Predictive value of proliferation, differentiation and apoptosis as intermediate markers for colon tumorigenesis

In order to determine the prognostic significance of proliferation, differentiation, and apoptosis as intermediate markers for colon tumor development, these indices were measured during the promotion phase of tumorigenesis. Two hundred and sixty male Sprague-Dawley rats were provided with one of two fats (corn oil and fish oil) and two fibers (pectin and cellulose) plus or minus the carcinogen azoxymethane (AOM) and killed at two time points (18 and 36 wk) in a 2x2x2x2 factorial design. In vivo cell proliferation was measured immunohistochemically using incorporation of bromodeoxyuridine into DNA. Differentiation was assessed by binding of Dolichos biflorus agglutinin (DBA) to colonocytes. Apoptosis was measured by immunoperoxidase detection of digoxigenin-labeled genomic DNA. Adenocarcinoma incidence results at week 36 were 70.3% for corn oil + AOM and 56.1% for fish oil + AOM treatment (P < 0.05); no main effect of fiber was observed. At week 18, AOM treatment increased the number of cells per crypt column in the proximal colon compared with saline controls (P = 0.0358) and increased the proliferative zone in the distal colon compared with controls (P = 0.0073). However, changes in cell proliferation at week 18 did not predict the beneficial effect of fish oil versus corn oil. In contrast, DBA binding (the marker for differentiation) was higher in fish oil versus corn oil fed animals in both the proximal and distal colon and in each portion of the crypt (P = 0.0001). There were a greater number of apoptotic cells/crypt column in the proximal colon (P = 0.0019) and distal colon (P = 0.0358) with fish oil compared with corn oil, and indices of apoptosis also predicted certain fat/fiber interactions. Measurements of differentiation and apoptosis had greater prognostic value to detect dietary effects on tumor incidence than did measurements of cell proliferation.      

Dietary fish oil reduces O6-methylguanine DNA adduct levels in rat colon in part by increasing apoptosis during tumor initiation.

There is epidemiological, clinical, and experimental evidence that dietary fish oil, containing n-3 polyunsaturated fatty acids, protects against colon tumor development. However, its effects on colonocytes in vivo remain poorly understood. Therefore, we investigated the ability of fish oil to modulate colonic methylation-induced DNA damage, repair, and deletion. Sprague Dawley rats were provided with complete diets containing either corn oil or fish oil (15% by weight). Animals were injected with azoxymethane, and the distal colon was removed 3, 6, 9, or 12 h later. Targeted apoptosis and DNA damage were assessed by cell position within the crypt using the terminal deoxynucleotidyl transferase-mediated nick end labeling assay and quantitative immunohistochemical analysis of O6-methylguanine adducts, respectively. Localization and expression of the alkyl group acceptor, O6-methylguanine-DNA-methyltransferase, was also determined. Lower levels of adducts were detected at 6, 9, and 12 h in fish oil- versus corn oil-fed animals (P < 0.05). In addition, fish oil supplementation had the greatest effect on apoptosis in the top one-third of the crypt, increasing the apoptotic index compared with corn oil-fed rats (P < 0.05). In the top one-third of the crypt, fish oil feeding caused an incremental stimulation of apoptosis as adduct level increased. In contrast, a negative correlation between apoptosis and adduct incidence occurred with corn oil feeding (P < 0.05). Diet had no main effect (all tertiles combined) on O6-methylguanine-DNA-methyltransferase expression over the time frame of the experiment. The enhancement of targeted apoptosis combined with the reduced formation of O6-methylguanine adducts may account, in part, for the observed protective effect of n-3 polyunsaturated fatty acids against experimentally induced colon cancer.     

Morphodensitometric analysis of protein kinase C beta(II) expression in rat colon: modulation by diet and relation to in situ cell proliferation and apoptosis

