells per crypt. In most cases, higher numbers of stem cells per crypt correspond to lowering the chance of colon cancer initiation (except if mutation rates associated with daughter cells are a lot lower than those associated with stem cells). Finally, we argue that the evolutionarily optimum which corresponds to a large number of stem cells per crypt, pushes the onset of cancer to an older age, but it actually acts against older individuals by increasing their chance of developing cancer.     

Sucrose enhancement of the early steps of colon carcinogenesis in mice

The association of refined sugars and colorectal cancers andpolyps in three recent case-control studies led us to investigatethe effects of sucrose, fructose and glucose on colonic epithelialproliferation and sensitivity to carcinogenesis. CF1 and C57BL/6Jmice were used; proliferation was assessed as vincristine-accumulatedmitotic figures per crypt section; sensitivity to carcinogenesiswas assessed as the number of aberrant crypt foci (ACF) percolon observed following the colon carcinogen, azoxymethane(AOM, 3 mg/kg and 5 mg/kg). Oral gavages of sucrose and fructosein CF1 mice (10 g/kg) increased colonic proliferation 16 h later(2.8±0.6 and 4.1±0.7 (mean±SEM) accumulatedmitotic figures/crypt section), compared with glucose and water(1.0+0.2 and 0.4±0.1). Sucrose and fructose given 14h prior to the AOM (5 mg/kg) increased the sensitivity of thecolon to carcinogenesis (18.4±1.5 and 13.1±1.8ACF/colon), compared with glucose and water (11.4±2.0and 8.6±1.1). Similar results were observed with C57BL/6Jmice. We conclude that dietary sucrose and fructose may representrisk factors for colorectal cancer through a direct effect ofthe sugars on colonic epithelial proliferation.     

Silk protein, sericin, suppresses colon carcinogenesis induced by 1,2-dimethylhydrazine in mice

Male 5-week old ICR mice were examined for the effect of feeding silk protein, sericin on colon carcinogenesis. In experiment 1, mice were fed the diets supplemented with 1.5% or 3% sericin for five weeks, and given weekly injections of 1,2-dimethylhydrazine (DMH) for the initial three weeks. Supplemental sericin caused a dose-dependent decrease in the development of colonic aberrant crypt foci. In experiment 2, mice were fed the diet supplemented with 3% sericin for 115 days, and given weekly injections of DMH for the initial ten weeks. The incidence and number of colon tumors were suppressed by consumption of sericin. The results suggest a potential usefulness of sericin as a chemopreventive agent for colon carcinogenesis.     

大肠癌旁粘膜细胞增殖模式与术后复发机制的探讨

目的：探讨大肠癌旁粘膜细胞增殖模式变化与肿瘤术后复发的关系。方法：用免疫组化染色方法对７８例大肠癌标本的癌组织、癌旁粘膜及１２例正常大肠粘膜进行ｐ５３蛋白、增殖细胞核抗原（ＰＣＮＡ）检测,并进行随访。结果：癌组织ＰＣＮＡ高表达明显高于癌旁（Ｐ＜００１）,癌旁ＰＣＮＡ高表达与隐窝增殖区扩大、上移,明显影响术后第１年局部复发死亡率（Ｐ＜００１）,癌组织ｐ５３蛋白阳性表达率６１５４％,癌旁为２６９２％,癌及癌旁ｐ５３阳性表达不影响细胞增殖活性及术后第一年内的复发死亡率（Ｐ＞００５）。结论：大肠癌的复发可能与大肠癌的发生一样是正常隐窝增殖分化失调的顺序过程。ＰＣＮＡ可直观表现其增殖活性及增殖模式。ｐ５３突变及表达是多中心性的,癌旁ｐ５３突变点的存在是肠癌局部复发的潜在因素。     

Effects of androgen manipulations on chemically induced colonic tumours and on macroscopically normal colonic mucosa in male Sprague-Dawley rats

