Beef induces and rye bran prevents the formation of intestinal polyps in ApcMin mice: relation to ß-catenin and PKC isozymes

Marja Mutanen1, Anne-Maria Pajari and Seija I. Oikarinen

Department of Applied Chemistry and Microbiology (Nutrition), PO Box 27, University of Helsinki, Helsinki, FIN-00014, Finland


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Epidemiological studies suggest that high consumption of red meat and saturated fat and low consumption of fiber are associated with an increased risk of colon cancer. Therefore, we studied whether diets high in red meat or high in different grain fibers as well as inulin, polydisperse ß(2->1) fructan, could affect the formation of intestinal polyps in ApcMin mice. Min mice were fed the following high-fat (40% of energy) diets for 5–6 weeks; a high-beef diet and a casein-based diet without added fiber or casein-based diet with 10% (w/w) oat, rye or wheat bran, or 2.5% (w/w) inulin. One group had a normal low-fat AIN93-G diet. The mice fed the rye-bran diet had the lowest number of polyps in the distal small intestine [15.4 ± 8.7 (mean ± SD)], and in the entire intestine (26.4 ± 12.1). The rye-bran group differed significantly (P = 0.001–0.004) from the beef group (36.6 ± 9.4 and 52.8 ± 13.2). In addition, the beef group differed significantly from the AIN93-G group (P = 0.009) and also from the wheat-bran group (21.0 ± 6.1 and 35.0 ± 8.2; P = 0.02) in the distal small intestine. The inulin group (32.9 ± 14.3 and 49.3 ± 16.3), on the other hand, was close to the beef group and it differed significantly from the rye-bran group in the distal small intestine. The number of animals bearing tumors in the colon + caecum was only 33% in the rye-bran group when compared with 89% in the beef and 100% in the inulin groups. The mice fed the rye-bran and beef diets had the lowest levels of cytosolic ß-catenin (0.60 ± 0.42 and 0.67 ± 0.26) and they differed significantly (P = 0.040 and 0.062) from the mice fed the oat-bran diet (1.46 ± 0.43). No differences between groups in expression of protein kinase C (PKC) {alpha}, ßII, {delta} and {zeta} were found. The four PKC isozymes were positively correlated with cytosolic ß-catenin levels (r = 0.62–0.68; P < 0.0001).

Abbreviations: APC, adenomatous polyposis coli; Min, multiple intestinal neoplasia; PKC, protein kinase C.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The incidence of colon cancer is increasing in all industrialized countries. Epidemiological evidence suggests that diet is clearly involved, but the mechanisms through which food components either promote or inhibit colon carcinogenesis are still far from clear. Colorectal tumorigenesis involves activating mutations in proto-oncogenes, such as ras and c-myc (1), as well as mutations that inactivate tumor suppressor genes (2) including the adenomatous polyposis coli (APC) gene. These genetic events, in turn, lead to epigenetic changes in signal transduction pathways, which regulate apoptosis, cell differentiation and proliferation (3). Diet may affect cancer development either at the level of mutations or more likely the different steps of cell signalling pathways. Several signalling pathways are related to colon carcinogenesis, of which the two central pathways are those that involve APC-ß-catenin and cyclooxygenase (4).

Mutation in the APC gene is an early event in development of colorectal tumor (5) and germline mutations of APC lead to the familial adenomatous polyposis (FAP) syndrome. Apart from causing the FAP syndrome, the inactivation of the APC gene is an important initiation event for the development of sporadic adenomas and carcinomas. Mutations in the APC gene can be detected in up to 80% of sporadic colorectal carcinomas (6). Thus, APC gene mutations, resulting in the expression of truncated tumor-suppressor protein, are important initiation events for colorectal tumorigenesis. The APC gene codes a huge multifunctional cytoplasmic protein that, as a part of the APC–ß-catenin–GSK-3ß–microtubule complex, controls the post-translational stability of ß-catenin. ß-Catenin, in turn, can bind cell-adhesion protein E-cadherin, at the plasma membrane as well as the DNA-binding protein, Tcf/Lef transcription factors, in the nucleus. These two pathways control normal cell architecture and growth (7) and it has been suggested that mutation in the APC gene lead to disturbance of the equilibrium in these pathways with subsequent abnormal cell growth. Currently, it is assumed that especially intracellular accumulation of ß-catenin results in abnormal gene transcription that promotes tumor development (8). Accumulation of ß-catenin may arise as a consequence of loss of function of either the APC gene or the APC–ß-catenin–GSK-3ß complex. There is actually some in vitro evidence that GSK-3ß may be phosphorylated by some protein kinase C (PKC) isoforms with its subsequent specific inactivation (9).