We have recently demonstrated that overexpression of PKC beta(II) renders transgenic mice more susceptible to carcinogen-induced colonic hyperproliferation and aberrant crypt foci formation. In order to further investigate the ability of PKC beta(II) to modulate colonocyte cytokinetics, we determined the localization of PKC beta(II) with respect to cell proliferation and apoptosis along the entire colonic crypt axis following carcinogen and diet manipulation. Rats were provided diets containing either corn oil [containing n-6 polyunsaturated fatty acids (PUFA)] or fish oil (containing n-3 PUFA), cellulose (non-fermentable fiber) or pectin (fermentable fiber) and injected with azoxymethane (AOM) or saline. After 16 weeks, an intermediate time point when no macroscopic tumors are detected, colonic sections were utilized for immunohistochemical image analysis and immunoblotting. Cell proliferation was measured by incorporation of bromodeoxyuridine into DNA and apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling. In the distal colon, PKC beta(II) staining was localized to the upper portion of the crypt. In comparison, proximal crypts had more (P < 0.05) staining in the lower tertile. AOM enhanced (P < 0.05) PKC beta(II) expression in all regions of the distal colonic crypt (upper, middle and lower tertiles). There was also an interaction (P < 0.05) between dietary fat and fiber on PKC beta(II) expression (corn/pectin > fish/cellulose, fish/pectin > corn/cellulose) in all regions of the distal colonic crypt. With respect to colonic cell kinetics, proliferation paralleled the increase in PKC beta(II) expression in carcinogen-treated animals. In contrast, apoptosis at the lumenal surface was inversely proportional to PKC beta(II) expression in the upper tertile. These results suggest that an elevation in PKC beta(II) expression along the crypt axis in the distal colon is linked to enhancement of cell proliferation and suppression of apoptosis, predictive intermediate biomarkers of tumor development. Therefore, select dietary factors may confer protection against colon carcinogenesis in part by blocking carcinogen-induced PKC beta(II) expression.     

Induction of tumors in the colon and liver of the immunodeficient (SCID) mouse by 2-amino-3-methylimidazo[4,5-f ]quinoline (IQ)-modulation by long-chain fatty acids

We have recently shown that immunodeficient (SCID) mice, which lack functional T and B cells, are highly susceptible to low dose site specific induction of colon aberrant crypt foci (ACF), surrogates for colon tumors, by 2-amino-3-methylimidazo[4,5-f ]quinoline (IQ). To test whether long-term exposure to a high dose in the diet might prove carcinogenic to the SCID mouse colon, in contrast to other mice strains tested to date, the compound was administered at 300 p.p.m. in the diet to female 6-7-week-old SCID mice for 32 weeks. IQ induced high numbers of ACF, hyperplastic polyps, dysplasia, and colon adenomas, as well as hepatocellular altered foci and liver adenomas. Induction of colon tumors did not correlate with the main sites where ACF developed, the proximal colon, however, being seen mainly in the mid and distal colon. Induction of colon tumors correlated significantly with the incidence of dysplasia, crypt height, the mitotic index, cell proliferation and numbers of 8-hydroxydeoxyguanosine (8-OHdG)-positive cells in the colon crypt, particularly in mid and distal colon. Administration of 20% omega-6 polyunsaturated fatty acids (corn oil), omega-3 polyunsaturated fatty acids (perilla oil), or monounsaturated fatty acids (olive oil) simultaneously with IQ in the diet resulted in: (i) inhibition of colon and liver tumor induction by corn and perilla oil, whereas olive oil showed no effects; (ii) no reduction in total numbers of ACF by corn oil or perilla oil but significant suppression in the olive oil treated group; (iii) inhibition of tumor development particularly by omega-3 polyunsaturated fatty acids in perilla oil, correlating significantly with decreased cell proliferation in both colon and liver and a marked decrease in crypt heights and mitotic indices. Selective reduction in the numbers of 8-OHdG-positive nuclei, mainly in the middle and distal colon crypts, was also found to correlate with tumor inhibition. Thus, the results indicate carcinogenicity of IQ in the colon of the SCID mouse and preventive effects of polyunsaturated fatty acids.     

Tissue-specific attenuation of endogenous DNA I-compounds in rats by carcinogen azoxymethane: possible role of dietary fish oil in colon cancer prevention.