Epidemiological and experimental studies suggest that androgens influence colonic carcinogenesis. We investigated the effects of hormonal manipulations (surgical and chemical castration, hormone substitution) on colonic tumour development, tumour and mucosal histopathology, and epithelial proliferation in macroscopically normal colonic mucosa in male rats, after induction of chemical colon carcinogenesis by subcutaneous injections of azoxymethane (AOM). Chemical castration with cyproterone acetate, but not surgical castration, resulted in increased colonic tumorigenesis, which was accompanied by decreased crypt length, decreased number of cells per crypt, and increased crypt epithelial mitotic index in the right colon. Chemically castrated rats also had crypt hyperplasia and increased numbers of dysplastic foci in the left colon which were not seen with surgical castration. By contrast, rats given testosterone after surgical castration showed decreased colonic tumorigenesis with an increased proportion of tumours in the left colon and lower percentage of tumours with invasion. The grossly normal mucosa of the testosterone-substituted castrated rats showed decreased crypt length in the right colon similar to the other groups of castrated rats, but no significant increase in mitotic index. Our results suggest that the anti-androgenic progestin cyproterone is a potent enhancer of colonic tumorigenesis and epithelial proliferative abnormalities after AOM administration. Exogenous testosterone after castration alters tumour distribution and characteristics and suppresses epithelial proliferative abnormalities. Finally, androgen effects on the colonic mucosa are more prominent in the right than in the left colon, suggesting different influences of hormones on the epithelium of these anatomical sites.     

The trophic effect of dietary fibre is not associated with a change in total crypt number in the distal colon of rats

Soluble fibres, such as guar gum, promote and wheat bran or methylcellulose protect from chemically induced colon carcinogenesis, relative to the effect of a fibre-free diet in rats. Mechanisms are poorly understood. Whereas all fibres are trophic to the colonic epithelium, the heterogeneity of effects on carcinogenesis may reflect different effects on the total number of crypts and, therefore, the size of the stem cell population. This study aimed to assess this hypothesis. Sprague-Dawley rats were fed one of fibre-free diets with or without 10% wheat bran, methylcellulose or guar gum for 4 weeks. The distal colons were stained with methylene blue and quantified for the number and density of crypts using an image analysis system. Epithelial proliferative kinetics was measured stathmokinetically. Methodology for quantifying crypts was valid and reproducible. Rats fed a fibre-free diet had atrophic distal colon, as shown by a decrease in crypt column height and a lower mitotic index. Fibre supplementation prevented the atrophy and was associated with crypt mouth areas that were 30-60% larger than those in the fibre-free group (P < 0.001, ANOVA), with the methylcellulose group being the largest (1.16 microm(2)). The crypt density of the fibre-free group was 16-19% greater than those in fibre fed groups (P + 0.006), due to the smaller size of the crypts. However, there was no difference in the total number of crypts across the four dietary groups (P > 0.1). Distal colons in all of the dietary groups contained approximately 10(5) crypts. In conclusion, although variation in the amount or type of dietary fibre exerts heterogeneous effects on the growth of the colonic epithelium and on colon carcinogenesis, the total number of crypts in the distal colon remains constant. It is, therefore, unlikely that fibres influence carcinogenic events by altering the size of the stem cell population.     

A new biomarker that predicts colonic neoplasia outcome in patients with hyperplastic colonic polyps

The most frequently occurring lesions in the colon are the hyperplastic polyps. Hyperplastic polyps have long been considered as lesions with no malignant potential and colonoscopy for these patients is not recommended. However, recent works suggest that hyperplastic polyps may represent precursor lesions of some sporadic colorectal cancers. Until now, no biomarker allows to identify the subset of hyperplastic polyps that may have a malignant potential. Because the hormone precursor progastrin has been involved in colon carcinogenesis, we investigated whether its expression in hyperplastic polyps predicts the occurrence of colonic neoplasm after resection of hyperplastic polyps. We retrospectively analyzed progastrin expression in hyperplastic polyps from 74 patients without history of colorectal pathology. In our study, 41% of patients presenting an initial hyperplastic polyp subsequently developed adenomatous polyps, recognized as precursor lesions for colorectal adenocarcinomas. Progastrin was overexpressed in the hyperplastic polyps in 40% of the patients. We showed a significant association between progastrin overexpression and shortened neoplasm-free survival (P = 0.001). Patients with high overexpression of progastrin had a 5-year neoplasm-free survival rate of 38% as compared with 100% for the patients with low progastrin expression. In addition, we established a predictive test on the basis of progastrin staining and patients' age that predicts occurrence of neoplasm after developing a first hyperplastic polyp with a sensitivity of 100% [95% confidence interval (CI), 79%-100%] and a specificity of 74% (51%-90%). We show that progastrin expression evaluation in hyperplastic polyps is an efficient prognostic tool to determine patients with higher risk of metachronous neoplasms who could benefit from an adapted follow-up.     

