Colorectal Tumor Development Research Articles

Evolution of DNA ploidy state and DNA index in colorectal adenomas and carcinomas using the crypt isolation technique: new hypothesis in colorectal tumorigenesis.

The evolution of DNA diploid, aneuploid and multiploid (diploid and aneuploid) states that represent DNA types that are independent of genetic alterations in colorectal tumors were examined. Changes in the DNA index (DI) accompanying tumor development from adenoma to carcinoma were assessed. In colorectal adenomas and early cancers, the DNA was diploid or multiploid. A pure aneuploid state was observed in advanced carcinomas only, whereas the aneuploid DI values of adenomas were characterized by two distinct peaks. The DI values for the carcinomas were randomly distributed. However, in advanced carcinomas, aneuploid carcinomas tended to have lower DI whereas aneuploid populations within multiploid carcinomas tended to have higher DI. Early cancers were subdivided into two groups: a cancer region associated with an adenomatous region (group A tumors) and a cancer region that exhibited an absence of or a very limited adenomatous region (group B tumors). Group A tumor DI were lower than group B. It is suggested that low DI adenomas might transform into group A tumors, which consequently progress to advanced aneuploid carcinomas. In addition, group B tumors might derive predominantly from high DI adenomas or from group A tumors by high DI evolution, and might progress into advanced multiploid carcinomas. Therefore, the evolution of the DNA index might play an important role in the development of colorectal tumors.

Expression of E2F-4 gene in colorectal adenocarcinoma and corresponding covering mucosa: an immunohistochemistry, image analysis, and immunoblot study.

E2F-4 is a transcription factor involved in the transition of the cell from the resting state (G0/G1) to the proliferative stage (S). It has been associated with the p107 and p130 members of the Rb-family and it is responsible for many important growth suppressive functions. E2F-1, one member of the E2F family, has a similar structure to E2F-4; however, both have different mechanisms of action in regulating cell-cycle progression. Although E2F-4 acts mainly as a repressor in the early part of the cell cycle, E2F-1 has the ability to function as both an oncogene and a tumor suppressor gene. In an attempt to identify the role of E2F-4 as a potential mediator of cell proliferation, differentiation, tumorigenesis, and apoptosis in colorectal mucosa comparing with that of E2F-1, the authors examine 20 patients with human colon cancer and their corresponding histologically healthy mucosa by using immunohistochemical methods, computerized quantitative image analysis, and immunoblot analysis. Immunohistochemical studies were performed with formalin-fixed, paraffin-embedded sections stained with a monoclonal antibody against the E2F-4 protein. Apoptosis levels were determined by in situ assay. Positivity was scored by a Computerized Image Analyzer to detect the relative amount of the protein. Immunoblot analysis was performed on protein extracts from snap-frozen tissues of the same specimens. The results show that the expression of E2F-4 was greater in the tumor cells than in their corresponding benign epithelium as determined by immunohistochemical staining and image analysis. This was confirmed by semiquantitative IB analysis of the E2F-4 protein. The labeling index (LI) of E2F-4 in the tumors was inversely proportional to the LI of apoptotic cells. Within these cases, 12 cases showed a very high E2F-4 LI corresponding to low apoptosis LI. Three cases with relatively lower levels of E2F-4 LI were characterized with high apoptotic rates. These data suggest that E2F-4 gene overexpression plays a role in the development of colorectal tumors and appears to play a role in suppressing apoptosis.

Mutation and expression of the beta-catenin-interacting protein ICAT in human colorectal tumors.

Aberrant activation of Wnt signaling caused by mutations in the tumor suppressor adenomatous polyposis coli or beta-catenin is a critical event in the development of human colorectal tumors. We have recently identified the ICAT gene, which encodes a small protein that interacts with beta-catenin and represses Wnt signaling. We examined the prevalence of mutations in the entire ICAT coding sequence and intronic splice donor and acceptor regions of ICAT by PCR-SSCP and also the expression of the ICAT gene by RT-PCR. The ICAT gene was mapped to chromosome 1p36.1-p36.2, which is implicated in the pathogenesis of various types of cancers. However, no mutations in ICAT were detected among 128 colorectal tumors. Instead, ICAT was found to be overexpressed in almost half of colorectal carcinomas. Cases exhibiting ICAT overexpression showed a significantly higher incidence of well-differentiated adenocarcinoma and positive lymphatic permeation. Our results suggest that ICAT is not the putative tumor suppressor on 1p36.1-p36.2, although aberrant overexpression of ICAT may play a role in the pathogenesis of colorectal carcinomas.