PKC isoenzymes are involved in diverse biological processes, including cellular proliferation and differentiation. PKC has also been implicated in propagating signals for apoptosis (10). Colorectal cancer tissue has lower PKC activity in comparison with that in normal mucosa, and levels of isozyme expression also differ (11). Some experimental studies in vivo with animals have also revealed that in colonic mucosa dietary manipulation affects both PKC activity and the distribution and levels of different isozymes. Animal cancer models suggest that PKC ßII, especially, is upregulated and PKC {alpha}, {delta} and {zeta} are downregulated (12,13) in colon tumorigenesis. Fish oil seems to block carcinogen-induced downregulation of PKC isoenzymes (14), which may explain the known protective association between fish intake and the risk of colon cancer.

Multiple intestinal neoplasia (Min) mice are a widely used cancer model and provide a good carcinogenesis model for both sporadic and inherited forms of colorectal cancer. This provides the opportunity to study the role of diet on the pathogenesis of neoplasm in a model in which the initial molecular defects are the same in both human and mouse. In Min mice, the mutation is a heterozygous nonsense mutation at codon 850 of the Apc gene (15), which is a homologue of human APC. Similarly in humans and Min mice, loss of function of both APC/Apc alleles is associated with development of multiple intestinal tumors (16). Furthermore, several current, independent lines of evidence suggest that non-steroidal anti-inflammatory drugs (NSAIDs) are effective chemopreventive agents in human colon carcinogenesis. NSAIDs decrease tumor load also in Min mice indicating that Min mice provide a genetically relevant and practical model for dietary studies. Indeed, some dietary factors like fish oil fatty acids (17), fat (18) and food mutagens (19) have already been shown to affect polyp incidence and size in this animal model. However, in these studies the level of ß-catenin, the key protein in the APC–ß-catenin pathway has not been measured.

Epidemiological evidence strongly suggests that diets rich in plant-derived foods, including grains, fruits and vegetables, and low in red meat and fat are associated with a reduced risk for several cancers, including colon cancer. In healthy rats, we have recently shown that beef and inulin, a new `dietary fiber' used in many food applications and having a positive `health promoting claim' due to its hypolipidaemic effects (20), increased colonic PKC activity. Furthermore, we found that different cereal brans had clearly distinct effects on colonic PKC isozyme levels (21,22). To better understand these earlier results we carried out this study with Min mice to see whether these same dietary factors, i.e. red meat, inulin and cereal brans, affect the development of intestinal polyps in Min mice. To further elucidate the mechanisms by which these dietary factors may modulate tumor formation, we also analyzed the levels of ß-catenin and four PKC isozymes in the intestinal mucosa of Min mice.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
The Laboratory Animal Ethics Committee of the Faculty of Agriculture and Forestry, University of Helsinki approved the study protocol. Male C57BL/6J Min mice, 5–7 weeks of age and initial body mass of 23 g, were obtained from the Jackson Laboratory (Bar Habor, ME). Animals were housed in plastic cages in a temperature- and humidity-controlled animal facility, with 12 h light/dark cycle. After 1 week acclimatization period on a standard rodent chow, the animals were stratified by body mass and age and assigned randomly to the experimental diets, seven to nine mice/group. They had free access to the semi-synthetic diets and tap water for 5–6 weeks. The body weights of the animals were recorded weekly.

Diets
The high fat (40% energy) experimental diets were composed in a way that the diets contained similar amounts of protein and carbohydrate with respect to energy intake (Table IGo). The fat used in the diets was a mixture of butter, rapeseed oil and sunflower seed oil providing the intake of saturated, monounsaturated and polyunsaturated fatty acid in the ratio 3:2:1. It corresponded to the intake of these fatty acids in the Western-type diet. The non-fiber and beef diets were without any fiber, and the others contained 10% (w/w) rye, wheat or oat bran or 2.5% (w/w) polydisperse ß(2->1) fructan, inulin (Raftiline®, Orafti, Tienen, Belgium). The bran-supplemented diets were prepared by diluting the fiber-free high-fat diet with addition of brans at 100 g/kg diet. In this way, the nutrient intake as percentage energy was kept constant in all diets. The amount of beef added to the beef diet was calculated to replace casein as a protein source. Prior to addition to the diet, the beef was minced and freeze-dried. One group had a normal low-fat AIN93-G diet. The diets were stored at –20°C and kept at 4°C only for the use within 1 week.


