Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other malignancies of the same tissue type, often in a nearby location. Such field defects have been indicated in colon cancer. The molecular abnormalities that are responsible for a field defect in the colon should be detectable at high frequency in the histologically normal tissue surrounding a colonic adenocarcinoma or surrounding an adenoma with advanced neoplasia (well on the way to a colon cancer), but at low frequency in the colonic mucosa from patients without colonic neoplasia.Using immunohistochemistry, entire crypts within 10 cm on each side of colonic adenocarcinomas or advanced colonic neoplasias were found to be frequently reduced or absent in expression for two DNA repair proteins, Pms2 and/or ERCC1. Pms2 is a dual role protein, active in DNA mismatch repair as well as needed in apoptosis of cells with excess DNA damage. ERCC1 is active in DNA nucleotide excision repair. The reduced or absent expression of both ERCC1 and Pms2 would create cells with both increased ability to survive (apoptosis resistance) and increased level of mutability. The reduced or absent expression of both ERCC1 and Pms2 is likely an early step in progression to colon cancer.DNA repair gene Ku86 (active in DNA non-homologous end joining) and Cytochrome c Oxidase Subunit I (involved in apoptosis) had each been reported to be decreased in expression in mucosal areas close to colon cancers. However, immunohistochemical evaluation of their levels of expression showed only low to modest frequencies of crypts to be deficient in their expression in a field defect surrounding colon cancer or surrounding advanced colonic neoplasia.We show, here, our method of evaluation of crypts for expression of ERCC1, Pms2, Ku86 and CcOI. We show that frequency of entire crypts deficient for Pms2 and ERCC1 is often as great as 70% to 95% in 20 cm long areas surrounding a colonic neoplasia, while frequency of crypts deficient in Ku86 has a median value of 2% and frequency of crypts deficient in CcOI has a median value of 16% in these areas. The entire colon is 150 cm long (about 5 feet) and has about 10 million crypts in its mucosal layer. The defect in Pms2 and ERCC1 surrounding a colon cancer thus may include 1 million crypts. It is from a defective crypt that colon cancer arises.     

Applicability of different antibodies for the immunohistochemical localization of CFTR in respiratory and intestinal tissues of human and murine origin.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, which has a major role as a chloride (Cl(-)) channel. Although perhaps all functions of CFTR are still not fully characterized, localization studies are necessary to understand the consequences of the more than 1000 mutations thus far identified. Our aim was to determine the histological localization of CFTR on respiratory and colon epithelia of human and murine origin with a panel of several antibodies produced against different CFTR epitopes, using an indirect immunofluorescence method. Our results on human tissues confirm the apical localization of CFTR in ciliated cells of the respiratory mucosa and show that in colon tissue CFTR is observed in both apical and basolateral membranes of epithelial cells from colon crypts. However, poor tissue preservation of colon biopsies after immunohistochemistry (IHC) raises doubts about the latter localization. Contrary to human, mouse colon epithelium (not biopsed) presents good tissue preservation and evidences many cylindrical surface cells with high apical expression of CFTR. For the antibodies' sensitivity, we demonstrate that MATG1061, 24-1, M3A7, and MPCT-1 give good results, allowing the histological localization of CFTR protein of both human and murine origin.     

TRAIL up-regulation must be accompanied by a reciprocal PKCε down-regulation during differentiation of colonic epithelial cell: implications for colorectal cancer cell differentiation

