Long term effects of high fat and sucrose diets on obesity and lymphocyte proliferation in mice.

OBJECTIVE: To clarify the effect of prolonged feeding of a high-fat and sucrose, and to clarify the effect of sucrose instead of other carbohydrate on obesity and immunity in C57BL/6J mice. METHODS: We investigated the development of obesity and immune cell function in four groups of mice fed high-fat, high-fat plus high-sucrose, high-sucrose, and control diet for 7 months. RESULTS: Mice fed high-fat and high-fat plus high-sucrose groups developed severe obesity. Body weight, adipose tissue weight, serum leptin, blood glucose, and insulin were significantly higher, while the level of serum soluble leptin receptor was significantly lower in mice fed high-fat and high-fat plus high-sucrose diets than in mice fed the control or high-sucrose diets. Splenocyte proliferation stimulated by T-cell mitogen (PHA, ConA, and anti-CD 3 antibody) and B-cell mitogen (LPS) was significantly lower in both obese, high-fat and high-fat plus high-sucrose groups than in control and high-sucrose groups. However, these parameters did not differ between high-fat and high-fat plus high-sucrose groups. CONCLUSIONS: Long-term feeding of high-fat diet and high-fat plus high-sucrose diet similarly induced severe obesity in C57BL/6J mice. Not only T-cell, but also B-cell function may be impaired in mice made severely obese by the high-fat or high-fat plus high-sucrose diets.

Distribution of preneoplastic lesions and tumors, and beta-catenin gene mutations in colon carcinomas induced by 1,2-dimethylhydrazine plus dextran sulfate sodium.

Mucin-depleted foci (MDF) are considered as useful biomarkers in rat colon carcinogenesis. The purpose of the present study was to examine the mechanism(s) underlying rat colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH) plus 1% Dextran Sulfate Sodium (DSS). Twelve male F344 rats were given subcutaneous injections (40mg/kg body) of DMH twice a week. They received DSS in the drinking water for 1 week after the first injection of DMH and then were maintained on tap water. The rats were sacrificed at 10 and 14 weeks after the first injection of DMH. Colon tissues were divided into 10 segments from anus to cecum (A/J) and stained with Alcian blue (AB) to identify MDF. We found that MDF and tumors were induced in the rat colon after treatment with DMH plus DSS and that the number of MDF in each segment of the colon was significantly correlated with that of tumors (p=0.006). In addition, we found that the beta-catenin protein was accumulated in cytoplasm and nuclei of MDF and the frequent beta-catenin gene mutations in the colon tumors. These results suggest that MDF is closely related to rat colon carcinogenesis induced by DMH plus DSS.

Modifying effects of Terminalia catappa on azoxymethane-induced colon carcinogenesis in male F344 rats.

The modifying effects of dietary administration of an herb, Terminalia catappa (TC), were investigated on rat colon carcinogenesis induced by a carcinogen azoxymethane (AOM). The number of aberrant crypt foci (ACF) and beta-catenin accumulated crypts (BCACs) in the colon, and proliferating cell nuclear antigen (PCNA) labelling index in the colonic epithelium were examined in a total of 36 male F344 rats. All animals were randomly divided into five experimental groups (4-10 rats in each group). At 6 weeks of age, rats in groups 1, 2 and 3 were given s.c. injections of AOM once a week for 2 weeks at a concentration of 20 mg/kg body weight. One week before the first injection of AOM, rats in groups 2 and 3 were fed a diet containing 0.02 and 0.1% TC, respectively, throughout the experiment. Rats in group 4 were fed a diet containing 0.1% TC. Rats in group 5 were served as untreated controls. All animals were sacrificed at the experimental week 5 after the start of the experiment. Oral administration of TC at both doses significantly decreased the numbers of both ACF/colon/rat (P<0.05 for 0.02% TC, P<0.005 for 0.1% TC) and BCAC/cm/rat (P<0.05 for both 0.02 and 0.1% TC), when compared with the control group (group 1). Colonic PCNA labelling index in groups 2 and 3 was also significantly lower than that in group 1 (P<0.001 for 0.02% TC, P<0.005 for 0.1% TC). These results suggest that TC has a potent short-term chemopreventive effect on biomarkers of colon carcinogenesis and this effect may be associated with the inhibition of the development of ACF and BCACs.