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Table I. Nutrient composition of experimental diets (g/kg diet)a
 
Assessment of adenomas
After the feeding period, the mice were killed by CO2 asphyxiation. The small intestine, caecum and colon were removed, measured, opened along the longitudinal axis and rinsed with ice-cold saline. The small intestine was divided into five sections. The caecum and colon were kept together. The small intestine and colon + caecum were then spread flat on a microscope slide and a number; diameter and location of adenomas were determined with an inverse light microscope with a screen at a magnification of x2.5. The diameter of adenomas was scored with a millimeter scale placed on the screen. For each group, the mean number of polyps per animal (± SD), the mean polyp diameter per animal (± SD) and the mean number of polyps per section were calculated.

Tissue preparation and immunoblotting
The mucosa in each section of the intestine was scraped off with a microscope slide and snap frozen in liquid nitrogen. Extraction of cytosolic and particulate proteins from the mucosal sample and rat brain homogenate (positive control) as well as western blotting was carried out as described previously (A.-M.Pajari, S.Olkarinen and M.Mutanen, submitted for publication). The representative western blot on ß-catenin is shown in Figure 1Go.



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Fig. 1. Representative immunoblot of Min mice small intestine membrane and cytosol extracts probed with anti-ß-catenin antibody. Lane 1, cytosol extract (30 µg); lane 2, membrane extract (30 µg); lane 3, rat brain homogenate (10 µg). ß-Catenin was detected as 91 kDa protein band. Immunoreactive band in the low molecular weight (70 kDa) might present a degradation product.

 
Antibodies
Protein levels of PKC {alpha}, ßII, {delta} and {zeta} as well as ß-catenin were analyzed in the distal part of the small intestinal mucosa from high-fat, cereal bran and inulin groups. Polyclonal rabbit anti-PKC {alpha}, ßII, {delta} and {zeta}, alkaline phosphatase-conjugated anti-rabbit secondary antibody and the blocking peptides for each PKC isozyme were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal mouse anti-ß-catenin was purchased from Transduction Laboratories (Lexington, KY) and alkaline phosphatase-conjugated anti-mouse secondary antibody from Zymed (San Francisco, CA). The antibodies for PKC {alpha} and {zeta} isozymes react, to a lesser extent, with ß and {iota}({lambda}) isozymes, respectively. PKC ßII and {delta} antibodies are non-cross-reactive with other PKC isoforms. ß-Catenin antibody does not recognize other catenins. Monoclonal mouse anti-ß-catenin from Santa Cruz Biotechnology (Santa Cruz, CA) was used to confirm the specificity of the ß-catenin signal and blocking peptide of each PKC was used according to the manufacturers' instructions to control the specificity of PKC antiserums.

Statistical analysis
The analysis of variance with the Tukey's post-hoc test was used for all comparisons. The correlations between number of adenomas and isozyme levels were analyzed by a correlation analysis (Pearson). The Systat statistical computer package (Systat Inc., version 5.2) was utilized for all the statistical analyses.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The mean ± SD final body weights of the animals were 25.8 ± 3.3, 29.5 ± 1.1, 30.9 ± 4.3, 27.6 ± 3.8, 26.7 ± 5.7, 24.7 ± 2.4 and 27.2 ± 2.6 g for the AIN93-G, non-fiber, rye, wheat, oat, inulin and beef diet groups, respectively (P = 0.02 between the rye and inulin diet groups). Most of the polyps were developed in the distal small intestine (Table IIGo). The mice fed the rye-bran diet had the lowest number of polyps in the distal small intestine (15.4 ± 8.7, mean ± SD) and in the entire intestine (26.4 ± 12.1). The rye-bran group differed significantly (P = 0.001–0.004) from the beef group (36.6 ± 9.4 and 52.8 ± 13.2). In addition, the beef group differed significantly from the AIN93-G group (P = 0.009) and also from the wheat-bran group (21.0 ± 6.1 and 35.0 ± 8.2; P = 0.02) in the distal small intestine. The inulin group (32.9 ± 14.3 and 49.3 ± 16.3), on the other hand, was close to the beef group and it differed significantly from the rye-bran group in the distal small intestine. There was no statistically significant difference in tumor size between the dietary groups (Table IIIGo) even though the rye-bran diet tended to produce less large adenomas than the other diets. Furthermore, no difference in distribution of adenomas along the length of the intestine was found. In the colon and caecum, adenoma number did not differ significantly, but the number of animals bearing tumors was only 33% in the rye-bran group when compared with 89% in the beef group and 100% in the inulin group (Table IIGo).