Dietary sphingolipids in colorectal cancer prevention.

Sphingolipids are widespread membrane components that are found in all eukaryotic cells. They consist of a long chain sphingoid-base, usually sphingosine, which is acylated at the 2-amino position, forming a ceramide. All together, sphingolipids may represent the most structurally diverse category of lipids in nature. There is no known nutritional requirement for sphingolipids. Nonetheless, studies with experimental animals have shown that consumption of sphingolipids inhibits colon carcinogenesis, reduces serum low-density lipoprotein cholesterol and elevates high-density lipoproteins, which suggest that they are 'functional' components of food. In animal models (CF1 mice) sphingomyelin supplementation reduces the number of aberrant colonic crypt foci by approximately 70% and, with longer feeding, reduces the number of colonic adenocarcinomas. A possible mechanism of action of sphingolipids in suppressing colon carcinogenesis is that exogenously supplied sphingolipids bypass a sphingolipid signalling defect that is important in cancer (for example, a loss of cellular sphingomyelin turnover to produce ceramide and sphingosine). Indirect evidence suggests that sphingolipids can inhibit colon cancer in humans: sphingosine and ceramide induce apoptosis in a human adenocarcinoma cell line and feeding sphingolipids to Min mice reduces the number of colon tumours.     

Computer modeling implicates stem cell overproduction in colon cancer initiation.

On the basis of our investigation of the premalignant crypt phenotype in familial adenomatous polyposis patients, the hypothesis is developed that tumor initiation in the colon is caused by crypt stem cell overproduction. A novel kinetic model for the colonic crypt was used to investigate how the earliest tissue abnormality (altered crypt labeling index) arises in these patients who have a mutant APC genotype. Only an increase in crypt stem cell number, not changes in the rate of cell cycle proliferation, differentiation, or apoptosis of the non-stem cell population, simulated this abnormality. This suggests that APC regulates the number of stem cells in the colonic crypt and when the cells become mutant, an expansion of the crypt stem cell population results.     

Molecular genetics of ulcerative colitis-associated colon cancer in the interleukin 2- and beta(2)-microglobulin-deficient mouse

Mice deficient in beta(2)-microglobulin and interleukin 2 (beta(2)m(null) x IL-2(null)) spontaneously develop colon cancer in the setting of chronic ulcerative colitis (UC). We investigated mutations of the Apc and p53 genes and microsatellite instability in colonic adenocarcinomas arising in this model. Mutations of the Apc and p53 genes in the regions corresponding to mutation hot spots in human colorectal cancer were determined by sequencing in 11 colonic adenocarcinomas. Microsatellite instability was determined in matched normal and neoplastic DNA at five loci. All 11 adenocarcinomas harbored Apc mutations. Of these 11 tumors, 5 harbored truncating mutations. A total of 67 Apc mutations were found in these 11 tumors; 59 were missense mutations, whereas 8 were frameshift or nonsense mutations. Six of the 11 adenocarcinomas harbored p53 mutations. A total of seven p53 mutations were found in these 11 tumors; all mutations were transitions, 4 of which were C:G-->T:A transitions occurring in codon 229 at cytosine-guanine dinucleotides. Nine adenocarcinomas exhibited microsatellite instability in at least one of the five loci examined; 1 tumor had microsatellite instability in two loci. Molecular genetics, as well as clinical features, of colon cancer in the beta(2)m(null) x IL-2(null) mice are similar to those of human UC-associated colorectal cancer. As such, this model appears to be an excellent animal model to study UC-associated colorectal carcinogenesis.     