Expression of CD10 by stromal cells during colorectal tumor development

CD10 is a cell surface metalloprotease expressed by a variety of normal cell types, including lymphoid precursor cells, germinal center B lymphocytes, and some epithelial cells. We noticed that stromal cells of some cancers are positive for CD10. In this study, we investigated the role of CD10 produced by the stromal cells of colorectal neoplasms in the progression of colorectal neoplasms. Immunohistochemical examination of CD10 and p53 was performed in 169 colorectal epithelial neoplasms representing various stages of carcinogenesis. The results were correlated with the morphologic characteristics of the neoplasms. There was no expression of CD10 in the stromal cells of normal colorectal tissue. CD10-positive stromal cells were present adjacent to the tumor cells in 16 of 73 adenomas with mild or moderate dysplasia. More frequent expression of CD10 by the stromal cells was detected in adenomas with severe dysplasia (12 of 17), intramucosal carcinomas (10 of 16), and invasive carcinomas (50 of 63) than in adenomas with mild or moderate dysplasia (P < 0.0001). Expression of CD10 by > 10% of the stromal cells was detected only within the area of the invasive growth front of invasive carcinomas, not in adenomas and in only 1 of the intramucosal carcinomas. The difference between invasive and non invasive tumors was significant (P < 0.0001). The stromal expression of CD10 was significantly associated with the accumulation of p53 and a larger tumor size. These results indicate that CD10 expression is an integral part of colorectal carcinogenesis. CD10 expression seems to contribute to the invasion and thus probably facilitates metastasis. HUM PATHOL 33:806-811. Copyright 2002, Elsevier Science (USA). All rights reserved.

Natural history of colorectal nonpolypoid adenomas: a prospective colonoscopic study and relation with cell kinetics and K-ras mutations

Serial colonoscopic observations were prospectively conducted to elucidate the natural history of nonpolypoid tumors. Furthermore, to clarify whether cell kinetic status affects the tumor development, proliferative indices, apoptotic indices, and K-ras codon 12 point mutations on biopsy specimens were investigated. Seventy-five colorectal tumors, 13 polypoid and 62 nonpolypoid type (56 flat elevated and six depressed type) were studied. Proliferating and apoptotic cells were detected with anti-Ki-67 antibody and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method, respectively. Point mutations at K-ras codon 12 were examined by enriched polymerase chain reaction-based restriction fragment length polymorphism assay. The average follow-up period was 22 months (range 1-50). The lesions of subsequent exophytic growth, unchanged shape, depressed growth, and disappearance were observed in 0%, 92%, 0%, and 8% of polypoid type, in 39%, 39%, 13%, and 9% of flat elevated type, and in 33%, 67%, 0%, and 0% of depressed type, respectively. There was no significant difference in tumor size between initial and follow-up colonoscopy. Nonpolypoid tumors apparently changed to the exophytic growth during 2 yr or more. The tumors with exophytic growth had significantly higher proliferative indices/apoptotic indices ratios than those with unchanged morphology and disappearance/depressed growth (p < 0.05, respectively). K-ras codon 12 point mutations did not correlate with tumor development. Cell kinetic status plays an important role in determining minute colorectal tumor development, but not K-ras codon 12 mutations. Minute nonpolypoid adenomas frequently tend to grow slowly, and nearly 40% of those become the exophytic growth with time. Most of minute nonpolypoid tumors seem to follow the adenoma-carcinoma sequence.

