Global gene expression analysis of rat colon cancers induced by a food-borne carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
Kyoko Fujiwara,
Masako Ochiai,
Tsutomu Ohta1,
Misao Ohki1,
Hiroyuki Aburatani2,
Minako Nagao,
Takashi Sugimura and
Hitoshi Nakagama3
Biochemistry Division and 1 Medical Genetics Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan and 2 Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan
3 To whom correspondence should be addressed. Tel: +81 3 3547 5239; Fax: +81 3 35422530; Email: hnakagam{at}gan2.res.ncc.go.jp
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Abstract
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Colon cancers develop after accumulation of multiple genetic and epigenetic alterations in colon epithelial cells. To shed light on global changes in gene expression of colon cancers and to gain further insight into the molecular mechanisms underlying colon carcinogenesis, we have conducted a comprehensive microarray analysis of mRNA using a rat colon cancer model with the food-borne carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Of 8749 genes or ESTs on a high density oligonucleotide microarray, 27 and 46 were over- and underexpressed, respectively, by
3-fold in colon cancers in common in two rat strains with distinct susceptibility to PhIP carcinogenesis. For example, genes involved in inflammation and matrix proteases and a cell cycle regulator gene, cyclin D2, were highly expressed in colon cancers. In contrast, genes encoding structural proteins, muscle-related proteins, matrix-composing and mucin-like proteins were underexpressed. Interestingly, a subset of genes whose expression is characteristic of Paneth cells, i.e. the defensins and matrilysin, were highly overexpressed in colon cancers. The presence of defensin 3 and defensin 5 transcripts in cancer cells could also be confirmed by in situ mRNA hybridization. Furthermore, Alcian blue/periodic acid Schiff base (AB-PAS) staining and immunohistochemical analysis with an anti-lysozyme antibody demonstrated Paneth cells in the cancer tissues. AB-PAS-positive cells were also observed in high grade dysplastic aberrant crypt foci, which are considered to be preneoplastic lesions of the colon. Our results suggest that Paneth cell differentiation in colon epithelial cells could be an early morphological change in cryptic cells during colon carcinogenesis.
Abbreviations: AB-PAS, Alcian blue/periodic acid Schiff base; ACF, aberrant crypt foci; DIG, digoxigenin; APC, adenomatous polyposis coli; EST, expressed sequence tagged; H&E, hematoxylin and eosin; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
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Introduction
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The development of colon cancers comprises multiple steps requiring the accumulation of genetic and epigenetic alterations in colon epithelial cells, and these changes further affect expression of a variety of downstream genes and may cause considerable changes in gene expression profiles in cancer cells as a consequence. Inactivation of the adenomatous polyposis coli (APC) gene, ß-catenin, K-RAS, SMAD2, SMAD4, p53 and mismatch repair genes by genetic alterations, for example, play key roles (1,2). Furthermore, alterations of gene expression profiles by perturbation of CpG island methylation in promoter regions and/or the histone acetylation/deacetylation status of chromatin also have a substantial impact on colon carcinogenesis (2).
Oral administration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), one of the most abundant heterocyclic amines produced while cooking meat and fish (3,4), induces aberrant crypt foci (ACF) (5,6), putative preneoplastic lesions of the colon (7,8), in experimental animals within a short period and colon adenomas and adenocarcinomas after 1 or 2 years in rats, preferentially in males (9). A number of studies have revealed that PhIP-induced rat colon cancers resemble human neoplasms with regard to observed histological features and genetic alterations (1014). There are several advantages with the use of animal cancer models to dissect the molecular basis of colon carcinogenesis. For example, inbred experimental animals share a common genetic background within the strain and, furthermore, carcinogenesis experiments using these animals can be carried out under well-controlled conditions. Genetic and/or epigenetic alterations in colon cancers induced in experimental animals are therefore expected to be more uniform compared with those in humans with diverse genetic backgrounds. Colon cancers induced by PhIP indeed demonstrate ß-catenin accumulation in both cytoplasm and nucleus (13) and ß-catenin mutations are observed at codons 32, 34, 36 or 38 in exon 2, the majority being G
T transversions (12,13). In the Apc gene, 5'-GGGA-3' sites in exons 14 and 15 and a 5'-agGGGGG-3' site at the junction of intron 10 and exon 11 are mutation hot-spots (10,13). Using a model system, we have recently revealed sequential progression from dysplastic ACF to colon cancer (14,15). Although the PhIP-induced rat colon cancer model has provided cancer researchers with a powerful tool for dissecting molecular events involved in the formation of colon cancers with relevance to human colon carcinogenesis, extensive studies aimed at the elucidation of early genetic events in colon cancer development have hitherto not been conducted.