I-compounds are bulky covalent DNA modifications that are derived from metabolic intermediates of nutrients. Some I-compounds may play protective roles against cancer, aging, and degenerative diseases. Many carcinogens and tumor promoters significantly reduce I-compound levels gradually during carcinogenesis. Colon cancer is the second leading cause of cancer death in the United States, whereas cancer of the small intestine is relatively rare. Here we have studied levels of I-compounds in DNA of colon and duodenum of male Sprague-Dawley rats treated with azoxymethane. The effects of dietary lipids (fish oil or corn oil) on colon and duodenal DNA I-compounds were also investigated. Rats fed a diet containing fish oil or corn oil were treated with 15 mg/kg azoxymethane. Animals were terminated 0, 6, 9, 12, or 24 hours after injection. I-compound levels were analyzed by the nuclease P1-enhanced (32)P-postlabeling assay. Rats treated with azoxymethane displayed lower levels of I-compounds in colon DNA compared with control groups (0 hour). However, I-compound levels in duodenal DNA were not diminished after azoxymethane treatment. Animals fed a fish oil diet showed higher levels of I-compounds in colonic DNA compared with corn oil groups (mean adduct levels for fish and corn oil groups were 13.35 and 10.69 in 10(9) nucleotides, respectively, P = 0.034). Taken together, these results support claims that fish oil, which contains a high level of omega-3 polyunsaturated fatty acids, may have potent chemopreventive effects on carcinogen-induced colon cancer. The fact that duodenal I-compounds were not diminished by azoxymethane treatment may have been due to the existence of tissue-specific factors protecting against carcinogenesis. In conclusion, our observations show that endogenous DNA adducts may serve not only as sensitive biomarkers in carcinogenesis and cancer prevention studies, but are also helpful to further our understanding of the chemopreventive properties of omega-3 fatty acids and mechanisms of carcinogenesis.     

Dietary n-3 PUFA increases the apoptotic response to 1,2-dimethylhydrazine, reduces mitosis and suppresses the induction of carcinogenesis in the rat colon

The effect of dietary fish oil on colonic crypt cell apoptosis and proliferation was examined in male Wistar rats, 24 and 48 h after administration of 1,2-dimethylhydrazine (DMH), and its influence on the induction of aberrant crypt foci (ACF) in the distal colon was assessed. Rats (125-150 g) fed a high-fat semi-synthetic diet containing corn oil (CO) were given DMH (30 mg/kg body wt) or a sham injection of EDTA/NaCl. Animals were then fed either the CO diet or a diet in which fish oil (EPA 18.7%; DHA 8%) was substituted for corn oil. Subgroups of rats (n = 5) were killed after 24 and 48 h, and crypt cell apoptosis and proliferation were quantified by morphological criteria in isolated intact crypts from the mid and distal colon. Consumption of the fish oil diet (FO) was associated with increased apoptotic cell death (P < 0.001) and suppression of proliferation (P < 0.05) in colonic crypts both 24 and 48 h after DMH. In a second experiment, animals were given three injections of DMH or sham injections of carrier at weekly intervals. For 48 h after each injection animals were fed either the CO or FO diet, but otherwise maintained on the CO throughout. The number and crypt multiplicity of ACF in the distal colon were determined after 18 weeks, and animals given the FO diet for the 48 h period following carcinogen administration were found to have significantly fewer ACF than rats fed the CO diet (P < 0.05). The data demonstrate that the fatty acid composition of the diet is an important determinant in the induction of carcinogenesis by DMH. The proliferative and apoptotic response of the colonic crypt to carcinogen and fish oil, coupled with the reduced incidence of ACF, suggest n-3 PUFA can protect against the carcinogenic effects of DMH by mediating changes in the balance proliferation and cell death.     

Carcinogen and dietary lipid regulate ras expression and localization in rat colon without affecting farnesylation kinetics.

Epidemiological and experimental data suggest that dietary fiber and fat are major determinants of colorectal cancer. However, the mechanisms by which these dietary constituents alter the incidence of colon cancer have not been elucidated. Evidence indicates that dominant gain-of-function mutations short-circuit protooncogenes and contribute to the pathogenesis of cancer. Therefore, we began to dissect the mechanisms whereby dietary fat and fiber, fed during the initiation, promotion and progression stages of colon tumorigenesis, regulate ras p21 localization, expression and mutation frequency. Male Sprague-Dawley rats (140) were provided with corn oil or fish oil and pectin or cellulose plus or minus the carcinogen azoxymethane (AOM) in a 2 x 2 x 2 factorial design and killed after 34 weeks. We have previously shown adenocarcinoma incidence in these animals to be 70.3% (52/74) for corn oil + AOM and 56.1% (37/66) for fish oil + AOM (P < 0.05). Total ras expression as well as ras membrane:cytosol ratio was 4- to 6-fold higher in colon tumors than in mucosa from AOM- or saline-injected rats. Expression of ras in the mucosal membrane fraction was 13% higher for animals fed corn oil compared with fish oil feeding (P < 0.05), which is noteworthy since ras must be localized at the plasma membrane to function. The elevated ras membrane:cytosol ratio in tumors was not due to increased farnesyl protein transferase activity or prenylation state, as nearly all detectable ras was in the prenylated form. Phosphorylated p42 and p44 mitogen activated protein kinase (ERK) expression was two-fold higher in tumor extracts compared with uninvolved mucosa from AOM- and saline-injected rats (P < 0.05). The frequency of K-ras mutations was not significantly different between the various groups, but there was a trend toward a greater incidence of mutations in tumors from corn oil fed rats (85%) compared with fish oil fed rats (58%). Our results indicate that the carcinogen-induced changes in ras expression and membrane localization are associated with the in vivo activation of the ERK pathway. In addition, suppression of tumor development by dietary n-3 polyunsaturated fatty acids may be partly due to a combined effect on colonic ras expression, membrane localization, and mutation frequency.     