Effects of epigallocatechin-3-gallate on growth, epidermal growth factor receptor signaling pathways, gene expression, and chemosensitivity in human head and neck squamous cell carcinoma cell lines.

The antitumor effects of the green tea compound epigallocatechin-3-gallate (EGCG) have not been studied in detail previously in head and neck squamous cell carcinoma (HNSCC) cells. Overexpression of the epidermal growth factor receptor (EGFR) occurs frequently in HNSCC, which is an adverse prognostic factor. Therefore, we examined in detail the molecular effects of EGCG on two human HNSCC cell lines, YCU-N861 and YCU-H891, focusing on the EGFR signaling pathway. The 70% lethal dose (IC(70)) of EGCG for both cell lines was 10 microg/ml. Treatment with EGCG increased the proportion of cells in the G(1) phase of the cell cycle and induced apoptosis. In cells treated with EGCG, there was a decrease in the cyclin D1 protein, an increase in the p21(Cip1) and p27(Kip1) proteins, and a reduction in the hyperphosphorylated form of pRB, changes that may account for the arrest in G(1). EGCG also caused a decrease in the Bcl-2 and Bcl-X(L) proteins, an increase in the Bax protein, and activation of caspase 9, suggesting that EGCG induces apoptosis via a mitochondrial pathway. Treatment with EGCG inhibited phosphorylation of the EGFR, signal transducer and activator of transcription3 (Stat3), and extracellular regulated kinase (ERK) proteins and also inhibited basal and transforming growth factor-alpha-stimulated c-fos and cyclin D1 promoter activity. EGCG at 0.1 microg/ml (a concentration found in serum after oral administration) markedly enhanced the growth-inhibitory effects of 5-fluorouracil. Taken together, these findings provide insights into molecular mechanisms of growth inhibition by EGCG and suggest that this naturally occurring compound may be useful, when used alone or in combination with other agents, in the chemoprevention and/or treatment of HNSCC.

Different mutation status of the beta-catenin gene in carcinogen-induced colon, brain, and oral tumors in rats.

Mutations in the region corresponding to the N-terminal phosphorylation sites (codons 1-51) of the rat beta-catenin gene (Ctnnb1) were investigated in rat colon tumors induced by 1-hydroxyanthraquinone (1-HA) plus methylazoxymethanol (MAM) acetate, by using polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis. The beta-catenin gene was also screened for mutations in rat brain and oral tumors induced by ethyl nitrosourea (ENU) and 4-nitroquinoline 1-oxide (4-NQO), respectively. In colon tumors, beta-catenin gene mutations were found in two of three adenomas (67%) and 26 of 28 adenocarcinomas (93%), with a total incidence of 90% (28 of 31 adenomas plus adenocarcinomas). Eight (29%) were (34)G-->T (second position), eight (29%) were (32)G-->A (first position), five (18%) were (34)G-->A (first position), five (18%) were (41)C-->T (second position), one (4%) was (34)G-->A (second position), and one (4%) was (32)A-->G (second position), mutations, resulting in the substitutions of Gly(34)-->Val, Asp(32)-->Asn, Gly(34)-->Arg, Thr(41)-->Ile, Gly(34)-->Glu, and Asp(32)-->Gly, respectively. The (34)G-->T (second position) mutations found in this study were unique compared to those found in other carcinogen-induced rat colon carcinogenesis models. In contrast, beta-catenin gene mutations were not found in either the brain or oral tumors. These results suggest that mutations in the beta-catenin gene in rat tumors occur in specific tissues or organ sites and in a carcinogen-specific manner. Thus, the mutation spectrum in the beta-catenin gene is organ- and chemical carcinogen-specific.

Highly sensitive determination of plasma cytokines by time-resolved fluoroimmunoassay; effect of bicycle exercise on plasma level of interleukin-1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha), and interferon gamma (IFN gamma).