In the present study we therefore performed a global gene expression analysis of rat colon cancers induced by PhIP using high density oligonucleotide microarrays (GeneChip; Affymetrix, Santa Clara, CA). To eliminate detection of strain-specific changes, but rather to detect specific gene expression profiles essential for colon cancer development, two rat strains, F344 and ACI, were subjected to analysis, the former being the more susceptible to PhIP-induced colon carcinogenesis. A considerable number of genes were found to be differentially expressed in colon cancers compared with normal counterpart epithelium, including examples characteristic of Paneth cells. Global changes in gene expression profiles are also discussed in comparison with those reported in human colon cancers. Another focus is on the appearance of Paneth cells in ACF, especially in dysplastic ones, and its biological significance.
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Materials and methods
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Animals and diets
PhIP was purchased from the Nard Institute (Osaka, Japan) in the form of PhIP-HCl and added to AIN-93G basal diet (7% w/w soybean oil; Dyets, Bethlehem, PA) at a concentration of 400 p.p.m. A high fat diet (AIN-93G basal diet supplemented with 23% w/w hydrogenated vegetable oil) was also purchased from Dyets. Five-week-old male F344 and ACI strain rats were purchased from CLEA Japan (Tokyo, Japan) and housed 3 per cage in an air-conditioned animal room with a 12 h light/dark cycle. Prior to the experiment, all the animals were acclimatized to the housing environment and the AIN-93G basal diet for 1 week.
Experimental protocol and tissue samples
Starting at the age of 6 weeks, rats were fed a diet containing PhIP following an intermittent PhIP feeding protocol (13). At experimental week 60 all animals were killed and colons were removed. When colon cancers with polypoid growth were detected by the naked eye, cancerous parts were resected with a razor blade, bisected and one half was embedded in O.C.T. compound (Tissue-Tek; Sakura Finetechnical Co., Tokyo, Japan), frozen and stored at 80°C until use for frozen section preparation and RNA extraction. The remaining halves were fixed in neutral 10% formalin overnight at 4°C and embedded in paraffin blocks according to standard procedures. Normal counterparts were collected from the surrounding normal parts of the colon and separately embedded in O.C.T. compound and samples were snap-frozen in liquid nitrogen and stored at 80°C until use for RNA extraction. In separate experiments using the intermittent PhIP feeding protocol, ACF were assayed at experimental weeks 18 and 25, after fixation of tissue in formalin and embedding in paraffin blocks as described above.
High density oligonucleotide microarray analysis
Twelve colon cancer tissues, six each from F344 and ACI rats, and 12 normal counterparts were collected by digging them out of frozen O.C.T. blocks using 18 gauge needles. Total RNA was extracted from
1 mg of tissue with TRIZOL reagent (Invitrogen, Carlsbad, CA). Two of six colon cancer tissues from F344 rats, however, did not provide sufficient amounts of good quality RNA. The remaining four samples from F344 and six from ACI rats were subjected to the following experiments. cRNA was synthesized, labeled with biotin and hybridized to high density oligonucleotide microarrays, Rat Genome U34A (RG U34A; Affymetrix), as described previously. The average hybridization intensity for each array was scaled to 1000 to reliably compare multiple arrays. Prior to statistical analysis, genes were filtered according to the following criteria. For genes overexpressed in cancers, for example, they should have present (P) or marginal (M) calls in at least half of the colon cancer samples of the respective rat strains. For genes underexpressed in cancers, in contrast, they should have P or M calls at least in half of the normal counterpart samples. To assess statistical differences in gene expression between colon cancers and normal tissues, average signal intensity and standard variation were calculated for each group and GeneSpring 4.3 (Silicon Genetics, Redwood City, CA) was employed for the MannWhitney U-test. The significant P value was set at 0.05. Then, genes which were differentially expressed between cancer and normal tissue at
3-fold were selected and subjected to further analysis, including Venn diagrams, hierarchical clustering analysis, functional classification and comparison with expression profiles of human colon cancers. Permutation analysis was also carried out to assess the statistical significance of genes differentially expressed between the two rat strains.