Chemopreventive effects of dietary mustard oil on colon tumor development

Fatty acid composition of dietary fat is one of the detrimental factors in colon cancer development. Fats containing omega 6-polyunsaturated fatty acids (e.g. corn oil) enhance and omega 3-polyunsaturated fatty acids (e.g. fish oil) reduce chemically-induced colon cancer in animal studies. The purpose of this study is to investigate the effects of dietary mustard oil (containing omega 3-polyunsaturated fatty acid) on azoxymethane-induced colon cancer in rats and compare with corn and fish oil treated groups. Colon tumor incidence and multiplicity were found to be 90, 75, and 50% and 1.7, 0.8, and 0.4 tumors/rat in corn, fish and mustard oil treated groups respectively. Omega-3 polyunsaturated fatty acid levels were highest in serum and colon microsomal fractions of the fish oil group followed by the mustard oil group. Corn oil group had the highest level of omega 6-polyunsaturated fatty acid levels in serum and colon microsomal fractions. The results indicate that dietary mustard oil is more effective in preventing colon cancer in rats than dietary fish oil.     

Fish oil increases mitochondrial phospholipid unsaturation,upregulating reactive oxygen species and apoptosis in rat colonocytes

We have shown that a combination of fish oil (high in n-3 fatty acids) with the butyrate-producing fiber pectin, upregulates apoptosis in colon cells exposed to the carcinogen azoxymethane, protecting against colon tumor development. We now hypothesize that n-3 fatty acids prime the colonocytes such that butyrate can initiate apoptosis. To test this, 30 Sprague-Dawley rats were provided with diets differing in the fatty acid composition (corn oil, fish oil or a purified fatty acid ethyl ester diet). Intact colon crypts were exposed ex vivo to butyrate, and analyzed for reactive oxygen species (ROS), mitochondrial membrane potential (MMP), translocation of cytochrome C to the cytosol, and caspase-3 activity (early events in apoptosis). The fatty acid composition of the three major mitochondrial phospholipids was also determined, and an unsaturation index calculated. The unsaturation index in cardiolipin was correlated with ROS levels (R = 0.99; P = 0.02). When colon crypts from fish oil and FAEE-fed rats were exposed to butyrate, MMP decreased (P = 0.041); and translocation of cytochrome C to the cytosol (P = 0.037) and caspase-3 activation increased (P = 0.032). The data suggest that fish oil may prime the colonocytes for butyrate-induced apoptosis by enhancing the unsaturation of mitochondrial phospholipids, especially cardiolipin, resulting in an increase in ROS and initiating apoptotic cascade.     

The effects of dietary omega-3 fatty acids on the DU-145 transplantable human prostatic tumor

Two omega-3 fatty acids present in fish oil are effective inhibitors of some models of mammary and colon tumorigenesis in rodents. The present studies were conducted to determine if eicosapentaenoic and docosahexaenoic acids can modify the growth of DU-145 human prostatic tumor cells in nude mice. Two experimental diets tested contained either 23.52% corn oil or 20.52% fish oil, plus 3% corn oil (w/w). In the fish oil-fed group of mice: (a) tumor growth was significantly inhibited; (b) tumor cells in histological sections were smaller but more connective tissue was present; (c) immunochemical staining for human prostatic acid phosphatase was less intense, and (d) tumor content of PGE2 was smaller than in the 23.52% corn oil-fed group. Fatty acid composition of phosphoglyceride and neutral lipid fractions of liver, prostate, and tumor tissue reflect the dietary intake of omega-3 and omega-6 fatty acids. These results are consistent with a role for omega-3 fatty acids in the inhibition of growth of human prostatic tumor cells in nude mice by dietary modification.     