Natural history of colorectal nonpolypoid adenomas: a prospective colonoscopic study and relation with cell kinetics and K- ras mutations

OBJECTIVE: Serial colonoscopic observations were prospectively conducted to elucidate the natural history of nonpolypoid tumors. Furthermore, to clarify whether cell kinetic status affects the tumor development, proliferative indices, apoptotic indices, and K- ras codon 12 point mutations on biopsy specimens were investigated. METHODS: Seventy-five colorectal tumors, 13 polypoid and 62 nonpolypoid type (56 flat elevated and six depressed type) were studied. Proliferating and apoptotic cells were detected with anti-Ki-67 antibody and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method, respectively. Point mutations at K- ras codon 12 were examined by enriched polymerase chain reaction-based restriction fragment length polymorphism assay. RESULTS: The average follow-up period was 22 months (range 1–50). The lesions of subsequent exophytic growth, unchanged shape, depressed growth, and disappearance were observed in 0%, 92%, 0%, and 8% of polypoid type, in 39%, 39%, 13%, and 9% of flat elevated type, and in 33%, 67%, 0%, and 0% of depressed type, respectively. There was no significant difference in tumor size between initial and follow-up colonoscopy. Nonpolypoid tumors apparently changed to the exophytic growth during 2 yr or more. The tumors with exophytic growth had significantly higher proliferative indices/apoptotic indices ratios than those with unchanged morphology and disappearance/depressed growth ( p < 0.05, respectively). K- ras codon 12 point mutations did not correlate with tumor development. CONCLUSIONS: Cell kinetic status plays an important role in determining minute colorectal tumor development, but not K- ras codon 12 mutations. Minute nonpolypoid adenomas frequently tend to grow slowly, and nearly 40% of those become the exophytic growth with time. Most of minute nonpolypoid tumors seem to follow the adenoma-carcinoma sequence.

Mutations in APC, Kirsten-ras, and p53--alternative genetic pathways to colorectal cancer.

Colorectal cancer is one of the most significant causes of cancer death. A genetic model for colorectal cancer has been proposed in which the sequential accumulation of mutations in specific genes, including adenomatous polyposis coli (APC), Kirsten-ras (K-ras), and p53, drives the transition from healthy colonic epithelia through increasingly dysplastic adenoma to colorectal cancer. We have characterized tumor mutation spectra in a large cohort of colorectal cancer patients. In marked contrast to the predictions of the sequential model of mutation accumulation, only 6.6% of tumors were found to contain mutations in APC, K-ras, and p53, with 38.7% of tumors containing mutations in only one of these genes. The most common combination of mutations was p53 and APC (27.1%), whereas mutations in both p53 and K-ras were extremely rare. Statistical analysis (two-sided Fisher's exact test) confirmed that mutations in K-ras and p53 co-occurred less frequently than expected by chance (P < 0.01, Fisher's exact test). This finding suggests that these mutations lie on alternate pathways of colorectal tumor development. The heterogeneous pattern of tumor mutations in our patient cohort suggests that multiple alternative genetic pathways to colorectal cancer exist and that the widely accepted genetic model of cancer development is not representative of the majority of colorectal tumors.

Association of a common polymorphism in the human GH1 gene with colorectal neoplasia.

Growth hormone (GH) may be associated with the development of colorectal tumors directly and/or indirectly via an increased plasma level of insulin-like growth factor-I (IGF-I), which has been associated with colorectal cancer risk. Because a T-to-A polymorphism in the human GH1 gene at position 1663 is putatively associated with lower levels of GH and IGF-I, we investigated the relationship of this polymorphism to the risk of colorectal neoplasia. We analyzed data from two case-control studies conducted in Hawaii: a population-based study of 535 case patients with colorectal adenocarcinoma and 650 control subjects and a sigmoidoscopy screening-based study with 139 case patients with adenoma and 202 control subjects. All subjects were tested for the GH1 polymorphism. Logistic regression was used to adjust for known risk factors. Plasma IGF-I and IGF binding protein-3 (IGFBP-3) levels were measured in a subset of 293 subjects in the adenoma study (135 case patients and 158 control subjects). All statistical tests were two-sided. Adjusted odds ratios (ORs) for colorectal cancer associated with T/T, T/A, and A/A genotypes were 1.00, 0.75 (95% confidence interval [CI] = 0.58 to 0.99), and 0.62 (95% CI = 0.43 to 0.90), respectively (P(trend) =.006). Adjusted ORs for adenoma were 1.00, 0.76 (95% CI = 0.46 to 1.24), and 0.62 (95% CI = 0.31 to 1.22), respectively (P(trend) =.17). Data from both studies consistently showed that the A allele was associated with a lower risk of colorectal neoplasia than the T allele, although the association with adenoma was not statistically significant. These associations were consistently suggested in Caucasians and Native Hawaiians but not in Japanese. The ratio of plasma IGF-I/IGFBP-3 was lower in individuals with the A allele than in individuals with the T allele (P =.01). The human T1663A GH1 gene polymorphism, which may confer lower levels of GH and IGF-I, appears to be associated with a decreased risk of colorectal cancer.

