Ki-ras proto-oncogene mutations in sporadic colorectal adenomas: relationship to histologic and clinical characteristics.

BACKGROUND & AIMS: [corrected] The goal of this study was to examine the relationship between Ki-ras mutations in colorectal adenomas and characteristics of both the subject (age, gender, and family/personal history of colonic neoplasia) and the adenoma (multiplicity, size, location, and histologic features). METHODS: Ki-ras mutations were detected by direct sequencing in 738 adenomatous polyps removed at baseline from 639 participants in a nutritional trial of adenoma recurrence. RESULTS: Ki-ras mutations were detected in 17.2% of the adenomas. Ki-ras mutations were unrelated to gender, family, or personal history of colonic neoplasia, location within the colorectum, or adenoma multiplicity, but were more common in older subjects (P = 0.01 for trend), in larger adenomas (P < 0.0001 for trend), in adenomas with villous histology (odds ratio [OR], 3.2; 95% confidence interval [CI], 2.1-4.9 vs. tubular), and in adenomas with high-grade dysplasia (32.0% vs. 13.6%; OR, 3.0; 95% CI, 1.9-4.6 vs. low-grade dysplasia). Multivariate analysis showed Ki-ras mutations to be independently associated with subject age (P = 0.01 for trend), tubulovillous/villous histology (OR, 2.3; 95% CI, 1.5-3.7), and high-grade dysplasia (OR, 1.9; 95% CI, 1.2-3.1). Adenoma size was not independently related to Ki-ras mutation. CONCLUSIONS: Ki-ras mutations are associated with the histologic features of adenoma progression (villous histology and high-grade dysplasia) rather than with adenoma growth.

Expression of beta-catenin and full-length APC protein in normal and neoplastic colonic tissues.

Mutations of the APC gene are thought to be early events in the process of colorectal carcinogenesis. Although the complete function(s) of the APC gene product is not known, it has been shown that the APC protein interacts with beta-catenin in a multi-protein complex to regulate the level of expression of beta-catenin. Loss of normal APC protein function can lead to an accumulation of beta-catenin in the cytosol and the nucleus. Immunohistochemical methods were used to determine the relationship between APC and beta-catenin protein expression in human colonic tissues (150 normal, 9 hyperplastic, 58 adenomas and 83 carcinomas) and 12 paired samples of normal and cancer tissue in mouse colon. In all samples of normal human and mouse colonic mucosa and in human hyperplastic polyps both APC and beta-catenin immunoreactivity were present in colonocytes. APC expression was cytoplasmic, with maximal immunoreactivity in the goblet cells. beta-Catenin expression was predominantly localized to the plasma membrane, with no nuclear immunoreactivity. APC immunoreactivity was absent in all of the mouse adenocarcinomas and 83% of the human colon cancers. All of the human and mouse carcinomas had nuclear and cytoplasmic beta-catenin expression. In contrast, only 29% of the 58 colonic adenomas were completely negative for APC immunoreactivity. Regardless of the presence or absence of APC, all of the adenomas had cytoplasmic and nuclear beta-catenin immunoreactivity. Many colonic adenomas retain expression of full-length APC protein whereas it is usually lost in colorectal cancers. Regardless of the status of APC protein expression, beta-catenin protein was found in the cytoplasm and nucleus of all neoplastic colonic mucosa. The dissociation between loss of expression of APC and accumulation of beta-catenin in the nucleus suggests that inactivation of both alleles of the APC gene may not be required for beta-catenin nuclear accumulation in colonic adenomas.

Risk factors for Ki-ras protooncogene mutation in sporadic colorectal adenomas.

The Ki-ras protooncogene frequently is mutated in colorectal adenocarcinomas, but the etiology of this molecular event is uncertain. We investigated the association between variables known or suspected to be related to risk for colorectal cancer and the occurrence of Ki-ras mutations in colorectal adenomas. This study was conducted among 678 male and female participants, 40-80 years of age, enrolled in a phase III trial testing the effects of a wheat bran fiber supplement on adenoma recurrence. Exposure information on the risk factors of interest was assessed through self-administered questionnaires. Mutations in codons 12 and 13 of the Ki-ras protooncogene were analyzed in baseline adenomas 0.5 cm or larger by PCR amplification followed by direct sequencing. Eighteen percent (120 of 678) of the participants had one or more adenoma(s) with Ki-ras mutations. A higher risk of Ki-ras mutations was associated with increasing age and a lower intake of total folate. The odds ratio (OR) for Ki-ras mutations for individuals >72 years of age was 1.98 [95% confidence interval (CI) = 1.19-3.27; P for trend = 0.008] compared with those less than 65 years of age. Compared with individuals in the lower tertile of total folate, those in the upper tertile had an approximately 50% lower risk of having Ki-ras mutation-positive adenomas (OR = 0.52; 95% CI = 0.30-0.88; P for trend = 0.02). There was a suggestion of a stronger inverse association of total folate with G-->T transversions (OR = 0.41; 95% CI = 0.20-0.87) than G-->A transitions (OR = 0.61; 95% CI = 0.31-1.21), although the CIs for the associations overlap. The results of these analyses suggest that the protective effect of folate in colon cancer observed in published studies may be mediated through folate's effect on Ki-ras mutations.