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Table II. Effect of beef, cereal brans and inulin on tumor development in the small intestine and colon of Min mice, mean and SD
 

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Table III. Effect of beef, cereal brans and inulin on tumor size (% from total) in the small intestine of Min mice, mean and SD
 
ß-Catenin level was measured in cytosolic and particulate fractions of the distal small intestinal mucosa. The mice fed the rye-bran and beef diets had the lowest levels of cytosolic ß-catenin (0.60 ± 0.42 and 0.67 ± 0.26, respectively) and they differed significantly (P = 0.04 and 0.062) from the mice fed the oat-bran diet (1.46 ± 0.43) (Figure 2Go). Cytosolic ß-catenin levels in the non-fiber (0.82 ± 0.74) and wheat-bran (0.96 ± 0.39) groups were between the two extremes.



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Fig. 2. Effect of beef, cereal brans and inulin on cytosolic ß-catenin levels in the small intestine of Min mice (1, non-fiber; 2, inulin; 3, rye; 4, wheat; 5, oat; 6, meat diet). Inulin diet differed significantly (P < 0.05) from the non-fiber, rye and beef diets, and the oat diet differed significantly (P < 0.05) from the rye and meat diets. Results are expressed as sample band intensity (optical density of the specific ß-catenin band multiplied by band area) divided by rat brain band intensity.

 
Protein levels of four PKC isoforms, {alpha}, ßII, {delta} and {zeta} were analyzed in the distal small intestinal mucosa of the Min mice. PKC {alpha} and {delta} isoforms were analyzed in cytosolic fraction and PKC ßII and {zeta} isoforms both in particulate and cytosolic fractions. There were surprisingly large variations in the levels of PKC isoforms between the animals of each group, and no significant differences in PKC isozyme expression between the groups could be found (Table IVGo). All PKC isozymes analyzed were positively correlated with cytosolic ß-catenin levels (r = 0.62–0.68; P < 0.0001).


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Table IV. PKC Isozyme expression in distal small intestine of Min mice on the beef, cereal brans and inulin diets (relative units), mean and SD
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study demonstrates a promoting effect of a high-beef diet and, on the other hand, a cancer protective effect of rye- and wheat-bran diets on intestinal tumor development in Min mice. The results are fairly well in line with epidemiological evidence (2224). However, we could see no promoting effect of fat, since adenoma number in mice fed the high-fat Western-type diet was nearly identical with mice fed the low-fat AIN93-G diet. Although there are some discrepancies regarding the effect of fat as such on the number of intestinal tumors in Min mice (18,25), recent epidemiological findings do not show an independent effect of fat on colon cancer risk in humans after adjustment for total energy intake (26). Overall, the consistency of our results with the epidemiological data supports the relevance of the Min mice model for studies on diet and colon carcinogenesis.

The mice fed the high-beef diet had the highest number of tumors in the small intestine as well as in the colon. Only few earlier studies have determined the effect of red meat on colon tumor development in carcinogen-treated rats and none in Min mice. The results of earlier studies have been complicated to interpret, probably because a complex interaction between meat, a source of protein, and the type and amount of fat in the diet. Thus, depending on the other dietary ingredients, both positive and negative results with regard to red meat have been published (2730). One possible candidate in mediating the tumor-promoting effect of red meat is a high level of heterocyclic aromatic amines in meat diets (31,32). These compounds are formed when meat is cooked, and they have been shown to be potent mutagens in the Ames assay. This cannot, however, be the case in our study, since the beef used was freeze-dried instead of cooking. The other possibility is an increased formation of endogenous N-nitroso compounds during the beef diet. N-nitroso compounds are effective alkylating agents and may therefore cause mutations in the K-ras gene. Ras mutations are common in colorectal cancer and are formed after Apc mutation (33). We did not measure N-nitroso compounds in our study, but based on the results from a human study with 600 g of red meat per day (34), concentration of these compounds could have been increased also in Min mice fed the high-beef diet.