A highly sensitive time-resolved fluoroimmunoassay of human plasma cytokines is described. The cytokines such as interleukin-1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha) are known to be acute inflammatory cytokines and it has been reported that these cytokines are secreted into blood by physical exercise. In this study, a sandwich-type immunoassay of cytokines was established using a europium chelate BHHCT-Eu3+ as a powerful labeling material. The minimum detection limits of cytokines, i.e. IL-1 alpha, TNF alpha, and interferon gamma (IFN gamma) were about 1/10 smaller than those of enzyme-linked immunosorbent assay currently used. By this immunoassay we investigated cytokine increase/decrease in plasma which was thought to derive from the myocytes damaged by bicycle exercise. Healthy young men performed two kinds of bicycle ergometer exercises, under conditions of an incremental and a constant loading. Blood samples were taken before, during, and after exercises, and the concentration levels of plasma IL-1 alpha, TNF alpha, and IFN gamma were determined. In the case of incremental exercise, IL-1 alpha increased significantly at the first stage but decreased to the basal level from the second stage, in spite of heavier exercise. In the case of 30 min constant exercise, the level of plasma IFN gamma increased in recovery period, 2 h after the light-exercise. TNF alpha level was significantly higher in a heavy-exercise. The concentration of IL-1 alpha peaked at the early stage of the incremental exercise; this fact has not been reported in previous studies. This cytokine is unique in showing a sudden increase during the early stage, while others increase after the exercise. Our highly sensitive assay made it possible to detect a slight change in plasma cytokines.

Mobilization of NK cells by exercise: downmodulation of adhesion molecules on NK cells by catecholamines.

The change of plasma catecholamine concentration correlates with the change of natural killer (NK) activity and NK cell number in peripheral blood mononuclear cells (PBMC) during and after moderate exercise. We studied the causal relation between exercise-induced catecholamine and expression of adhesion molecules on NK cells during and after exercise. The expression of CD44 and CD18 on CD3(-)CD56(+) NK cells was significantly reduced during exercise (P < 0.01). When PBMC were stimulated with 10(-8)M norepinephrine in vitro, the expression of these adhesion molecules on CD3(-)CD56(+) NK cells was downmodulated within 30 min. The binding capacity of NK cells to a CD44 ligand, hyaluronate, was reduced by the stimulation with norepinephrine (P < 0.01). The intravenous injection of norepinephrine in mice decreased the expression of CD44 and CD18 on CD3(-)NK1.1(+) cells (P < 0.01) and increased the number of CD3(-)NK1.1(+) cells in PBMC (P < 0.01). These findings suggest that exercise-induced catecholamines modulate the expression of adhesion molecules on NK cells, resulting in the mobilization of NK cells into the circulation.

Chromosomal mapping of genes controlling development, histological grade, depth of invasion, and size of rat stomach carcinomas.

Rat stomach cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are widely used as a model of differentiated-type human stomach cancers. ACI/N (ACT) rats are susceptible and BUF/Nac (BUF) rats are resistant to MNNG-induced stomach carcinogenesis, and the presence of an autosomal gene with a dominant BUF allele has been suggested. In this study, we performed a carcinogenicity test by giving MNNG in drinking water to 117 male ACI x (ACIxBUF)F1 backcross rats. Each of 100 effective rats was diagnosed for its "carcinoma development" and when it was bearing stomach carcinoma(s), for histological grade, depth of invasion, and size and number of tumors. Carcinoma development was diagnosed based both on the age of the rat and on the presence of stomach carcinoma(s). Linkage analysis was performed with the genotypes of 161 loci, covering 1637 cM of the rat genome. Contrary to our original expectations, the most influential gene was the one on chromosome (chr.) 15, Gastric cancer susceptibility gene 1 (Gcs1), which confers susceptibility to stomach carcinogenesis (LOD, 3.8) with a dominant BUF allele by promoting conversion from adenomas to carcinomas. Two resistance genes on chr. 4 and chr. 3, Gastric cancer resistance gene 1 (Gcr1) and Gcr2, were shown to confer dominant resistance (LOD, 2.7 and 2.6, respectively). Gcs1, Gcr1, and Gcr2 exerted additive effects on the development of stomach carcinomas. A gene on chr. 16, Gcr3, was indicated to reduce the depth of invasion (LOD, 2.2) and sizes of tumors (LOD, 1.9). No linkage was obtained using the number of tumors. These findings show that the coordinate effect of a susceptibility gene, Gcs1, and two resistance genes, Gcr1 and Gcr2, is responsible for the development of MNNG-induced stomach carcinomas and that Gcr3 is responsible for the growth of a stomach carcinoma, reflected in the depth of invasion and in the tumor size.