Arachidonic and Linoleic Acid Metabolism in Mouse Intestinal Tissue: Evidence for Novel Lipoxygenase Activity

Previous studies in our laboratory revealed a high expression of 15-lipoxygenase-1 in human colorectal carcinomas, suggesting the importance of lipoxygenase in colorectal tumor development. In this report, we have investigated the metabolism of arachidonic and linoleic acid by intestinal tissues of Min mice, an animal model for intestinal neoplasia. The polyp and normal tissues from Min mice intestine were homogenized, incubated with arachidonic or linoleic acid, and analyzed by reverse-, straight-, and chiral-phase HPLC. Arachidonic acid was converted to prostaglandins E2 and F2α. Little 12- or 15-hydroxyeicosatetraenoic acid was detected. Cyclooxygenase (COX)-2 was detected in polyps and the adjacent normal tissues by Western immunoblotting, but neither COX-1 nor leukocyte-type 12-lipoxygenase, the murine ortholog to human 15-lipoxygenase-1, was detected. These tissue homogenates converted linoleic acid to an equal mixture of 9(S)- and 13(S)-hydroxyoctadecadienoic acid (HODE). Inhibition of lipoxygenase activity with nordihydroguaiaretic acid blocked HODEs formation, but the COX inhibitor indomethacin did not. Degenerative-nested PCR analyses using primers encoded by highly conserved sequences in lipoxygenases detected 5-lipoxygenase, leukocyte-type 12-lipoxygenase, platelet-type 12-lipoxygenase, 8-lipoxygenase, and epidermis-type lipoxygenase-3 in mouse intestinal tissue. All of these PCR products represent known lipoxygenase that are not reported to utilize linoleic acid preferentially as substrate and do not metabolize linoleic acid to an equal mixture of 9(S)- and 13(S)-HODE. This somewhat unique profile of linoleate product formation in Min mice intestinal tissue suggests the presence of an uncharacterized and potentially novel lipoxygenase(s) that may play a role in intestinal epithelial cell differentiation and tumor development.

Alterations of the Dpc4 tumor suprressor gene in sporadic colon carcinoma: Identification of novel somatic mutation in tumor from Croatian patient

Dpc4 is functionally inactivated in about 10-20% of colorectal mutations. The role of DPC4 alterations in development and/or progression of colorectal tumors, we analyzed on 60 samples of Croatian patients. We investigated allelic losses and mutations in exons 8, 10 and 11. In this work we used polymerase chain reaction (PCR) as basic methods and variable nucleotide tandem re- peat (VNTR) analysis for three flanking markers (D18S474, D18S363, D18S46). LOH analysis was prepared on the acrylamide-Bis-acrylarnide gels with Spreadex polymer NAB, a novel gel matrix for DNA electrophoresis. In restriction fragment polymorphism (RFLP) analysis we used Mnll, Mael, and BspHI restriction site. Single strand conformation polymorphism (SSCP) was done onto GMA gels in denaturing conditions. Among 60 analyzed cases of colorectal cancers even 58% (97% ) were informative. LOH at any of three flanking markers was detected in 37 (64%) of informative tumor DNA's. Nine- teen cases of LOH found by D18S474 marker, 11 cases found by D18S363 marker, and 7 cases found by D18S46 marker, respectively. In RFLP analysis neither exarnined samples of colon carcinoma were positive for mutation in exon 8 and 10. Only one tumor had mutation in exon 11, and sequence analysis veri!ied new mutation -deletion of 20 bp (from 133-153 bp).. PCR-SSCP examination of 60 samples did not indicate other different types of SSCP variants. The malignant progression is a consequence more than one genetic change, and inactivation of DPC4 had a role 1n a multistep process of colon carcinoma progression.