Wheat and especially rye bran may have specific effects on neoplastic process in Min mice. The fiber intake of mice consuming the rye- or wheat-bran diets only slightly exceeded the recommended human daily intake, indicating that results are well comparable with the human situation. The protective role of wheat bran in colon tumor formation is consistently demonstrated in animal models (3537), including one study with Apc716 mice (35). The composition of rye bran is close to that of wheat bran, and a recent study with carcinogen-treated rats demonstrated that rye supplementation decreases colonic tumor formation (38). Wheat and rye brans may modulate the neoplastic process through several mechanisms. The most studied one is the production of short-chain fatty acids, especially butyrate (36). In rats fed diets identical to those used in this study, we found that both the rye- and wheat-bran diets led to the highest butyrate:propionate ratio (1.97 and 1.90, respectively) in the colon contents, and the non-fiber and inulin diets to the lowest ones (0.51–0.53) (unpublished observations). If butyrate production plays a role in adenoma formation in Min mice it can explain our results in the wheat, rye and inulin groups, but not in the non-fiber group. Actually, not all studies confirm the protective role of butyrate in colon carcinogenesis (37,39,40). Zoran et al. (37) confirmed the protective role of wheat on colon carcinogenesis in rats independently of luminal butyrate concentration. Wheat bran could also mediate its positive effects inside the colon through its ability to bind bile acids and to shorten fecal transit time due to bulking effect. These effects have been attributed to its high cellulose content. In this study the non-fiber diet and the AIN93-G diet containing 5% of cellulose produced as small an amount of adenomas as did the wheat-bran diet and, thus, in Min mice, cellulose does not seem to play a role. Furthermore, all diets including the non-fiber diet were high in fat (40% of energy intake) and presumably bile acid production was also considerable. The formation of adenomas was not, however, attenuated in the non-fiber diet, casting doubt on the hypothesis concerning the carcinogenic effects of bile acids in vivo.

Apart from the fiber component, rye and wheat brans contain considerable amounts of phytoestrogens (41), which have a structural similarity to mammalian estrogen and possess a possible cancer preventive effect through binding to estrogen receptors (42). The experimental evidence supporting the preventive role of phytoestrogens in colon tumorigenesis is, however, not very strong. Isoflavones, the phytoestrogens in soy, protected against chemically induced tumors in rats (43), but had no protective effect in two studies in Min mice (38,44). Our results and also those obtained with azoxymethane-treated rats in which tumor incidence was significantly reduced in a rye-bran group when compared with soy or control groups (38) strongly emphasize the need to further clarify the responsible compound in rye bran. Furthermore, even though the groups did not differ significantly in adenoma number in the colon, the rye-bran group had the lowest level and only three animals out of seven had adenomas while in the other groups most of the animals carried adenomas (Table IIGo). This gives further support of rye bran over the other cereal brans as a chemopreventive dietary component.

Inulin, a fructo-oligosaccharide that is suggested to enhance growth of Bifidogenic bacteria in the colon (45,46) did not show any tumor-preventing effect in the Min mice. Short-chain fructo-oligosaccharides (scFOS) have been studied previously only once with Min mice (47). The authors concluded that scFOS decreased tumor formation in the colon when compared with the control diet. Interestingly, all the diets tested, i.e. resistant starch, wheat bran and the control diet gave almost identical numbers of tumors in the small intestine (47). This, and the fact that there were only few tumors in the colon, indicates the possibility of detecting a chance result about a positive effect of scFOS on colon carcinogenesis. On the other hand, colonic preneoplastic aberrant crypt foci formation was reduced when inulin (10% w/w) was added to a basal AIN-76 control diet containing cellulose (48). However, the low fat concentration and the presence of cellulose in the AIN-76 diet may not optimally reflect a human high-risk diet. Oat bran is clearly more fermentable inside the lumen than wheat or rye bran and actually close to inulin in this respect (39). This phenomenon may explain the similarity of the results in adenoma formation between inulin and oat-bran groups. In chemically induced cancer models, oat bran has shown promotive, not protective effects on colon carcinogenesis (49) and, thus, our results are in line with them in this respect. In our study, the mean body weight was lowest in the inulin group at the end of the feeding period. However, no difference in body weights between the groups could be seen 1 week earlier, indicating that the food consumption during the last week of the experiment was probably lower in the inulin group than in the other groups. Whether this was due to the adenoma burden and whether it had some effect on the polyp formation, can only be speculated.