Differential-effects of omega-3 and omega-6 Fatty-acids on primary tumor-growth and metastasis

The amount and type of dietary lipid can significantly influence spontaneous tumor development and tumor progression. To determine the effect of fish oil (rich in omega-3 polyunsaturated fatty acids) and corn oil (rich in omega-6 polyunsaturated fatty acids) on primary tumor growth, metastasis and carcass weight, 45 female Lewis/Wistar rats with subcutaneous mammary tumor implants (MAC-33) were randomized to 1 of 3 diets with 30% lipid consisting of: (i) corn oil alone, (ii) combined 50%:50% corn oil:fish oil, or (iii) fish oil alone. Primary tumor weight was significantly reduced in animals which were fed fish oil or corn oil alone compared to animals given combined corn oil:fish oil diet. Biochemical analysis (protein, DNA, RNA) of the primary revealed no difference between dietary groups. Cell cycle analysis of the primary tumor showed no difference in percent G(0)-G(1), S, G(2)-M or growth fraction (% S + G(2)-M) between dietary groups. In contrast, lung metastasis, was reduced in animals fed the combined corn oil:fish oil diet. Thus, dietary, lipid intake can significantly influence primary tumor growth and tumor metastasis. Differential effects of omega-3 and omega-6 polyunsaturated fatty acids occur on primary tumor growth and development of distant pulmonary metastases in this animal model.     

Down-regulation of PLK3 gene expression by types and amount of dietary fat in rat colon tumors.

Epidemiological studies suggest that high intake of dietary fat rich in saturated fatty acids increases the colon cancer risk whereas dietary fish oil high in omega-3 fatty acids reduces the colon cancer risk. Previously, we reported that consumption of omega-6 fatty acid rich diets such as corn oil strongly promotes azoxymethane (AOM)-induced colon carcinogenesis in rats as compared to ingestion of a diet with equivalent amount of fat containing fish oil (HFFO) or low-fat diet (LFCO). Expression of PLK3 (Polo-like kinase-3, previously named Prk) is negatively correlated with the development of certain cancers. Ectopic expression of human PLK3 results in cell cycle arrest or induces apoptosis. To understand the role of PLK3 in colon carcinogenesis and to study the effect of types and amount of dietary fat on the expression levels of PLK3 in colon tumors, we analyzed the colon tumors and mucosa of rats administered the diets containing fish oil and corn oil for PLK mRNA expression. Here we report that expression of PLK3 was down-regulated in rat colon tumors. Quantitative polymerase chain reaction demonstrated that PLK3 mRNA levels were significantly lower in carcinogen (azoxymethane)-induced rat colon tumors than their uninvolved normal colonic mucosa. Among the normal mucosa isolated from rats fed on diets with various levels of fat (LFCO, or high fat diet with corn oil, HFCO, or supplemented with fish oil, HFFO), no significant changes in PLK3 mRNA expression was detected. Tumors isolated from rats fed with HFCO diet contained a very low level of PLK3 mRNA expression. Interestingly, tumors from rats fed the HFFO diet did not exhibit as dramatic down-regulation of PLK3 as the tumors of animals fed the HFCO diet. Furthermore, our results also indicate that the ectopic expression of a kinase active PLK3 construct induced apoptosis in HT-29 colon carcinoma cells. These observations suggest for the first time that a decreased activity of PLK3 may play a key role in colon tumor development as well as in HFCO-induced colon tumorigenesis.     

Associations between tissue-specific DNA alkylation,DNA repair and cell proliferation in the colon and colon tumour yield in mice treated with 1,2-dimethylhydrazine