Colitis-related rat colon carcinogenesis induced by 1-hydroxy-anthraquinone and methylazoxymethanol acetate (Review)

Patients with long-standing ulcerative colitis (UC) have an increased risk for developing colorectal cancer (CRC) compared to the general population. For investigation of the mechanisms and prevention of UC and UC-related CRC, establishment of a promising animal model for such disease is important. 1-hydroxyanthraquinone (1-HAQ) present in certain medicinal plants such as Rubia tinctorum L. is a genotoxic and rodent colon carcinogen. Long-term feeding of 1-HAQ induced hyper-cell proliferation in rat colonic crypts with ulcerative changes, crypt abscess, severe inflammation and erosion before the occurrence of tumors, which are similar to those found in human UC. In addition, 1-HAQ has a synergistic effect with methylazoxymethaol (MAM) acetate on colon carcinogenesis. The polymerase chain reaction-single strand conformation polymorphism analysis revealed no mutations in Ki-ras and p53 in colonic neoplasms induced by MAM acetate + 1-HAQ, MAM acetate alone or 1-HAQ alone. Also, no mutations of APC were found in these tumors. These findings are similar to those found in human ulcerative colitis-associated colon cancer in contrast with sporadic colon cancers. A previous study revealed that induced colonic tumors had beta-catenin mutation with high frequency, suggesting tumor development by activation of the beta-catenin-Tcf signaling pathway. Increased expression in TNF-alpha and IL-1alpha was found in these induced colonic neoplasms, and the expression was more remarkable in colonic mucosa of rats exposed to MAM acetate + 1-HAQ, MAM acetate or 1-HAQ when compared with that in untreated rats. Thus, these cytokines may act as growth factors in rat colon carcinogenesis by MAM acetate and 1-HAQ and the synergistic effect of 1-HAQ with MAM acetate might be related to the biological effects of the cytokines expressed in the inflammatory conditions induced by 1-HAQ.     

Oncogenic K-ras promotes early carcinogenesis in the mouse proximal colon

Oncogenic K-ras mutations are frequently observed in colon cancers and contribute to transformed growth. Oncogenic K-ras is detected in aberrant crypt foci (ACF), precancerous colonic lesions, demonstrating that acquisition of a K-ras mutation is an early event in colon carcinogenesis. Here, we investigate the role of oncogenic K-ras in neoplastic initiation and progression. Transgenic mice in which an oncogenic K-ras(G12D) allele is activated in the colonic epithelium by sporadic recombination (K-rasLA2 mice) develop spontaneous ACF that are morphologically indistinguishable from those induced by the colon carcinogen azoxymethane (AOM). Similar neoplastic changes involving the entire colon are induced in transgenic mice constitutively expressing K-ras(G12D) throughout the colon (LSL-K-ras(G12D)/Villin-Cre mice). However, the biochemistry and fate of K-ras-induced lesions differ depending upon their location within the colon in these mice. In the proximal colon, K-ras(G12D) induces increased expression of procarcinogenic protein kinase C beta II (PKC beta II), activation of the MEK/ERK signaling axis and increased epithelial cell proliferation. In contrast, in the distal colon, K-ras(G12D) inhibits expression of procarcinogenic PKC beta II and induces apoptosis. Treatment of K-rasLA2 mice with AOM leads to neoplastic progression of small ACF to large, dysplastic microadenomas in the proximal, but not the distal colon. Thus, oncogenic K-ras functions differently in the proximal and distal colon of mice, inducing ACF capable of neoplastic progression in the proximal colon, and ACF with little or no potential for progression in the distal colon. Our data indicate that acquisition of a K-ras mutation is an initiating neoplastic event in proximal colon cancer development in mice.     

Short term infusion of glycine-extended gastrin(17) stimulates both proliferation and formation of aberrant crypt foci in rat colonic mucosa.