IQ (2-amino-3-methylimidazo[4,5-f]quinoline)-induced aberrant crypt foci and colorectal tumour development in rats fed two different carbohydrate diets.

In most aberrant crypt foci (ACF) and colorectal tumour studies, chemical carcinogens not normally found in food have been used as initiators. In the present study the food-related compound, IQ (2-amino-3-methylimidazo[4,5-f]quinoline), has been used. A diet high in refined carbohydrates has been associated with enhanced development of ACF and colorectal cancer in humans. The present study was designed as an integrated part of our earlier published ACF study and follows the animals until tumour development. The aim of the study was to investigate (1) the effect of a refined carbohydrate-rich diet on the development of IQ-induced ACF over time and (2) possible correlation between early and late ACF and/or colorectal tumour development. The study showed that a feeding regimen with continuous doses of 0.03% IQ in the diet for 14 weeks, followed by 32 weeks without IQ was able to induce tumours in the rat colon, liver, skin and Zymbal gland. The data demonstrate that a sucrose-rich diet enhance ACF development. A correlation between the outcome of early and late ACF was seen. However, as the tumour incidence of this study was very low it was not possible to obtain a meaningful correlation between ACF development and colorectal tumour incidence.

Co-overexpression of DEAD box protein rck/p54 and c-myc protein in human colorectal adenomas and the relevance of their expression in cultured cell lines.

The RCK gene was cloned through a study of the breakpoint of the t(11;14)(q23;q32) chromosomal translocation observed in a human B-cell lymphoma and overexpression of the protein (rck/p54) due to the translocation was shown to be associated with malignant transformation. The rck/p54 protein belongs to the DEAD box protein/RNA helicase family, which has a variety of functions such as translation initiation, pre-mRNA splicing and ribosome assembly. It is considered that rck/p54 protein may have significant effects on the mRNA structure of genes associated with cell proliferation, facilitating protein synthesis. Expression of rck/p54 in colorectal adenomas, which are a premalignant lesion of colorectal cancer, was examined by Western blot analysis and immunohistochemistry. The rck/p54 protein was found to be overexpressed in tumor tissues resected from 17 of 26 cases (65.4%) of colorectal adenomas and 13 of 14 c-myc-positive cases (92.8%) also co-overexpressed rck/p54 protein. Thus, a significant correlation between rck/p54 and c-myc co-overexpression was found (Spearman's rank correlation, P = 0.0018). We demonstrate that overexpression of rck/p54 in two different cell lines, COS 7 and human colorectal cancer cell line SW480, caused an increase in c-myc protein levels by enhancement of its translation efficiency and/or stabilization of its mRNA. These results suggest that rck/p54 of the DEAD box protein/RNA helicase family may contribute to cell proliferation and carcinogenesis in the development of human colorectal tumors at the translational level by increasing synthesis of c-myc protein.

1,2-Dimethylhydrazine-induced colon carcinoma and lymphoma in msh2(-/-) mice.