Putative risk factors (DNA damage) and risk modifying factors (DNA repair and cell proliferation) were examined in an experimental mouse model in which treatment with dimethylhydrazine (6.8 mg/kg DMH i.p. once weekly) for up to 20 weeks induces colon tumours in a site specific manner with 0, 43 and 87% of animals having proximal, mid and distal colon tumours respectively at the highest cumulative dose. Levels of the pro-carcinogenic DNA adduct, O(6)-methylguanine (O(6)-MeG), in colonic DNA were found to vary with time after final treatment and with location within the colon but not with total DMH dose. O(6)-MeG levels were generally lowest in proximal colon DNA and highest in distal colon DNA. Steady state O(6)-MeG levels were obtained at the highest cumulative DMH dose with O(6)-MeG levels in mid and distal colon DNA being 5 and 10 times higher those in proximal colon DNA. O(6)-alkylguanine-DNA alkyltransferase (MGMT) activity, and cell proliferation indices in the colon were also found to vary with time after final treatment but not with either location within the colon or total DMH dose. O(6)-MeG levels, MGMT activity and cell proliferation indices at specific time points as well as basal MGMT activity were not associated with differences in tumour yield within the colon. However tumour yield was associated with the cumulative amount of O(6)-MeG present in DNA over the treatment period and with the treatment induced cumulative increase in cell proliferation, particularly within regions of the colon crypt where stem cells reside but not with cumulative changes in MGMT activity. Results are consistent with an increased cancer risk arising from an increased mutation load in the target stem cell population due to increased adduct formation/persistence and cell proliferation but also suggest that other cell specific factors may help to determine tumourigenic response.     

Effect of dietary fat on growth of MCF-7 and MDA-MB231 human breast carcinomas in athymic nude mice: relationship between carcinoma growth and lipid peroxidation product levels

Human breast carcinoma cell lines MCF-7 and MDA-MB231 were transplanted s.c. to female athymic nude mice at 3-4 weeks of age. At 7-10 days after transplantation, the mice were divided into groups and fed for 6-8 weeks one of the following semi-purified diets containing different amounts and types of fat, i.e. 5% corn oil, 20% corn oil, 20% butter, 19% beef tallow/1% corn oil and 19% fish (Menhaden) oil/1% corn oil. In addition experiments, the fish oil diets were supplemented with antioxidants (vitamin E, 8 g or 2000 IU/kg diet plus tertiary butyl hydroquinone, TBHQ, 4 g/kg diet) or ferric citrate (3 g/kg diet). Tumor peroxidation product levels were assessed by measuring 2-thiobarbituric acid reactants (TBA assay). At the termination of the studies (6-8 weeks of diet feeding) mean human breast carcinoma volume (MCF-7 and MDA-MB231) was the largest in mice fed the 20% corn oil diet, intermediate in mice fed the butter or beef tallow diets and the least in mice fed the fish oil diet. The difference in mean tumor volumes among mice fed the 20% corn oil diet and those fed the fish oil diet was significant (P less than 0.01). When comparing low (5% corn oil) and high (20% corn oil) fat diets, numerical increases in human breast carcinoma volume (MCF-7 and MDA-MB231) were consistently observed in the high-fat diet groups but these differences were not always significant. Tumor lipid peroxidation product levels were determined on the MDA-MB231 tumors; tumor lipid peroxidation levels were significantly (P less than 0.01) increased only in mice fed the fish oil diets. Supplementation of the fish oil diets with antioxidants (vitamin E + TBHQ) significantly reduced the level of tumor peroxidation products and significantly increased tumor volume (P less than 0.05). When tumor lipid peroxidation product levels in the fish oil plus antioxidant fed mice were reduced to the level of that observed in the tumors of the corn oil fed mice, no significant differences in tumor volumes were observed in these two groups. In contrast, supplementation of the fish oil diets with ferric citrate, significantly (P less than 0.05) increased tumor lipid peroxidation product levels and decreased tumor volume. Thus, the type of dietary fat can clearly affect the growth of human breast carcinomas (MCF-7 and MDA-MB231) maintained in athymic nude mice.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distal colorectal cancers with microsatellite instability (MSI) display distinct gene expression profiles that are different from proximal MSI cancers