Histological analysis
For hematoxylin and eosin (H&E) staining, paraffin sections were prepared at 3.5 µm thickness following standard procedures. Histological evaluation of colonic lesions was performed as described previously (13). For Alcian blue (pH 2.5)/periodic acid Schiff base (AB-PAS) staining to evaluate the presence of Paneth cells, both frozen (10 µm thickness) and paraffin (3.5 µm thickness) sections were used. The staining was carried out according to conventional methods.
In situ mRNA hybridiztion for defensin genes
In situ mRNA hybridization was carried out as described previously (16,17) under contract by Genostaff (Tokyo, Japan) using frozen sections prepared at 10 µm thickness. A 293 bp cDNA fragment of the rat neutrophil defensin 3 gene was amplified by PCR with primers 5'-CTCCCTGCATACGCCAAAG-3' (forward) and 5'-AACAGAGTCGGTAGATGCG-3' (reverse) and a 335 bp cDNA fragment of the defensin 5 gene with primers 5'-AACTTGTCCTCCTT TCTGCC-3' (forward) and 5'-AACATCAGCATCGGTGGCC-3' (reverse). Amplified fragments were cloned into pCRII (Invitrogen) and digoxigenin (DIG)-labeled RNA probes were generated by an in vitro transcription method using DIG-labeling mix (Roche Molecular Biochemicals, Tokyo, Japan). Hybridized probes were detected by an IgG antibody against the DIG label and visualized with NBT/BCIP solution (Roche Molecular Biochemicals). Nuclear counterstaining was performed with Kernechtrot Stain Sol (Muto Chemical, Tokyo, Japan).
Semi-quantitative RTPCR
Extracted RNA was transcribed to cDNA using an oligo(dT)1218 primer and SuperScriptTM II reverse transcriptase (Invitrogen) and the cDNAs produced were divided into aliquots in tubes and stored at 20°C until analyzed. Each aliquot of cDNA was subjected to semi-quantitative reverse transcription (RT)PCR with the primer sequences listed in Table I. A set of semi-quantitative RTPCR reactions for representative genes was carried out within 1 day to avoid the effects of degradation of cDNA templates. For reference, expression of the ß-actin and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) genes was also quantified for each sample. PCR amplification was carried out at 94°C for 30 s, 60°C for 30 s and 72°C for 1 min using Advantage Taq (Clontech, Palo Alto, CA) under the conditions recommended by the manufacturer. PCR cycles were set at 25 for ß-actin and G3PDH, 35 for
-defensin NP4 and ß-defensin 2 and 30 cycles for the other genes. PCR products were also analyzed by gel electrophoresis on a 2% agarose gel in 0.5x TBE (89 mM Tris, 89 mM boric acid, 1.9 mM EDTA). The amounts of PCR products were quantified by analysis performed on a Macintosh iBook G3 computer using the public domain NIH Image program (developed at the US National Institutes of Health and available on the Internet by anonymous ftp from zippy.nimh.nih.gov. or on floppy disk from the National Technical Information Service, Springfield, VA, part no. PB95-500195GEI). PCR reactions for individual genes were repeated twice, all of which gave similar results, and representative data are shown.
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Results
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Gene expression profiles in colon cancers of two rat strains
Of 8749 genes or ESTs on the RG U34A array, 89 and 97 were overexpressed
3-fold in colon cancers of F344 and ACI rats, respectively, and 109 and 183 were underexpressed by
3-fold, as shown in the Venn diagrams (Figure 1A and B; GeneChip data are available at http://www.ncc.go.jp/jp/nccri/divisions/02bioc/02bioc.html). As illustrated in Figure 1(A), 27 were overexpressed
3-fold in common in both F344 and ACI lesions. Although 62 genes were shown to be preferentially overexpressed in F344-derived colon cancers, 33 were also overexpressed
2-fold, in both strains (Figure 1A). Similarly, 32 of 70 genes overexpressed
3-fold in ACI rats were also overexpressed in the F344 strain by
2-fold. Collectively, of 159 genes which showed
3-fold expression in cancer tissues in either of the rat strains (Figure 1A), 92 (57.8%) showed
2-fold expression in colon cancers in both strains. Similarly, 158 of 246 (64.2%) genes demonstrated
2-fold expression in colon cancers in both strains (Figure 1B). We have recently reported that a considerable number of genes are differentially expressed in normal parts of colon epithelial cells between F344 and ACI rats after PhIP treatment (18) and the repertoire of differentially expressed genes may partly account for the different susceptibilities of the two rat strains. In contrast, differences in cancer tissues were relatively small compared with those in normal tissues. Permutation analysis was unable to detect apparent differences in the number of differentially expressed genes between cancer tissues of F344 and ACI rats, although significant differences were present in normal colon epithelium (data not shown). Hierarchical clustering analysis using the entire list of over- and underexpressed genes in cancers could not elucidate any cluster(s) of genes specific for either strain (data not shown).