Beta-catenin (Ctnnb1) gene mutations in diethylnitrosamine (DEN)-induced liver tumors in male F344 rats.

Alterations in multiple phosphorylation sites on exon 3 of the beta-catenin gene have recently been implicated in hepatocarcinogenesis in humans as well as mice. To identify genetic alterations which could be involved in the chemical-induced hepatocarcinogenesis of rats, we analyzed the status of the sites in the beta-catenin gene (Ctnnb1) of liver neoplasms induced by diethylnitrosamine (DEN) in male F344 rats, using the polymerase chain reaction-single strand conformation polymorphism method. In the present investigation, we examined 35 hepatocellular neoplasms (28 adenomas and 7 carcinomas) for the expression of mutations in the region of the beta-catenin gene. Point mutation at codon 32, 35, 37 or 41, which has been reported in human and mouse liver cell carcinomas and/or other cancers, was recognized in eleven (31%) out of 35 lesions (8 adenomas and 3 carcinomas). Our results indicate that Ctnnb1 mutations may contribute to hepatocarcinogenesis in rats. Our finding that Ctnnb1 mutation was present in adenomas as well as carcinomas also suggests that the mutation is a relatively early event in DEN-induced hepatocarcinogenesis in rats.

Indomethacin inhibits circulating PGE2 and reverses postexercise suppression of natural killer cell activity.

Natural killer (NK) cells are important in combating viral infections and cancer. NK cytolytic activity (NKCA) is often depressed during recovery from strenuous exercise. Lymphocyte subset redistribution and/or inhibition of NK cells via soluble mediators, such as prostaglandin (PG) E2 and cortisol, are suggested as mechanisms. Ten untrained (peak O2 consumption = 44.0 +/- 3.5 ml. kg-1. min-1) men completed at 2-wk intervals a resting control session and three randomized double-blind exercise trials after the oral administration of a placebo, the PG inhibitor indomethacin (75 mg/day for 5 days), or naltrexone (reported elsewhere). Circulating CD3(-)CD16(+)/56(+) NK cell counts, PGE2, cortisol, and NKCA were measured before, at 0.5-h intervals during, and at 2 and 24 h after a 2-h bout of cycle ergometer exercise (65% peak O2 consumption). During placebo and indomethacin conditions, exercise induced significant (P < 0.0001) elevations of NKCA (>100%) and circulating NK cell counts (>350%) compared with corresponding control values. With placebo treatment, total NKCA was suppressed (28%; P < 0.05) 2 h after exercise, and a postexercise elevation (36%; P = 0.02) of circulating PGE2 was negatively correlated (r = 0.475, P = 0.03) with K-562 tumor cell lysis. NK counts were unchanged in the postexercise period, but at this stage CD14(+) monocyte numbers were elevated (P < 0.0001). Indomethacin treatment eliminated the postexercise increase in PGE2 concentration and completely reversed the suppression of total and per CD16(+)56(+) NKCA 2 h after exercise. These data support the hypothesis that the postexercise reduction in NKCA reflects changes in circulating PGE2 rather than a differential lymphocyte redistribution.

Frequent mutations of the rat beta-catenin gene in colon cancers induced by methylazoxymethanol acetate plus 1-hydroxyanthraquinone.