Promoter methylation of the mismatch repair gene plays a key role in sporadic microsatellite instability (MSI) colorectal cancers. However, promoter methylation often occurs in proximal colon cancers, and molecular phenotypes underlying MSI cancers in distal colon have not been fully clarified. Our goal was to clarify the difference between MSI and microsatellite stability (MSS) cancers and, furthermore, to determine distinct characteristics of proximal and distal MSI cancers. By DNA microarray analysis of 84 cancers (33 MSI and 51 MSS), we identified discriminating genes (177 probe sets), which predicted MSI status with a high accuracy rate (97.6%). These genes were related to phenotypic characteristics of MSI cancers. Next, we identified 24 probe sets that were differentially expressed in proximal and distal MSI cancers. These genes included promoter methylation-mediated genes, whose expression was significantly down-regulated in proximal MSI cancers. Among discriminating genes between MSI and MSS, nine methylation-mediated genes showed down-regulation in MSI cancers. Of these, 7 (77.8%) showed down-regulation in proximal MSI cancers. Furthermore, methylation-specific PCR confirmed that frequency of hMLH1 promoter methylation was significantly higher in proximal MSI cancers (P = 0.0317). These results suggested that there is a difference between proximal and distal MSI cancers in methylation-mediated influence on gene silencing. In conclusion, using DNA microarray, we could distinguish MSI and MSS cancers. We also showed distinct characteristics of proximal and distal MSI cancers. The inactivation form of hMLH, per se, differed between proximal and distal MSI cancers. These results suggested that distal MSI cancers constitute a distinct subgroup of sporadic MSI cancers.     

Regional differences in normal and cancer-associated glycoconjugates of the human colon

Because inherent regional differences in colonic epithelium may determine the biologic behavior of tumors originating from different sites, and inasmuch as colonic epithelial cells secrete mucins that may reflect the state of cell differentiation, colonic goblet cell mucin was analyzed with the use of fluorescein isothiocyanate-conjugated lectins and fluorescence microscopy in normal fetal and adult mucosa and in cancers of the proximal and distal colon. In the adult proximal colon only the goblet cell mucin in the upper portion of the crypts was specifically labeled by the lectin Dolichos biflorus agglutinin (DBA), whereas this gradient was progressively lost distally; mucin in the upper and lower crypts of the sigmoid colon and rectum bound the label uniformly. In fetuses less than 22 weeks of age, DBA bound only to mucin in the crypts of the distal colon. Seven of 12 (58%) cancers originating from the proximal colon bound DBA, whereas only 2 of 23 (9%) from the distal colon bound this lectin (P less than .005). Logistic regression analysis suggested that this difference may reflect the occurrence of larger tumors in the proximal colon. Regional differences in the binding of Ulex europaeus agglutinin (UEA-I) to nonneoplastic mucosa was similar to that found by others; predominant binding occurred in the proximal colon in the adult. No difference was noted for UEA-I binding to tumors of the proximal (8 of 12; 66.6%) versus distal (11 of 23; 48%) colon (P = .48). These findings may reflect regional differences in normal and tumor-related carbohydrate structures in mucin of the human colon.     

Polyunsaturated fatty acids of the n -- 3 series influence intestinal crypt cell production in rats

The effects of saturated and polyunsaturated dietary lipidson intestinal mucosal cell proliferation were investigated inrats. Animals were randomly allocated to three groups of 10,and fed fibre-free diets containing lipid in the form of lard,corn oil or fish oil at a level of 80 g/kg. Total energy intakewas kept constant by pair-feeding. After 14 days the crypt cellproduction rates (CCPR) at two sites in the small intestine,one site in the caecum and two sites in the colon were determinedby the metaphase arrest technique, together with circulatinglevels of enteroglucagon and gastrin and parameters of mucosalmorphology. Consumption of fish oil led to lower CCPR comparedto corn oil at all sampling positions except the caecum (Treatment     

Suppression of lung tumor formation by the regulatory subunit of ribonucleotide reductase

The nucleotide metabolism enzyme ribonucleotide reductase is composed of a regulatory subunit (RRM1) and a catalytic subunit (RRM2). The RRM1 locus has frequent loss of heterozygosity in lung cancers, ectopic expression of RRM1 suppresses proliferation of ras-transformed mouse fibroblasts, and high levels of RRM1 expression are associated with a significant survival benefit in patients with lung cancer. In RRM1 transgenic human lung and colon cancer cell lines, we observed induction of G(2) cell cycle arrest, apoptosis, and efficient DNA damage repair. We generated strains of RRM1 transgenic mice and found that carcinogen-induced lung tumor formation was significantly suppressed. The tumor suppression was more pronounced in strains with high levels of RRM1 expression than in those with low levels of expression. DNA damage repair capacity in transgenic animals was determined, and RRM1 transgenic animals repaired chemically induced DNA damage with greater efficiency than control animals. We conclude that the regulatory subunit of ribonucleotide reductase has tumor suppressor activity that is mediated through efficient DNA damage repair.     