Genes overexpressed in colon cancers
As described above, 27 genes were overexpressed
3-fold in common in both F344 and ACI rats (Figure 1A). Despite our efforts to elucidate meaningful signal pathways in those 27 genes using the bioinformatics software package GenMapp (http://www.genmapp.org/), none could be identified. This could be due, at least in part, to insufficient coverage of genes in signal pathway databases for rats. Manual classification, however, was able to elucidate some interesting tendencies. Genes involved in inflammation, such as those encoding interleukin 1ß, small inducible cytokine subfamily A20 precursor, and proteases, such as matrilysin (Mmp7) and macrophage metalloelastase (Mme) were highly overexpressed in cancer tissues (Table II). A cell cycle regulator, cyclin D2, and the cancer-related gene retrotransposon virus-like 30 s sequence (VL30), which is known to be overexpressed in rodent hepatocellular tumors and lymphomas (19,20), were also highly expressed. Interestingly, a subset of genes encoding defensin and defensin-like proteins, which belong to the small cationic antimicrobial cytotoxic peptides (2123), were overexpressed
10-fold. Differential expression of some of the representative genes was confirmed by semi-quantitative RTPCR (Figure 2A).
Genes underexpressed in colon cancers
Forty-six genes were underexpressed
3-fold in common in colon cancers of both the F344 and ACI strains (Figure 1B). Genes encoding metabolic enzymes, such as alanine aminotransferase, minoxidil sulfotransferase and carbonic anhydrase IV, and signal transduction molecules were among the list (Table III). A considerable number of transcripts related to the structural proteins, i.e. skeletal and smooth muscle-related proteins, matrix-composing proteins and mucin-like proteins, were underexpressed in colon cancers. Down-regulation of mucin genes in colon cancers may reflect the drastic decrease in or complete loss of goblet cells in cancer tissues. Representative results of RTPCR analyses are depicted in Figure 2A.
Expression of oncogenes and tumor suppressor genes
Expression of tumor-related genes, including those considered to be involved in colon carcinogenesis, was also evaluated utilizing the Chip data. Signals for the Apc, bcl2, c-jun, erbB3, VHL and WT1 genes were below detectable levels. The p53 and c-fos genes were expressed at comparable levels in both colon cancers and normal parts of colon tissues (data not shown). Only the c-myc gene was expressed at a significantly higher level in cancer tissues than in normal counterparts in both rat strains, with 4.0- and 2.5-fold differences in F344 and ACI rats, respectively.
Comparison of gene expression profiles with human colon cancers
Some of the genes in the list have already been reported to be either over- or underexpressed in human colon cancers (24,25). Average fold changes between colon cancers and normal counterparts were calculated using combined data from both the F344 and ACI strains and genes with
5-fold differences are listed in Table IV and compared with human cases, referring to the literature (2431). The defensin
5 and defensin
6 genes were previously reported to be up-regulated in human colon cancers [SAGE data (25)]. In the present study, defensin NP3 (
3) and defensin NP1 (
1)-like molecule were revealed to be overexpressed, although they have not been reported to be overexpressed in human cases. The matrix proteases Mmp-7 and Mme [DNA chip data (24)] and Mash2, Mrp14 and cyclin D2 [SAGE data (25)] were also reported to be highly expressed in human colon cancers. In the case of underexpressed genes, many of them were also reported to be down-regulated in human cancers, such as mucin, guanylin, carbonic anhydrase IV and several muscle- and structure-related genes (24,32).