Recent evidence suggests that the beta-catenin gene (CTNNB1) acts as an oncogene, and some human colon tumors with an intact APC gene have activating mutations in CTNNB1. In this study, mutations in the region corresponding to N-terminal phosphorylation sites (codons 1-51) of the rat Ctnnb1 gene were investigated in 20 colon tumors associated with ulcerative colitis and induced with methylazoxymethanol acetate and 1-hydroxyanthraquinone. Ninety percent (18 of 20) of the tumors induced in male F344 rats harbored mutations, which were detected in three of four adenomas (75%) and 15 of 16 adenocarcinomas (94%). Of 18 total missense mutations, 13 (72%) were G-->A transitions at position 101, three were G-->A transitions at position 94, and two were C-->T transitions at position 122, resulting in the amino acid substitutions Gly34-->Glu, Asp32-->Asn, and Thr41-->Ile, respectively. Although there were no mutations in the Apc gene, as we previously reported in the same tumor samples, the results obtained in this study strongly implicate the Apc-beta-catenin-T-cell factor (Tcf) signaling pathway in methylazoxymethanol acetate, 1-hydroxyanthraquinone-induced colon carcinogenesis.

beta-Endorphin and natural killer cell cytolytic activity during prolonged exercise. is there a connection?

This study was designed to test whether a single 50-mg dose of the opioid antagonist naltrexone hydrochloride, ingested 60 min before 2 h of moderate-intensity exercise (i.e., 65% peak O2 consumption), influenced the exercise-induced augmentation of peripheral blood natural killer cell cytolytic activity (NKCA). Ten healthy male subjects were tested on four occasions separated by intervals of at least 14 days. A rested-state control trial was followed by three double-blind exercise trials [placebo (P), naltrexone (N), and indomethacin] arranged according to a random block design. The indomethacin exercise trial is discussed elsewhere (S. G. Rhind, G. A. Gannon, P. N. Shek, and R. J. Shepherd. Med. Sci. Sports Exerc. 30: S20, 1998). For both the P and N trials, plasma levels of beta-endorphin were increased (P < 0.05) at 90 and 120 min of exercise but returned to resting (preexercise) levels 2 h postexercise. CD3(-)CD16(+)CD56(+) NK cell counts and NKCA were significantly (P < 0.05) elevated at each 30-min interval of exercise compared with correspondingly timed resting control values. However, there were no differences in NK cell counts or NKCA between P and N trials at any time point during the two trials. Changes in NKCA reflected mainly changes in NK cell count (r = 0.72; P < 0.001). The results do not support the hypothesis that the enhancement of NKCA during prolonged submaximal aerobic exercise is mediated by beta-endorphin.

Generation of polymorphic markers tightly linked to the thymus enlargement loci by phenotype-directed representational difference analysis.

To obtain genetic markers linked to a specific genetic locus, genomic subtraction with a DNA pool of backcross or F2 intercross animals with a specific genotype at the locus is known to be effective. To determine whether the pooling strategy is also effective for isolation of genetic markers linked to a quantitative phenotype that can potentially be controlled by multiple genetic loci, we tested the ability of representational difference analysis (RDA) to isolate genetic markers linked to the thymus enlargement observed in the BUF/Mna (BUF) rat. This is known to be controlled by single major and minor genes, Ten1 and Ten2, on Chromosomes (Chrs) 1 and 13, respectively, both of which have dose effects on the normal WKY/Ncj (WKY) allele. DNA from an inbred WKY rat was used as the tester, and the driver was prepared from a DNA pool of 12 (WKY x BUF)F1 x BUF backcross rats with high thymus ratios (thymus weight/body weight), expected to have dominance of the BUF allele in the responsible loci. By two RDA series with the restriction enzymes BglII and BamHI, respectively, 28 polymorphic markers were isolated, and 8 of them were shown to be linked to Ten1, and one to Ten2. One of the 8 markers linked to Ten1 demonstrated no recombination in 18 rats with high thymus ratios. RDA with a DNA pool based on a quantitative phenotype (phenotype-directed RDA) can thus be considered an efficient approach for direct isolation of polymorphic markers linked to a quantitative trait.

Genetic alterations in a patient with Turcot's syndrome.