PKC isoenzymes play central roles in various cellular signalling pathways, participating in a variety of protein phosphorylation cascades that regulate/modulate cellular structure and gene expression. It has been firmly established that several isoforms of PKC have a role in the regulation of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) activity. Our interest in probing the role of the epsilon isoform of PKC in the colonic cell differentiation stems from the discovery that PKCε and TRAIL are involved in the differentiation of other cell types like hematopoietic stem cells. Although the role of PKCε and TRAIL in the gastrointestinal system is unclear, it has been observed that PKCε has oncogenic activity in colon epithelial cells (CEC), while TRAIL increases the death of intestinal epithelial cells during inflammation. Here we demonstrate a reciprocal expression of PKCε and TRAIL in human colon mucosa: CECs at the bottom of the colonic crypts show high levels of PKCε, being negative for TRAIL expression. On the contrary, luminal CECs are positive for TRAIL, while negative for PKCε. Indeed, TRAIL- and butyrate-induced differentiation of the human colorectal cancer cell line HT29 requires the decrease of PKCε expression, whose absence in turn increases cell sensitivity to TRAIL-induced apoptosis. Moreover, TRAIL preferentially promotes HT29 differentiation into goblet cells. Taken together, this data demonstrate that TRAIL and PKCε must be reciprocally regulated to ensure physiological CEC differentiation starting from the stem cell pool, and that the down-regulation of PKCε is however critical for the differentiation and apoptosis of cancer cells.     

Periostin is expressed in pericryptal fibroblasts and cancer-associated fibroblasts in the colon.

Periostin is a unique extracellular matrix protein, deposition of which is enhanced by mechanical stress and the tissue repair process. Its significance in normal and neoplastic colon has not been fully clarified yet. Using immunohistochemistry and immunoelectron microscopy with a highly specific monoclonal antibody, periostin deposition was observed in close proximity to pericryptal fibroblasts of colonic crypts. The pericryptal pattern of periostin deposition was decreased in adenoma and adenocarcinoma, preceding the decrease of the number of pericryptal fibroblasts. Periostin immunoreactivity appeared again at the invasive front of the carcinoma and increased along the appearance of cancer-associated fibroblasts. ISH showed periostin signals in cancer-associated fibroblasts but not in cancer cells. Ki-67-positive epithelial cells were significantly decreased in the colonic crypts of periostin-/- mice (approximately 0.6-fold) compared with periostin+/+ mice. In three-dimensional co-culture within type I collagen gel, both colony size and number of human colon cancer cell line HCT116 cells were significantly larger ( approximately 1.5-fold) when cultured with fibroblasts derived from periostin+/+ mice or periostin-transfected NIH3T3 cells than with those from periostin-/- mice or periostin-non-producing NIH3T3 cells, respectively. Periostin is secreted by pericryptal and cancer-associated fibroblasts in the colon, both of which support the growth of epithelial components.     

Function of CSE1L/CAS in the secretion of HT-29 human colorectal cells and its expression in human colon

The secretion of colorectal epithelium is important for maintaining the physiological function of colorectal organ. Herein, we report that cellular apoptosis susceptibility (CAS) (or CSE1L) protein regulates the secretion of HT-29 human colorectal cells. Polarity is essential for directed secretion of substances produced by epithelial cells to the external (luminal) compartment; CAS overexpression induced polarization of HT-29 cells. CAS was punctate stained in the cytoplasm of HT-29 cells, and CAS overexpression increased the translocation of CAS-stained vesicles to the cytoplasm near cell membrane and cell protrusions. CAS overexpression increased the secretion of carcinoembryonic antigen (CEA) and cathepsin D. Immunohistochemistry showed CAS was positively stained in the goblet cells of colon mucosa and cells in the crypts of Lieberkühn of human colon as well as the glands in metastatic colorectal cancer tissue. Our results suggest that CAS regulates the secretion of colorectal cells and may regulate the metastasis of colorectal cancer.     

Morphological, histochemical and immunohistochemical differences between tumorous and adjacent tissues in chemically induced colon cancer in rats.

Methods of morphological, histochemical and immunohistochemical analyses were used to further characterize differences between tumourous and adjacent grossly normal tissues in chemically-induced colon cancer in rats. Colon tumors were induced by the treatment of rats with 1,2-dimethylhydrazine or with N-methyl-N'-nitro-N-nitrosoguanidine alone or with subsequent treatment with deoxycholic bile acid. Tissues were studied morphologically (for the presence of goblet cells in the colon crypts, and the extent of infiltration of lymphocytes into the crypts and between them), histochemically (for the presence of positive reaction to neutral and acid mucopolysaccharides) and immunohistochemically (for the presence of tissue polypeptide antigen). All data were evaluated quantitatively, and index of tissue damage was calculated for both tumorous and non-tumorous tissues. Significant morphological differences were found between tumorous and adjacent apparently normal tissue. Histochemically and immunohistochemically, both types of tissue reacted very similarly to exposure to the carcinogens. Index of damage was significantly different from normal untreated colon in both kinds of tissue. It was suggested that precancerous state in tissue adjacent-to-tumor could be detected using the combination of these methods.     