Expression of defensin family genes in rat colon cancers
Defensin genes are composed of mainly two families,
and ß, categorized by sequence similarities (23,33). Genes in the former group are expressed mainly in neutrophils and some in the intestine, while the latter are ubiquitously expressed. defensin NP1 (
1)-like molecule and defensin NP3 (
3) was found to be highly expressed in colon cancers of both strains. In situ hybridization analysis revealed mRNAs of defensin NP3 (
3) and defensin
5 to be expressed exclusively in epithelial cells of colon cancers and not expressed in matrix cells (Figure 3A). Normal epithelium did not show any positive signals. Overexpression of these genes in colon cancer tissues was confirmed by RTPCR (Figure 2B). defensin
5, which is an intestinal-type defensin, but is not on the GeneChip, was also expressed exclusively in colon cancers. No expression was observed for defensin NP4 (
4), defensin ß1 (Figure 2B) or defensin ß-2 (data not shown) in either cancers or normal counterpart tissues.

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Fig. 3. Histological analysis of colon cancer tissues. (A) Frozen tissue sections were subjected to AB-PAS staining (a) and in situ hybridization with a defensin NP3 (b) or defensin 5 probe (c). Arrowheads in (b) and (c) indicate cells with defensin NP3 and defensin 5 transcripts, respectively. (B) Serial sections of paraffin embedded colon cancer tissues were subjected to H&E staining (a), AB-PAS staining (b) and immunostaining with an anti-lysozyme IgG antibody (c). As arrowheads indicate, Paneth cells can be recognized by the presence of typical pink granules (Paneth granules) by H&E staining. Paneth granules are also clearly visualized by AB-PAS staining (b) and by immunostaining for lysozyme protein (c).
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Presence of Paneth cells in cancers and preneoplastic lesions of the colon
Since intestinal-type defensins are known to be produced in Paneth cells (23,34), we examined the expression of other marker proteins specific to the Paneth cell lineage. H&E and AB-PAS staining revealed the presence of Paneth granules in colon cancer cells and lysozyme expression was also observed in cells with Paneth granules (Figure 3B). Furthermore, Paneth cells were observed in adenomas and, to our surprise, even in preneoplastic lesions. When examined by H&E and AB-PAS staining, three of eight colon cancers and two of three high grade dysplastic ACF observed at 18 or 25 weeks were demonstrated to contain Paneth cells within the lesion (Figure 4A). None of the non-dysplastic ACF demonstrated Paneth cell differentiation (Figure 4B).

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Fig. 4. Histological analysis of ACF. Serial sections of paraffin embedded high grade dysplastic (A) and non-dysplastic ACF (B) observed at experimental week 18. AB-PAS-positive Paneth granules are clearly evident in the high grade dysplastic ACF, but not in the non-dysplastic lesion. Another high grade dysplastic ACF collected at week 25 also gave similar results (data not shown). (a) H&E staining; (b) AB-PAS staining.
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Discussion
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With the present comprehensive gene expression analysis, conducted using PhIP-induced rat colon cancers in two rat strains, PhIP-induced colon cancers were found to possess somewhat common gene expression profiles in both F344 and ACI rats, despite the significant differences in gene expression between their normal colon epithelium (18). Furthermore, a subset of genes known to be overexpressed in human colon cancers was highly expressed in colon cancers of both rat strains. In addition, there are substantial similarities in a list of underexpressed genes between human (24) and rat tumors (present study). The histological features of the rat cancers also showed a high similarity with human cases, as described previously (10,11,13,14). Taking all the data together, the PhIP-induced colon carcinogenesis model in rats thus appears an appropriate and relevant system for investigation of human colon carcinogenesis.
High expression of cyclin D2 and c-myc may result from activation of the Wnt/ß-catenin signaling pathway as a consequence of ß-catenin accumulation, this being a common feature in rat and human tumors (2,13,35). An mRNA species for a mucin-like protein, homologous to mouse mucin 2, was here shown to be underexpressed in cancer tissues. Mucins are known to be abnormally expressed in neoplastic lesions of the colon of humans (36). Moreover, since mucin 2/ mice demonstrated reduced numbers of goblet cells and spontaneously developed adenomas in the small intestine (37), down-regulation of the mucin-like protein mRNA may play an important role in PhIP colon carcinogenesis in rats.