Symptomatic colonic polyps in childhood: not so benign.

BACKGROUND: The clinical spectrum of symptomatic polyps and the frequency of familial polyposis is not well defined in children. In the present study, a series of children with juvenile polyposis coli (JPC) and non-JPC polyps were studied. METHODS: Children with symptomatic colonic polyps and negative family history of polyps were ascertained by review of endoscopic records. Juvenile polyposis coli was defined as 10 or more juvenile polyps or any juvenile polyp in a relative of an index case of JPC. Polyps were tested for Ki-ras mutations, p53 overexpression, and aneuploidy. RESULTS: Seventy-eight children (age range, 0.4-18 years) were identified, all evaluated for lower gastrointestinal bleeding. Nine (12%) had JPC, 66 (84%) had isolated juvenile polyps, and 3 (4%) had other types of polyps. The JPC and non-JPC groups were similar in age (p = 0.4) and symptom duration (p = 0.3). The JPC group had more polyps (p = 0.0001), and greater likelihood of anemia (p = 0.01), polyps with adenomatous change (p = 0.03), and right-colon polyps (p = 0.001). In three of eight JPC families, polyps were identified in asymptomatic first-degree relatives. No abnormalities in Ki-ras, p53, or aneuploidy were identified. CONCLUSIONS: Juvenile polyposis coli is common in children with symptomatic polyps, and is associated with anemia, right-colon polyps, and adenomas. The risk of polyps and of colorectal cancer in relatives of persons with JPC requires further study.

Ras oncogene mutations in childhood brain tumors.

Although N-nitroso compounds (NNC) are ubiquitous in the human environment and are known neurocarcinogens in animal models, results of epidemiological studies have not yet convincingly associated NNCs with brain tumor occurrence in humans. Animal studies have suggested that specific codons (12, 13, and 61) in the ras family are mutable by exposure to NNCs. The purpose of this study was to measure the presence of mutations in the ras family of oncogenes in tissue from childhood brain (CB) tumors as a preliminary step toward investigating their potential use as biomarkers of chemical exposure. DNA was extracted from paraffin-embedded formalin-fixed CB tumors from tissues resected during neurosurgical operations. Using the PCR, designed RFLP-screening methods, and sequencing, we attempted to screen brain tumors from 46 children for the presence of H, K, and N-ras mutations at codons 12, 13, and 61. Screening for oncogene mutations using PCR, RFLP methods, and DNA sequencing was successfully completed for a high proportion of the available specimens. Astrocytoma specimens from three children for whom screening with PCR was successfully completed were found to contain CAA-->GAA point mutations in K-ras at codon 61. None of the specimens contained mutations at any of the other locations. These results, although preliminary, provide a potential clue for future mechanistic studies of CB tumors. The possible roles of NNCs in inducing this mutation, or of this mutation as an early or late event in tumor progression, however, remain unclear.

AOM-induced mouse colon tumors do not express full-length APC protein.

While evidence in both sporadic and inherited human colorectal cancer and MIN mice implicate the tumor suppressor gene, APC, in the causation of colorectal carcinogenesis, this gene has not been confirmed to be involved in rodent chemically-induced colon cancer models (RCCM). These experimental models are widely used to elucidate mechanisms involved in colon carcinogenesis (initiation, promotion and progression) as well as studies on chemoprevention (dietary and other) and intervention. To validate the RCCM as relevant models for sporadic human colorectal cancer, and to facilitate research on the role of the APC gene in colon carcinogenesis, we investigated the role of APC in azoxymethane (AOM)-induced colorectal tumors in mice. Using an antibody that recognizes the carboxy terminus of APC, we have characterized the pattern of staining observed in normal mouse intestinal tissue, in MIN mouse intestinal adenomas and in AOM-induced mouse colon tumors. The APC protein was localized in the cytoplasm of normal colonic epithelial cells. In the small intestine there was APC immunoreactivity along the villous and staining of the Paneth cells at the base of the glands. In the proximal and distal colonic crypts there appeared to be a gradient of staining which increased towards the luminal surface. This gradient was not as apparent in the small intestinal villi. Nuclei and mucus in the goblet cells showed no immunoreactivity. MIN mouse small bowel and colonic adenomas, known to have lost APC, stained negatively for APC. AOM-induced adenomas and carcinomas also consistently stained negatively using this antibody. This study demonstrates for the first time the loss of wild-type APC protein in AOM-induced mouse colon tumors and suggests that alterations in expression of this tumor suppressor gene, which is so commonly mutated in human colon cancer, is also involved in this animal model of colon cancer.

Effects of anticarcinogenic monoterpenes on phase II hepatic metabolizing enzymes.