Isoforms of the human PDZ-73 protein exhibit differential tissue expression

Patients with renal and colon cancer frequently develop IgG autoantibodies toward the NY-CO-38/PDZ-73 antigen, a protein of 652 amino acids (73 kDa) which contains three copies of the PDZ protein-protein interaction domain. The gene encoding PDZ-73 mapped to chromosome 11p15.4-p15.1. Additional tissue-specific isoforms were identified: PDZ-45, which lacks the third PDZ domain and the putative PEST protein degradation motif, is expressed in kidney, colon, small intestine, brain and testis; PDZ-54 and PDZ-59, which also lack the third PDZ domains, have unique carboxyl terminal amino acids and are expressed in brain, kidney, bladder, colon cancer and renal cancer; and a putative PDZ-37 isoform, containing only the third PDZ domain, that is expressed in the central nervous system. Immunohistochemical staining with anti-PDZ 73 monoclonal antibodies showed strong cytoplasmic reactivity in epithelial cells of the small intestine, colon and kidney tubules, with a prominent apical staining pattern in cells of the small intestine. The reactivity pattern of the antibodies with various tissues correlated with the mRNA expression pattern of the PDZ-45 isoform. The existence of multiple PDZ-73 isoforms with variations in tissue distribution, PDZ domains, protein degradation sequences and carboxyl terminal structure indicate that these isoforms have distinct tissue-specific functions.     

Application of confocal laser scanning microscopy to detect oxidative stress in human colon cells

Introduction Excess of intracellular reactive oxygen species in relation to antioxidative systems results in an oxidative environment which may modulate gene expression or damage cellular molecules. These events are expected to greatly contribute to processes of carcinogenesis. Only few studies are available on the oxidative/reductive conditions in the colon, an important tumour target tissue. It was the objective of this work to further develop methods to assess intracellular oxidative stress within human colon cells as a tool to study such associations in nutritional toxicology.

Methods We have measured H₂O₂-induced oxidative stress in different colon cell lines, in freshly isolated human colon crypts, and, for comparative purposes, in NIH3T3 mouse embryo fibroblasts. Detection was performed by loading the cells with the fluorigenic peroxide-sensitive dye 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (diacetoxymethyl ester), followed by in vitro treatment with H₂O₂ and fluorescence detection with confocal laser scanning microscopy (CLSM). Using the microgel electrophoresis (“Comet”) Assay, we also examined HT29 stem and clone 19A cells and freshly isolated primary colon cells for their relative sensitivity toward H₂O₂-induced DNA damage and for steady-state levels of endogenous oxidative DNA damage.

Results A dose-response relationship was found for the H₂O₂-induced dye decomposition in NIH3T3 cells (7.8-125 μM H₂O₂) whereas no effect occurred in the human colon tumour cell lines HT29 stem and HT29 clone 19A (62-1000 μM H₂O₂). Fluorescence was significantly increased at 62 μM H₂O₂ in the human colon adenocarcinoma cell line Caco-2. In isolated human colon crypts, the lower crypt cells (targets of colon cancer) were more sensitive towards H₂O₂ than the more differentiated upper crypt cells. In contrast to the CLSM results, oxidative DNA damage was detected in both cell lines using the Comet Assay. Endogenous oxidative DNA damage was highest in HT29 clone 19A, followed by the primary colon cells and HT29 stem cells.