Some of the genes found in the present study, including those encoding helixloophelix protein MASH2, organic cation transporter (OCT1A4), calcium-binding protein MRP14 and regulator of ubiquitous kinase (RUK), have not been reported so far to be differentially expressed in human colon cancers. Some of them are intriguing with regard to their biological functions, although there are few reports suggesting their involvement in human colon carcinogenesis. Considering the size (small), the non-invasive nature of rat lesions and the rare occurrence of p53 or K-ras mutations in PhIP-induced colon cancers (38,39), differential expression of these genes in cancer tissues may suggest that their alteration occurs at an early stage of human colon carcinogenesis. Alternatively, of course, this could simply be a rodent-specific phenomenon. Further analysis is warranted in the future to clarify this point.
Snyderwine et al. recently reported gene expression profiles in PhIP-induced mammary gland tumors (40). Expression of a few genes overlapped between mammary gland (40) and colon tumors (present study) induced by PhIP, except for the tubulin ß15 gene. In PhIP-induced rat mammary cancer, deregulation of cyclin D1/Cdk4 and phospho-Rb was postulated to play a central role (41). The results indicate that the molecular basis underlying PhIP carcinogenesis could differ from organ to organ, although cell proliferation may be accelerated by PhIP in both cases. In fact, ß-catenin mutations are frequently observed in rat colon cancers (1113), but only rarely in mammary tumors (42). Moreover, the mutation spectra found in PhIP-induced tumors also differ with the organ (43).
It is of great interest to note that Paneth cells were observed in colon cancers and even in much earlier lesions, dysplastic ACF. Paneth cells exist mainly in the small intestine and sometimes in colonic tumors, but are rarely found in normal colon epithelium (44,45). Yamada et al. have recently reported the presence of Paneth cells in crypts which accumulated ß-catenin (BCAC) induced by an alkylating agent, azoxymethane (46,47). Based on their observations and a previous report by Wilson et al. (48), Yamada and Mori (49) suggested a dysdifferentiating potential of BCAC and that Paneth cell differentiation could promote intestinal carcinogenesis. Taking the results together, the appearance of Paneth cells in colon cancers does not appear to be carcinogen-specific, but could be a common phenomenon in the development of colon cancers. Although the molecular mechanisms underlying the induction of Paneth cells in colon cancers remain to be clarified, activation of the Wnt/Apc/ß-catenin signaling pathway could be one causative event. Inhibition of ß-catenin/TCF signaling in colon cancer cell lines indeed results in G1 arrest or induction of markers which are characteristic of differentiated colon epithelial cells, as described previously (50). Mice deficient for the TCF4 transcription factor completely lack proliferating cells in the fetal small intestinal epithelium (51). The Wnt/APC/ß-catenin signaling pathway thus could be essential for maintenance of the differentiated or undifferentiated status of intestinal epithelial cells. Activation of the Wnt/APC/ß-catenin pathway may therefore affect the differentiation process and induce dysdifferentiation of colon epithelial cells as a consequence. Activation of this pathway is commonly observed in both PhIP- (1015) and azoxymethane-induced (35,52,53) colon cancers and also in preneoplastic lesions (15). Gene expression analysis of teratomas, derived from embryonic stem cells with null APC, showed up-regulation of defensin
genes compared with teratomas derived from embryonic stem cells with wild-type APC (54). Paneth cells were also observed in APC-deficient teratoma tissues (54). Again, it is highly plausible that activation of the Wnt/APC/ß-catein signaling pathway is a genetic causation of Paneth cell differentiation in colon cancer tissues. Although the biological consequences of Paneth cell differentiation (or metaplasia) for colon carcinogenesis remain to be clarified, it is possible that the appearance of Paneth cells reflects aberrant differentiation of colonic stem cells. Whatever the case, defensin could be utilized as a potential serological marker for early detection of colon cancers because of its nature as a secreted molecule.
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Acknowledgments
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This work was supported in part by a Grant-in-Aid for the Second Term of the Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labour and Welfare of Japan, and by a Grant-in-Aid for Scientific Research on Priority Areas of Cancer from the Ministry of Education, Science, Sport, Culture and Technology of Japan. K.F. was the recipient of a Research Resident Fellowship from the Foundation for Promotion of Cancer Research in Japan while this work was being conducted.
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Received October 24, 2003;
revised March 11, 2004;
accepted March 22, 2004.