Evidence is accumulating that gastrin precursors may act as growth factors for the colonic mucosa in vivo and for colorectal carcinoma cell lines in vitro. The effect of short term administration of synthetic gastrins on the colonic mucosa in vivo, however, has not been reported. The aim of our study was to determine whether continuous systemic infusion of glycine-extended gastrin(17) stimulated proliferation and accelerated carcinogenesis in the colorectal mucosa. A significant increase in colonic mucosal proliferation as assessed by metaphase index was seen in the caecum (23%, p < 0.02) and distal colon (27%, p < 0.001), but not the rectum, after treatment of intact rats with glycine-extended gastrin(17) for 1 week using implanted miniosmotic pumps. Defunctioning of the rectum reduced both the proliferative index and crypt height of the rectal mucosa of untreated rats. Treatment of rectally defunctioned animals with glycine-extended gastrin(17) for either 1 or 4 weeks resulted in a significant increase in both the proliferative index (40% and 93%, respectively) and crypt height (11% and 19%, respectively) of the rectal mucosa. The total number of aberrant crypt foci in intact rats treated with the procarcinogen azoxymethane plus glycine-extended gastrin(17) was increased by 48% compared to the value in controls treated with azoxymethane only (p = 0.01). We conclude that short term administration of glycine-extended gastrin(17) to mature rats not only has a proliferative effect upon colonic mucosa, but also increases the number of aberrant crypt foci formed in the colorectal mucosa after treatment with azoxymethane. Glycine-extended gastrin(17) could thus potentially act as a promoter of carcinogenesis.     

Damage-induced apoptosis in intestinal epithelia from bcl-2-null and bax-null mice: investigations of the mechanistic determinants of epithelial apoptosis in vivo

The influence of bcl-2 and bax expression on apoptotic cell death in mouse intestinal epithelia was assessed using homozygously null mice. Apoptosis was induced in vivo by the enterotoxin 5-fluorouracil (5FU) or by gamma-irradiation and its cell positional incidence was assessed. 5FU and gamma-radiation treated bax-null mice surprisingly showed no reductions in apoptotic yield in the small intestine or midcolon at 4.5 h at cell positions in which both agents had previously been shown to strongly induce p53 protein expression. The colonic epithelia of 5FU treated bcl-2-null mice showed elevated levels of apoptosis at 4.5 h: from 48 apoptotic events in wild-type mice to 273 in the nulls, scoring 200 half crypts. The increase occurred specifically in the cell positions considered to harbour colonic stem cells, at the base of crypts, where there is selective expression of bcl-2. There was a modest but significant increase in apoptosis in the small intestine of the bcl-2-null mice although the epithelia of wild-type mice here are not immunohistochemically positive for bcl-2 protein. These findings show that bcl-2 plays a key role in determining the sensitivity of colonic stem cells to damage-induced death but that bax is not responsible for the p53-dependent induction of apoptosis in this context.     

A novel inflammation-related mouse colon carcinogenesis model induced by azoxymethane and dextran sodium sulfate

To develop an efficient animal model for colitis-related carcinogenesis, male Crj: CD-1 (ICR) mice were given a single intraperitoneal administration (10 mg/kg body weight) of a genotoxic colonic carcinogen, azoxymethane (AOM), and a 1-week oral exposure (2% in drinking water) to a non-genotoxic carcinogen, dextran sodium sulfate (DSS), under various protocols. At week 20, colonic neoplasms (adenocarcinomas, 100% incidence with 5.60±2.42 multiplicity; and adenomas, 38% incidence with 0.20±0.40 multiplicity) with dysplastic lesions developed in mice treated with AOM followed by DSS. Protocols in which AOM was given during or after DSS administration induced a few tubular adenomas or no tumors in the colon. Immunohistochemical investigation of such dysplasias and neoplasms revealed that all lesions were positive for β-catenin, cyclooxygenase-2 and inducible nitric oxide synthase, but did not show p53 immunoreactivity. The results indicate that 1-week administration of 2% DSS after initiation with a low dose of AOM exerts a powerful tumor-promoting activity in colon carcinogenesis in male ICR mice, and may provide a novel mouse model for investigating colitis-related colon carcinogenesis and for identifying xenobiotics with modifying effects.