Conclusions Oxidative stress in colon cells leads to damage of macromolecules which is sensitively detected in the Comet Assay. The lacking response of the CLSM-approach in colon tumour cells is probably due to intrinsic modes of protective activities of these cells. In general, however, the CLSM method is a sensitive technique to detect very low concentrations of H₂O₂-induced oxidative stress in NIH3T3 cells. Moreover, by using colon crypts it provides the unique possibility of assessing cell specific levels of oxidative stress in explanted human tissues. Our results demonstrate that the actual target cells of colon cancer induction are indeed susceptible to the oxidative activity of H₂O₂.     

Analysis of the 10q11 cancer risk locus implicates MSMB and NCOA4 in human prostate tumorigenesis

Genome-wide association studies (GWAS) have established a variant, rs10993994, on chromosome 10q11 as being associated with prostate cancer risk. Since the variant is located outside of a protein-coding region, the target genes driving tumorigenesis are not readily apparent. Two genes nearest to this variant, MSMB and NCOA4, are strong candidates for mediating the effects of rs109939934. In a cohort of 180 individuals, we demonstrate that the rs10993994 risk allele is associated with decreased expression of two MSMB isoforms in histologically normal and malignant prostate tissue. In addition, the risk allele is associated with increased expression of five NCOA4 isoforms in histologically normal prostate tissue only. No consistent association with either gene is observed in breast or colon tissue. In conjunction with these findings, suppression of MSMB expression or NCOA4 overexpression promotes anchorage-independent growth of prostate epithelial cells, but not growth of breast epithelial cells. These data suggest that germline variation at chromosome 10q11 contributes to prostate cancer risk by influencing expression of at least two genes. More broadly, the findings demonstrate that disease risk alleles may influence multiple genes, and associations between genotype and expression may only be observed in the context of specific tissue and disease states.     

Large chromosome deletions,duplications, and gene conversion events accumulate with age in normal human colon crypts

Little is known about the types and numbers of mutations that may accumulate in normal human cells with age. Such information would require obtaining enough DNA from a single cell to accurately carry out reliable analysis despite extensive amplification; and complete genomic coverage under these circumstances is difficult. We have compared colon crypts, which are putatively clonal and contain ~2000 cells each, to determine how much somatic genetic variation occurs in vivo (without ex vivo cell culturing). Using high‐density SNP microarrays, we find that chromosome deletions, duplications, and gene conversions were significantly more frequent in colons from the older individuals. These changes affected lengths ranging from 73 kb to 46 Mb. Although detection requires progeny of a single mutant stem cell to reach niche dominance over neighboring stem cells, none of the deletions appear likely to confer a selective advantage. Mutations can become fixed randomly during stem cell evolution through neutral drift in normal human crypts. The fact that chromosomal changes are detected in individual crypts with increasing age suggests that either such changes accumulate with age or single stem cell dominance increases with age, and the former is more likely. This progressive genome‐wide divergence of human somatic cells with age has implications for aging and disease in multicellular organisms.     

Colon Cryptogenesis: Asymmetric Budding

The process of crypt formation and the roles of Wnt and cell-cell adhesion signaling in cryptogenesis are not well described; but are important to the understanding of both normal and cancer colon crypt biology. A quantitative 3D-microscopy and image analysis technique is used to study the frequency, morphology and molecular topography associated with crypt formation. Measurements along the colon reveal the details of crypt formation and some key underlying biochemical signals regulating normal colon biology. Our measurements revealed an asymmetrical crypt budding process, contrary to the previously reported symmetrical fission of crypts. 3D immunofluorescence analyses reveals heterogeneity in the subcellular distribution of E-cadherin and β-catenin in distinct crypt populations. This heterogeneity was also found in asymmetrical budding crypts. Singular crypt formation (i.e. no multiple new crypts forming from one parent crypt) were observed in crypts isolated from the normal colon mucosa, suggestive of a singular constraint mechanism to prevent aberrant crypt production. The technique presented improves our understanding of cryptogenesis and suggests that excess colon crypt formation occurs when Wnt signaling is perturbed (e.g. by truncation of adenomatous polyposis coli, APC protein) in most colon cancers.