Apoptotic and mitotic indices predict survival rates in lymph node-negative colon carcinomas.

An imbalance between apoptosis and mitosis is believed to underlie colon cancer development and progression. These processes regulate the growth of normal and neoplastic epithelia, and in tumors, may confer prognostic information. To test this hypothesis, we determined apoptotic and mitotic indices (AI, MI) by morphology in H&E sections of 154 lymph node-negative, sporadic colon carcinomas. The relationship of these indices to genetic (p53 and Bcl-2) and biological features (DNA ploidy and cell kinetics) and patient survival rates was determined. Tumor features were compared in proximal and distal tumors, given postulated differences in their pathogenesis. Bcl-2 and p53 proteins were examined using immunohistochemistry and DNA ploidy and proliferative indices (PIs) by flow cytometry. Tumor features were dichotomized for analysis of relapse-free survival and overall survival (OS) rates using a Cox proportional hazards model. Median patient follow-up was 8.8 years. The median AI and MI were 1.2% (0-7.6) and 0.40% (0-1.8), respectively, and did not differ by tumor site. AI correlated with histological grade (P = 0.03); MI correlated with PI (P = 0.02) and inversely with Bcl-2 in distal tumors (P = 0.02). p53 and Bcl-2 expression were detected in 52 and 53% of tumors, respectively. Distal tumor site was associated with aneuploidy (P = 0.001), p53 (P = 0.001), and PI > 15% (P = 0.002). In a univariate analysis, colon cancers with high MIs (>0.5%) had a poor prognosis (P = 0.04). Bcl-2 overexpression (>20% + tumor cells) was associated with more favorable OS (P = 0.04). The association of ploidy and PI with outcome was of borderline significance for all tumors; however, diploidy predicted better survival in proximal cancers. In distal cancers, low AIs (< or = 0.25%) and high MIs (>0.5%) were adverse prognostic markers. After adjustment for other variables, an increased MI predicted shorter OS with a hazard ratio (HR) for death of 2.70; 95% confidence interval (CI) was 1.23-5.91 (P = 0.01). Expression of Bcl-2 was associated with more favorable OS (HR, 0.46; 95% CI, 0.21-1.0; P = 0.06). In proximal cancers, Bcl-2 expression was the most important predictor of OS (HR, 0.17; 95% CI, 0.03-0.85; P = 0.03). In distal tumors, low AIs (HR, 3.33; 95% CI, 1.27-9.09; P = 0.01) and high MIs predicted poor survival. In conclusion, increased mitosis and low or absent Bcl-2 expression are significant risk factors for death in node-negative colon cancers, as are low rates of apoptosis in distal tumors. If validated prospectively, our results may identify patient subsets than can benefit from adjuvant chemotherapy.     

Modifying effect of tuna orbital oil rich in docosahexaenoic acid and vitamin D3 on azoxymethane-induced colonic aberrant crypt foci in rats

The modifying effect of dietary tuna (Thunnus thynnus orientalis) orbital oil rich in docosahexaenoic acid (DHA) and vitamin D3 (VD3) on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) was investigated in male F344 rats. Animals were given three weekly subcutaneous injections of AOM (15 mg/kg body weight) to induce ACF. The rats were fed the experimental diet containing 5% tuna orbital oil (low fish oil), 23.5% tuna orbital oil (high fish oil), 5% corn oil (low corn oil) or 23.5% corn oil (high corn oil) for 5 weeks, starting 1 week before the first dose of AOM. Animals were sacrificed 2 weeks after the last AOM injection to count colonic ACF and assay the expression of cyclooxygenase (COX)-1 and -2. High corn oil diet significantly increased the development of ACF, when compared with low corn oil diet (P<0.005). High fish oil diet also increased ACF formation compared with low fish oil diet (P<0.01), but the increase was smaller than high corn oil diet. The frequency of ACF was significantly lower in the rats fed high fish oil diet than high corn oil diet (P<0.02). Moreover, frequency of ACF consisted of 4 or more crypts in rats fed the high fish oil diet was significantly lower than that of rats given high corn oil diet. COX-1 and COX-2 expression did not significantly differ among the groups. These results suggest that fish oil derived from tuna, which contains high amounts of DHA and VD3, suppresses the formation and growth of ACF without affecting COX-1 and COX-2 expression, and may have a preventive effect on colon carcinogenesis.