Masumi Suzui

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.

Suppression of azoxymethane-induced rat colon aberrant crypt foci by dietary protocatechuic acid.

The modifying effect of dietary exposure to protocatechuic acid (PCA) on the development of azoxymethane (AOM)‐induced colonic aberrant crypt foci (ACF) was investigated in male F344 rats. The effects of PCA feeding on the silver‐stained nucleolar organizer regions protein (AgNORs) count in the colonic epithelial cells and on the ornithine decarboxylase (ODC) activity in the colonic mucosa were also estimated. Animals were given weekly s.c. injections of AOM (15 mg/kg body weight) for 3 weeks to induce ACF. These rats were fed diet containing 1000 or 2000 ppm PCA for 5 weeks, starting one week before the first dosing of AOM. All rats were killed 2 weeks after the last AOM injection, to measure the number of ACF, ODC activity, and AgNORs count per nucleus in the colon. In rats given AOM and PCA, the frequency of ACF/colon was significantly decreased compared with that in rats given AOM alone (P< 0.005 at 1000 and P< 0.05 at 2000 ppm). ODC activity in the colon of rats given AOM and PCA at both doses was also significantly lower than that of rats treated with AOM alone (P< 0.05). Similarly, the mean AgNORs count in rats fed PCA was significantly smaller than that of rats treated with AOM alone (P< 0.0001). Treatment with PCA alone did not affect these three biomarkers. These results provide further evidence that PCA could be a chemopreventive agent against rat colon carcinogenesis.

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.

Enhanced colon carcinogenesis induced by azoxymethane in min mice occurs via a mechanism independent of β-catenin mutation

The multiple intestinal neoplasia (min) mouse is a well-established cancer model in which loss of a single copy of the APC protein predisposes mice to the development of numerous tumors in the intestine. We have developed a novel variation of the min mouse model by using azoxymethane (AOM) to cause an increase in tumor incidence, number and size. Thus, treatment of min mice with AOM resulted in 2.6-, 6.3- and 5.9-fold increases in overall tumor incidence, multiplicity and size, respectively, when compared to wild type C57BL/6J mice treated with AOM. Furthermore, adenocarcinomas of the colon, which are otherwise relatively rare in min mice, increased in incidence (P<0.004), multiplicity (P<0.005), and size (P<0.02) in the AOM-treated min mice when compared to control untreated min mice. Of these adenocarcinomas, the number of poorly plus moderately differentiated adenocarcinomas was also significantly higher in the AOM-treated min mice (P<0.008). Thirty-seven histopathologically verified colon tumors (eight adenomas, five carcinoma in situ and 24 adenocarcinomas) induced in min mice and in C57BL/6J mice after treatment with or without AOM were analyzed for mutations in the beta-catenin gene or de novo mutations in the Apc gene. No mutations in the beta-catenin gene were found in any of colon tumors in min mice with or without treatment with AOM. However, mutations in either the beta-catenin gene or the Apc gene were found in tumors induced in C57BL/6J mice by AOM. These results suggest that mutations in the beta-catenin gene are less contributory to tumor development in min mice, as is the case in familial adenomatous polyposis (FAP) in humans. However, de novo mutations in either the Apc or beta-catenin gene can play a role in tumor development in C57BL/6J mice treated with AOM. The differences in mutation status between min and C57BL/6J mice may indicate different genetic pathways for developing colon tumors. These two experimental systems may, therefore, be useful animal models of human colon carcinomas in patients with FAP and in patients with sporadic colon carcinomas.

β‐Catenin (Ctnnb1) Gene Mutations in Diethylnitrosamine (DEN)‐induced Liver Tumors in Male F344 Rats

Alterations in multiple phosphorylation sites on exon 3 of the β‐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 β‐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 β‐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.

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. Mol. Carcinog. 19:25–30, 1997.

The mRNA overexpression of inflammatory enzymes, phospholipase A2 and cyclooxygenase, in the large bowel mucosa and neoplasms of F344 rats treated with naturally occurring carcinogen, 1-hydroxyanthraquinone

Inflammation has been considered to be related to carcinogenesis. Previously, we demonstrated that 1-hydroxyanthraquinone (1-HA), a naturally occurring carcinogen, induced severe inflammation such as ulcerative colitis in colonic mucosa. We also showed that indomethacin inhibited the tumorigenicity of 1-HA. In this study, we examined the expressions of major enzymes in arachidonic acid cascade related to inflammation in the colon mucosa of rats treated with 1-HA. After the treatment of 1% 1-HA diet, colon lesions were observed and RNA was extracted from mucosa and neoplasms. The mRNA expressions of group II phospholipase A2, cyclooxygenase-2 and 5-lipoxygenase, were examined by using a reverse transcriptase polymerase chain reaction. The expressions of phospholipase A2 and cyclooxygenase were significantly increased in non-neoplastic mucosa in rats treated with 1-HA compared with those in control rats. The expressions in the neoplasms induced by 1-HA were also increased. Phospholipase A2, especially, was much higher in the neoplasms than in non-neoplastic mucosa. However, the expression of 5-lipoxygenase showed no change in the non-neoplastic mucosa and neoplasms of rats treated with 1-HA, compared with that in control rats. These findings suggest that the inflammation induced by 1-HA may be related to the metabolites through a cyclooxygenase pathway, which indicates a prostaglandin synthesis, but not through a lipoxygenase pathway, which indicates a leukotriene synthesis in arachidonic acid cascade.

Genetic alterations in a patient with Turcot's syndrome

Turcots 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 adenocar‐clnoma). Germline mutations of the p53 gene, somatic mutations of the Ki‐ras, p53and APC genes, and microsatel‐lite Instability (MSI) was examined using polymerase chain reaction (PCR)‐slngle strand conformation polymorphism analysis, followed by PCR‐dlrect sequencing, and sequencing after subclonlng. No germline mutations of the p53 gene were found. Somatic mutations of Kl‐ras and APC genes were found in the colon adenocarcinoma but not in the brain tumor. No somatic mutation of the pS3 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 Kl‐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.