Defective mismatch repair (MMR) in humans is particularly associated with familial colorectal cancer, but defective repair in mice is generally associated with lymphoma in the absence of experimental exposure to carcinogens. Loss of MMR also confers resistance to the toxic effects of methylating agents. We investigated whether resistance to methylation contributes to increased susceptibility to colorectal cancer in mice by exposing mice with defects in the MMR gene msh2 to a methylating agent. Tumor incidence and time of death in msh2(+/+), msh2(+/-), and msh2(-/-) mice were analyzed after weekly exposure (until tumor appearance) to the methylating agent 1,2-dimethylhydrazine (DMH). Chemically induced and spontaneous tumors were characterized by frequency, type, and location. The tumor incidence in untreated and treated mice of each genotype was compared by a Mann-Whitney U test. Carcinogen-induced apoptosis in histologic sections of small and large intestines was also determined. All statistical tests were two-sided. Homozygous inactivation of the msh2 gene statistically significantly accelerated (P<.0001) death due to the development of DMH-induced colorectal tumors and lymphomas. Rates of death from DMH-induced colorectal adenocarcinoma were similar in msh2 heterozygous and wild-type mice, but only msh2 heterozygotes (msh(+/-)) developed additional, noncolorectal malignancies (notably trichofolliculoma [two of 21], angiosarcoma of the kidney capsule [two of 21], and lymphoma [one of 21]), suggesting that heterozygosity for msh2 slightly increases DMH susceptibility. DMH induced apoptosis in small intestinal and colonic epithelial crypts that was dependent on active msh2. Inactivation of msh2 allows the proliferation of gastrointestinal tract cells damaged by methylating agents. Furthermore, MMR constitutes a powerful defense against colorectal cancer induced by DNA methylation.

Distinct Regions of the Cadherin Cytoplasmic Domain Are Essential for Functional Interaction with Gα12 and β-Catenin

Heterotrimeric G proteins of the G(12) subfamily mediate cellular signals leading to events such as cytoskeletal rearrangements, cell proliferation, and oncogenic transformation. Several recent studies have revealed direct effector proteins through which G(12) subfamily members may transmit signals leading to various cellular responses. Our laboratory recently demonstrated that Galpha(12) and Galpha(13) specifically interact with the cytoplasmic domains of several members of the cadherin family of cell adhesion molecules (Meigs, T. E., Fields, T. A., McKee, D. D., and Casey, P. J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 519-524). This interaction causes beta-catenin to release from cadherin and relocalize to the cytoplasm and nucleus, where it participates in transcriptional activation. Here we report that two distinct regions of the epithelial cadherin (E-cadherin) tail are required for interaction with beta-catenin and Galpha(12), respectively. Deletion of an acidic, 19-amino acid region of E-cadherin abolishes its ability to bind beta-catenin in vitro, to inhibit beta-catenin-mediated transactivation, or to stabilize beta-catenin; causes subcellular mislocalization of beta-catenin; and disrupts cadherin-mediated cell adhesion. On the other hand, deletion of a distinct 11-amino acid region of E-cadherin dramatically attenuates interaction with Galpha(12); furthermore, Galpha(12) is ineffective in stimulating beta-catenin release from an E-cadherin cytoplasmic domain lacking this putative Galpha(12)-binding region. These findings indicate that Galpha(12) and beta-catenin do not compete for the same binding site on cadherin and provide molecular targets for selectively disrupting the interaction of these proteins with cadherin.

KAI1 , CAR , and Smad4 expression in the progression of colorectal tumor

The abnormal expression of several genes is involved in the development of colorectal tumors. The aim of this study was to examine the expression of KAI1, CAR, and Smad4, which has been associated with the progression of other cancers. We examined 12 adenomas, 38 primary carcinomas, 10 liver metastases, and 7 adenocarcinoma cell lines, by fluorescent multiplex reverse transcription-polymerase chain reaction (RT-PCR). KAI1 expression was higher in adenomas (2.55 +/- 0.61; P < 0.05) and primary carcinomas (3.30 +/- 0.42; P < 0.005) than in normal mucosa, but it was not correlated with tumor stage. CAR expression was lower in Dukes' B (1.28 +/- 0.53) than in Dukes' A (2.38 +/- 0.48) (P < 0.05), but increased with tumor progression; Dukes' C (1.45 +/- 0.25), Dukes' D (1.53 +/- 0.14), and metastases (2.09 +/- 0.39) (P < 0.05). Smad4 expression increased in adenomas (2.30 +/- 0.46; P < 0.05), but decreased with tumor progression; Dukes' A (2.14 +/- 0.37), Dukes' B (1.65 +/- 0.41), Dukes' C (1.57 +/- 0.25), Dukes' D (1.08 +/- 0.18), and metastases (0.82 +/- 0.21) (P < 0.05). These results suggest that the upregulation of CAR and down-regulation of Smad4 are associated with the progression of colorectal tumors, while the upregulation of these genes and of KAI1 seems to be involved in the early stage.