The monocyclic monoterpenoid compounds limonene and sobrerol have anticarcinogenic activity when fed during the initiation stage of dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinogenesis. Here we investigated the potential roles of hepatic glutathione-S-transferase (GST; EC 2.5.1.18) and uridine diphosphoglucuronosyl transferase (UDPGT; EC 2.4.1.17) in monoterpene-mediated chemoprevention. Diets containing the isoeffective anticarcinogenic terpenes, 5% limonene or 1% sobrerol, elevated hepatic GST activity > 2-fold when measured using the general substrate 1-chloro-2,4-dinitrobenzene and 3,4-dichloronitrobenzene for the GST dimer 3-3. However, there were no significant changes in hepatic GST activity when 1,2-epoxy-3-(p-nitrophenoxy)propane was used. We found that both terpene diets increased GST affinity-purified protein 1.5-fold and the HPLC subunit profile. Liver GST subunit 3 had the greatest increase followed by 1 and 4 with no change in subunit 2. Both terpene diets significantly increased the activity of the methylcholanthrene-inducible and the phenobarbital-inducible UDPGT isozymes. We propose that much of the anticarcinogenic activity of these monocyclic monoterpenes during the initiation phase of DMBA carcinogenesis is mediated through the induction of the hepatic detoxification enzymes GST and UDPGT.

Effects of monoterpenoids on in vivo DMBA-DNA adduct formation and on phase I hepatic metabolizing enzymes.

We have previously demonstrated the anticarcinogenic effects of monocyclic monoterpenes such as limonene when given during the initiation phase of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary cancer in Wistar-Furth (WF) rats. Here we investigated the possible mechanisms for this chemoprevention activity including limonene's effects on DMBA-DNA adduct formation and hepatic metabolism of DMBA. Twenty-four hours after carcinogen administration, there were approximately 50% of the total DMBA-DNA adducts found in control animals formed in the liver, spleen, kidney and lung of limonene-fed animals. While circulating levels of DMBA and/or its metabolites were not different in control and limonene-fed rats, there was a 2.3-fold increase in DMBA and/or DMBA-derived metabolites in the urine of the limonene-fed animals. Studies of the effects of limonene and sobrerol, a hydroxylated monocyclic monoterpenoid with increased chemoprevention activity, on phase I metabolizing enzymes revealed that these terpenoids modulated cytochrome P450 (CYP) and epoxide hydratase (EH) activity. The 5% limonene diet increased total CYP to the same extent as phenobarbital (PB) treatment when compared to control, while 1% sobrerol (isoeffective in chemoprevention to 5% limonene) did not. However, both 5% limonene and 1% sobrerol diets greatly increased the levels of microsomal EH protein and associated hydrating activities towards benzo[a]pyrene 4,5-oxide when compared to control and PB treatment. These changes also modified the rate and regioselectivity of in vitro microsomal DMBA metabolism when compared to PB treatment or control. Identification of the specific isoforms of CYP induced by these terpenoids was performed using antibodies to CYP isozymes in Western blot analysis and inhibition studies of microsomal DMBA metabolism. Five per cent limonene was more effective than 1% sobrerol at increasing the levels of members of the CYP2B and 2C families but was equally effective at increasing EH. Furthermore, both terpenoid diets caused increased formation of the proximate carcinogen, DMBA 3,4-dihydrodiol. While these terpene-induced changes in hepatic CYP and EH do not explain the anticarcinogenic mechanism of these chemopreventive agents, or the ability of limonene systemically to reduce DMBA-DNA binding, they do reveal novel and selective induction mechanisms of hepatic enzymes.

The prevention of nitrosomethylurea-induced mammary tumors by d-limonene and orange oil.

The monocyclic monoterpenoid d-limonene and its source, orange oil, were found to prevent rat mammary carcinomas induced by the direct-acting carcinogen nitrosomethylurea. This chemopreventive effect was limited to the promotion/progression stage in this carcinogenesis model.

Anti-carcinogenic activity of d-limonene during the initiation and promotion/progression stages of DMBA-induced rat mammary carcinogenesis.

d-Limonene was found to be effective in reducing the average number of rat mammary carcinomas that developed in 7,12-dimethylbenz[a]anthracene-treated rats when the terpene was fed during the initiation or during the promotion/progression stage of carcinogenesis. The time to the appearance of the first tumor was extended only when d-limonene was fed during the initiation stage. These effects could not be attributed to changes in mammary-relevant endocrine functions.

Mouse skin tumor promoting activity of orange peel oil and d-limonene: a re-evaluation.

Orange peel oil has previously been shown to be a promoter of mouse skin tumors. It has been assumed that this activity is due to its major (95%) constituent, d-limonene. We have tested both orange peel oil and purified d-limonene as skin tumor promoters in a two-stage skin carcinogenesis model in which tumors were initiated with 7,12-dimethylbenz[a]-anthracene. We confirmed that topically applied orange peel oil is a very weak promoter of both skin papillomas and carcinomas. However, this promotional activity could not be accounted for by topically applied d-limonene. We thus feel that one or more minor components of orange peel oil has promotional activity. Neither orange peel oil nor d-limonene had promotional activity when given via the diet.