Turcot's syndrome (TS) is a rare disorder associated with the development of both brain and colon neoplasms. Because of the very low incidence of the disease, its molecular basis remains unclear. Presented is a TS case of a 30-year-old Japanese male with a histopathologically confirmed diagnosis of both brain tumor (glioblastoma multiforme) and colon tumor (well-differentiated adenocarcinoma). Germline mutations of the p53 gene, somatic mutations of the Ki-ras, p53 and APC genes, and microsatellite instability (MSI) was examined using polymerase chain reaction (PCR)-single strand conformation polymorphism analysis, followed by PCR-direct sequencing, and sequencing after subcloning. No germline mutations of the p53 gene were found. Somatic mutations of Ki-ras and APC genes were found in the colon adenocarcinoma but not in the brain tumor. No somatic mutation of the p53 gene was present in either colon or brain tumors. Microsatellite instability of both colon and brain tumors was positive in two of four loci. These results indicate that the colon tumor of the TS patient carries the Ki-ras and APC gene mutations. The finding of MSI in both the brain and the colon tumors may support the hypothesis that alterations of DNA repair genes are involved in the tumor development of the TS patient.

High frequency of beta-catenin (ctnnb1) mutations in the colon tumors induced by two heterocyclic amines in the F344 rat.

Activating mutations in the beta-catenin (CTNNB1) gene corresponding to N-terminal phosphorylation sites in the protein have been implicated in the development of human colon cancer. To determine the possible involvement of such mutations during chemically induced colon carcinogenesis, we examined the corresponding region of Ctnnb1 in colon tumors induced in the F344 rat by two cooked meat heterocyclic amines, 2-amino-3-methylimidazo[4,5-f]quinoline and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). All of the colon tumors induced by 2-amino-3-methylimidazo[4,5-f]quinoline that were examined (5 of 5) and 4 of 7 PhIP-induced colon tumors had mutations within or flanking codons corresponding to important phosphorylation sites in beta-catenin. None of the colon tumors bearing Ctnnb1 mutations had genetic changes in the Apc gene, and those that contained wild-type Ctnnb1 were known from our previous work to contain Apc mutations. The results provide evidence for a major role of the beta-catenin/Apc pathway in the development of heterocyclic amine-induced colon tumors and give further weight to the view that regulation of beta-catenin is critical to the tumor suppressive effects of Apc during colon carcinogenesis. In contrast, Ctnnb1 mutations were completely absent in 23 PhIP-induced mammary tumors, in accordance with recent work showing that human breast carcinomas lack mutations in CTNNB1.

Expression of bcl-2, bax, and bcl-XL proteins in azoxymethane-induced rat colonic adenocarcinomas.

Using western blotting and immunochemical analysis, we investigated alterations in the expression of the apoptosis-related proteins bcl-2, bax, and bcl-X in colonic adenocarcinomas induced by subcutaneous injection of azoxymethane (AOM) (15 mg/kg body weight weekly for 2 wk) into male Sprague-Dawley rats. Expression of the apoptosis-repressor bcl-2 in the colonic tumors was significantly weaker (0.6-fold) than that in adjacent non-neoplastic mucosa. The expression of bax protein, an apoptosis accelerator, was significantly stronger (7.33-fold) in all the tumors than in the non-tumoral mucosa. bcl-XL protein, which functions as a repressor of apoptosis, was significantly upregulated (3.23-fold) in all the tumors when compared with the non-neoplastic mucosa. There was no significant difference between the expression of these proteins in the non-neoplastic mucosa of the AOM-treated rats and in the normal mucosa of saline-treated control rats. As determined by immunohistochemical analysis, the tumor cells had more bax and bcl-X protein. These findings indicate that the regulation of the apoptosis-related proteins bcl-2, bax, and bcl-XL was altered in the AOM-induced colonic neoplastic tissue. In terms of resistance to apoptosis, elevated levels bcl-XL protein may have considerable meaning in this experimental model as well as in human colorectal cancer.

Circulating levels of peripheral blood leucocytes and cytokines following competitive cycling.