Effect of dietary galacto-oligosaccharides on azoxymethane-induced aberrant crypt foci and colorectal cancer in Fischer 344 rats.

The aim of the present study was to investigate the effects of galacto-oligosaccharides (GOS, Elix'or) on the development of aberrant crypt foci (ACF) and colorectal tumours in rats treated with azoxymethane (AOM). Two groups of 102 male Fischer 344 rats were injected twice with AOM to induce colorectal tumours, and fed diets containing either a low [5% (w/w); LGOS] or a high [20% (w/w); HGOS] concentration of GOS. Four weeks after the last AOM injection, 18 animals from each group were killed and their colon was removed for scoring ACF. Half of the animals in the LGOS group were switched to an HGOS diet (L/HGOS) and half of those in the HGOS group to an LGOS diet (H/LGOS). Six weeks after the change in diet, nine animals per group were killed for scoring ACF. Ten months after the start of the study the remaining animals were killed for scoring colorectal tumours. The aberrant crypt multiplicity scored after 13 weeks and the colorectal tumour incidence in rats fed an HGOS diet were significantly lower than those in rats fed an LGOS diet. However, the induction of ACF by AOM, the proliferation rate and apoptotic index of the adenomas, and the size and multiplicity of colorectal tumours were not influenced by the amount of GOS in the diet. The aberrant crypt multiplicity, scored after 13 weeks, was predictive for the tumour outcome at the end of the study. It was concluded that an HGOS diet has a protective effect against the development of colorectal tumours in rats and that this protective effect is exerted during the promotion phase rather than the initiation phase of carcinogenesis.

POU Homeodomain Protein OCT1 Is Implicated in the Expression of the Caudal-related Homeobox Gene Cdx-2

The caudal homeobox gene Cdx-2 is a transcriptional activator for approximately a dozen genes specifically expressed in pancreatic islets and intestinal cells. It is also involved in preventing the development of colorectal tumors. Studies using "knockout" approaches demonstrated that Cdx-2 is haplo-insufficient in certain tissues including the intestines but not the pancreatic islets. The mechanisms, especially transcription factors, which regulate Cdx-2 expression, are virtually unknown. We found previously that Cdx-2 expression could be autoregulated in a cell type-specific manner. In this study, we located an octamer (OCT) binding site within the mouse Cdx-2 gene promoter. This site, designated as Cdx-2(P)OCT, is involved in the expression of the Cdx-2 promoter. Both pancreatic and intestinal cell lines were found to express a number of POU (OCT binding) homeodomain proteins examined by electrophoretic mobility shift assay. However, it appears that Cdx-2(P)OCT interacts only with OCT1 in the nuclear extracts of the intestinal cell lines examined, although it interacts with OCT1 and at least two other POU proteins that are to be identified in the pancreatic InR1-G9 cell nuclear extract. Co-transfecting OCT1 cDNA but not five other POU gene cDNAs activates the Cdx-2 promoter in the pancreatic InR1-G9 and the intestinal Caco-2 cell lines. In contrast, Cdx-2(P)OCT cannot act as an enhancer element if it is fused to a thymidine kinase promoter. Furthermore, Cdx-2(P)OCT-thymidine kinase fusion promoters cannot be activated by OCT1 co-transfection. Cell type-specific expression, cell type-specific binding affinity of POU proteins to the cis-element Cdx-2(P)OCT, and the DNA content-dependent activation of Cdx-2 promoter via Cdx-2(P)OCT by OCT1 suggest that POU proteins play important and complicated roles in modulating Cdx-2 expression in cell type-specific manners.