To clarify the mechanisms concerning diet and neoplastic process, we measured mucosal levels of ß-catenin, a target molecule for APC protein. Defect in function of the APC protein leads to elevation in free ß-catenin (50) that can bind to Tcf/Lef transcription factors and stimulate gene expression in the nucleus. In APC–/– colon carcinoma cells, one consequence of inactivation of the APC gene is constitutive activation of ß-catenin/Tcf-mediated transcription (8). Recently, the c-myc proto-oncogene was identified as a direct target for Tcf transcription factor (51). In this study, the mice having a rye-bran diet had the lowest level of cytosolic ß-catenin as well as the lowest number of adenomas, suggesting that the rye-bran diet prevented tumor formation by decreasing the cytosolic ß-catenin level. Furthermore, mice fed the inulin and oat-bran diets had the highest ß-catenin levels together with the high number of adenomas, which gives further support to the assumption that dietary fibers affect tumor formation in Min mice by modifying the level of cytosolic ß-catenin. This ß-catenin presumably represents the pool available for interacting with Tcf/Lef transcription factors. Interestingly, although the beef diet induced the highest number of adenomas, the cytosolic ß-catenin level stayed at the level found in the mice fed the rye-bran diet. Thus, the mechanism mediating the tumor promoting effect of beef in Min mice is probably different from that of fibers and does not involve the ß-catenin signaling pathway.

A number of human and animal studies suggest that epigenetic changes in PKC are involved in colon carcinogenesis. Colonic tumors of both human and animal origin possess a reduced PKC activity as well as alterations in levels of several isozymes (5254). Furthermore, recent study with transgenic PKC ßII mice shows that overexpression of PKC ßII is associated with colonic hyperproliferation, elevated ß-catenin levels and decreased GSK-3ß activity (55). In the present study, protein levels of four PKC isozymes did not differ between the dietary groups. In healthy rats, we have recently shown that different cereal brans as well as beef are capable of modifying colonic steady-state PKC activity and isozyme levels (21, A.-M. Pajari, S.Oikarinen and M.Mutanen, submitted for publication). The reason for not detecting any dietary effects on PKC isozyme levels in this study may be that the neoplastic process in Min mice is beyond the state in which diet can modulate PKC expression. It is, however, of interest that the cytosolic ß-catenin level was highly significantly correlated with all four of the PKC isozymes measured. This correlation supports the involvement of PKC in regulation of the ß-catenin pathway in vivo in Min mice. This finding supports the results obtained from the transgenic PKC ßII mice study (55) and also the study of Goode et al. (9) showing that PKC isoforms {alpha}, ß1 and {gamma} are capable of efficiently phosphorylating GSK-3ß and thus causing its subsequent inactivation.

In Min mice, continuous formation of new intestinal tumors proceeds up to the age of 77 days and then the total number of tumors remains relatively constant between the age of 77 and 115 days. The average tumor size increases by ~50% from day 77 to 115 (56). The mice in this study were 40–50 days old when the dietary treatment started and they were killed 35 days later, i.e. at the age of 75–85 days. Since the groups did not differ with respect to adenoma size, the diets did not affect the growth of tumors formed prior to the dietary treatment, but rather prevented appearance of new tumors. We could see differences between the dietary treatments even though the animals were exposed to the diets quite late in their neoplastic process. This emphasizes the important chemopreventive potential of diet in intestinal neoplasia.


    Notes
 
1 To whom correspondence should be addressed Email: marja.mutanen{at}helsinki.fi Back


    Acknowledgments
 
This work is part of the Finnish technology program `Innovation in Foods'.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received October 21, 1999; revised January 24, 2000; accepted February 14, 2000.