The objective of the study was to determine if prolonged and strenuous cycling leads to a polarized cytokine response, and/or unique mobilization of circulating leucocyte populations. Resting venous blood samples were collected from 6 amateur cyclists, 24 hr before, and at 10-25 min and 150 min after completion of a 250-km road race (race time: 404 +/- 3.5 min). Total leucocyte counts were significantly elevated following competition. Cell counts of CD3+CD8bright+ lymphocytes were depressed by 50% 150 min after competition. A significant increase in CD4+ cells expressing the IL-2R alpha chain was evident 150 min after competition. IL-6 concentrations were greatly increased, both at 10-25 min and 150 min after competition. Resting TNF-a concentrations were approximately doubled at both time points after competition. Plasma levels of IFN-gamma, IL-10 and IL-12 were below detection thresholds at all time points. These results suggest that performance of a 6.5 h competitive cycle-race does not induce a Type-1 or Type-2-dominated cytokine response, but one that is typical of a proinflammatory cytokine response.

No involvement of APC gene mutations in ulcerative colitis-associated rat colon carcinogenesis induced by 1-hydroxyanthraquinone and methylazoxymethanol acetate.

A rat model for human ulcerative colitis (UC) has been developed by using 1-hydroxyanthraquinone (1-HA) to cause severe inflammation of colonic mucosa. 1-HA also has synergistic effects on the carcinogenicity of methylazoxymethanol (MAM) acetate in the rat colon. In this study, four adenomas and 16 adenocarcinomas induced in male F344 rats by 1-HA and MAM acetate were examined for mutations in the entire coding regions and introns flanking coding exons of the APC gene by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) and PCR-restriction-SSCP analyses. No mutations were found. These results, together with our previous observations of a relative lack of Ki-ras gene mutations in the same tumors, are similar to those found in human UC-associated colon cancer, suggest a common pathway in these two systems, although they are different in their implication of p53 mutations. Therefore, this model may have some relevance and application to the study of colon cancer in human inflammatory bowel disease, which is not associated with APC mutations or with Ki-ras or p53 mutations.

Telomerase activity of normal tissues and neoplasms in rat colon carcinogenesis induced by methylazoxymethanol acetate and its difference from that of human colonic tissues.

Telomerase activity in tissues may be related to tumor development, especially malignant conversion, in humans. However, there are few reports about telomeres and telomerase activity in animals. In this study, we examined telomerase activity in rat colon carcinogenesis and in normal rat liver tissue and compared it with that of human colon cancer tissues. This is the first report concerning telomerase activity in rats. F344 rats were used, and colon neoplasms were induced with methylazoxymethanol acetate. There was telomerase activity in not only the induced colon neoplasms but also the colon mucosa and livers of untreated rats, in contrast with the results from normal human somatic tissues in previous reports. Indeed, we also observed negative results in normal human mucosa, despite the positive results in colon-cancer tissues. These findings suggest that there is a difference in the telomerase activities in humans and rats. Because rat telomeres are very long (20-100 kp, average 50 kp) compared with human telomeres (5-15 kp, average 12 kp), the difference in the telomere lengths of rats and humans might be related to their enzyme activities, although this is still unclear. Furthermore, because the inhibition of telomerase has been proposed as a novel cancer therapy for humans, the rat model presented here, in which telomerase is expressed in somatic tissues, may be useful for studies of telomerase inhibition, including inhibition by chemopreventive agents.

Different expression patterns of cyclins A, D1 and E in human colorectal cancer.

The expressions of cyclins A, D1 and E at the protein level were investigated by Western blotting in human colorectal carcinomas and in adjacent non-neoplastic colorectal mucosas. Cyclin E was higher in the cancer tissue than in the non-neoplastic mucosa in 92% patients (35 out of 38 cases). However, the cyclin A expression of the mucosa was higher than that of the cancer tissue in 63% (25 out of 40 cases) cases, and only 4 (10%) cancers had higher cyclin A expression. Eleven cancers (27%) demonstrated expression equivalent to that in the mucosa. Equal expression of cyclin D1 in cancer and mucosal tissues was found in 51% cases (20/39), lower expression of cyclin D1 by cancer tissues was demonstrated in 41% cases (16/39) and only three cancers showed higher expression than the mucosa. Proliferating-cell nuclear antigen immunohistochemistry revealed that the labeling index of the cancer tissue was 43.5 +/- 8.3% while that of the mucosa was only 14.8 +/- 5.1%. These results proved that colorectal cancers express high levels of cyclin E, consistent with a high rate of cell proliferation, whereas most of such cancer lose control of cyclin A and cyclin D1 expression.