Distal deletion of 1p in colorectal tumors: An initial event and/or a step in carcinogenesis? Study by fluorescence in situ hybridization interphase cytogenetics

Cytogenetics studies have suggested that short arm deletion in chromosome 1 is involved in triggering colorectal tumor development. To elucidate the role of 1p under-representation in the tumoral process, we investigated by fluorescence in situ hybridization interphase cytogenetics, using simultaneously centromeric and p36 telomeric probes for chromosome 1, 27 primary adenocarcinomas, 5 metastases, 5 adenomas and as control 4 normal mucous membranes. The 1p under-representation in paradiploid tumoral cells, interpreted as a 1p deletion, was observed in 8/27 adenocarcinomas, 2/5 metastases and 3/5 adenomas. Thus, in diploid cells 1p deletion was observed in some tumors independently of the stage of the process. The 1p under-representation in total number of examined cells, i.e., diploid and aneuploid, was observed in 14/16 grade B1–B2 tumors, in 5/8 grade C1–C2 tumors, and all grade D tumors (3/3) and all metastases (5/5). There were no correlations with location or histological characteristics of cancers, gender or age of patients. These results show high frequency of 1p under-representation in intestinal tumors, and lead to separate the under-representation of 1p in diploid cells, which correspond to a 1p deletion probably implicated in the initiation of the process, from the under-representation in aneuploid cells, which mainly may be the consequence of complex rearrangements in relation to extension of the malignant process.

Carcinoma of the Ampulla of Vater Associated With or Without Adenoma: A Clinicopathologic Analysis of 198 Cases With Reference to p53 and Ki-67 Immunohistochemical Expressions

Adenomatous areas are found frequently within or in the vicinity of carcinoma of the ampulla of Vater. This makes definite diagnosis difficult in the preoperative examination. The adenoma–carcinoma development hypothesis is generally accepted for colorectal tumors. Recently, a genetic alteration model during colorectal tumor development has attracted much attention, leading to various studies. We studied clinicopathologic features, prognostic factors, and the alteration of the p53 tumor suppressor gene using p53 immunohistochemical staining in pure adenomas, pure carcinomas, and carcinomas with adenomatous areas. A proliferative activity of the tumors using Ki-67 was also evaluated. Nine cases of pure adenoma and 198 cases of carcinoma of the ampulla of Vater were selected for this study. Among the 198 cases of the carcinoma, 83 cases (42%) had adenomatous areas. Positivity of p53 immunohistochemical staining was 0% in pure adenomas, 36% in the adenomatous areas of carcinomas with adenomatous areas and 62% in the carcinomatous areas of carcinomas with adenomatous areas, and 56% in pure carcinoma. Accumulation of p53 protein and the Ki-67 labeling index revealed no significant difference in prognosis. The clinicopathological factors examined were as follows: degree of invasion of the surrounding tissue, such as duodenal wall; pancreatic parenchyma; the presence or absence of lymphatic permeation; venous invasion; perineural invasion; the presence of regional lymph node metastasis; and TNM stage. Each of the clinicopathological factors showed a significant difference. Multivariate analysis revealed strong predictors for a worse prognosis: presence of lymphatic permeation, invasion of the pancreas, and perineural invasion. In conclusion, our results are consistent with the adenoma–carcinoma development hypothesis. It would seem that the molecular events leading to p53 accumulation in neoplasms of the ampulla of Vater occur relatively late during the oncogenetic process. Moreover, we think it may be useful to refer to the p53 overexpression in the diagnosis of ampullary tumors.

Frequent Altered Expression of Fragile Histidine Triad Protein in Human Colorectal Adenomas

Fragile histidine triad (FHIT) gene is involved in deletions on the short arm of chromosome 3 in various human cancers. We found that 47% of colorectal adenomas, which is a higher frequency than that of K-ras, showed altered expression of the Fhit protein by Western blot analysis. The amount of Fhit protein was inversely correlated with the degree of dysplasia. Importantly, 27% of low-grade dysplastic adenomas showed altered expression of Fhit protein. Additionally, expression of human Fhit protein in human colon carcinoma cell line SW480 exhibited a marked inhibition of growth and rendered SW480 cells highly susceptible to undergo apoptosis compared with control cells. These findings suggest that altered expression of the FHIT gene is a quite early aberration in the development of colorectal tumors and that Fhit protein may act as a tumor suppressor.