Epithelial Cells Of Normal Colon Research Articles

Expression of metastasis suppressor gene AES driven by a Yin Yang (YY) element in a CpG island promoter and transcription factor YY2.

We recently found that the product of the AES gene functions as a metastasis suppressor of colorectal cancer (CRC) in both humans and mice. Expression of amino‐terminal enhancer of split (AES) protein is significantly decreased in liver metastatic lesions compared with primary colon tumors. To investigate its downregulation mechanism in metastases, we searched for transcriptional regulators of AES in human CRC and found that its expression is reduced mainly by transcriptional dysregulation and, in some cases, by additional haploidization of its coding gene. The AES promoter‐enhancer is in a typical CpG island, and contains a Yin‐Yang transcription factor recognition sequence (YY element). In human epithelial cells of normal colon and primary tumors, transcription factor YY2, a member of the YY family, binds directly to the YY element, and stimulates expression of AES. In a transplantation mouse model of liver metastases, however, expression of Yy2 (and therefore of Aes) is downregulated. In human CRC metastases to the liver, the levels of AES protein are correlated with those of YY2. In addition, we noticed copy‐number reduction for the AES coding gene in chromosome 19p13.3 in 12% (5/42) of human CRC cell lines. We excluded other mechanisms such as point or indel mutations in the coding or regulatory regions of the AES gene, CpG methylation in the AES promoter enhancer, expression of microRNAs, and chromatin histone modifications. These results indicate that Aes may belong to a novel family of metastasis suppressors with a CpG‐island promoter enhancer, and it is regulated transcriptionally.

Profiling of the Tetraspanin Web of Human Colon Cancer Cells

Tetraspanins are integral membrane proteins involved in a variety of physiological and pathological processes. In cancer, clinical and experimental studies have reported a link between tetraspanin expression levels and metastasis. Tetraspanins play a role as organizers of multimolecular complexes in the plasma membrane. Indeed each tetraspanin associates specifically with one or a few other membrane proteins forming primary complexes. Thus, tetraspanin-tetraspanin associations lead to a molecular network of interactions, the "tetraspanin web." We performed a proteomic characterization of the tetraspanin web using a model of human colon cancer consisting of three cell lines derived from the primary tumor and two metastases (hepatic and peritoneal) from the same patient. The tetraspanin complexes were isolated after immunoaffinity purification using monoclonal antibodies directed against the tetraspanin CD9, and the associated proteins were separated by SDS-PAGE and identified by mass spectrometry using LC-MS/MS. This allowed the identification of 32 proteins including adhesion molecules (integrins, proteins with Ig domains, CD44, and epithelial cell adhesion molecule) (EpCAM), membrane proteases (ADAM10, TADG-15, and CD26/dipeptidyl peptidase IV), and signaling proteins (heterotrimeric G proteins). Importantly some components were differentially detected in the tetraspanin web of the three cell lines: the laminin receptor Lutheran/B-cell adhesion molecule (Lu/B-CAM) was expressed only on the primary tumor cells, whereas CD26/dipeptidyl peptidase IV and tetraspanin Co-029 were observed only on metastatic cells. Concerning Co-029, immunohistofluorescence showed a high expression of Co-029 on epithelial cells in normal colon and a lower expression in tumors, whereas heterogeneity in terms of expression level was observed on metastasis. Finally we demonstrated that epithelial cell adhesion molecule and CD9 form a new primary complex in the tetraspanin web.

Oestrogen receptor beta (ERβ) is abundantly expressed in normal colonic mucosa, but declines in colon adenocarcinoma paralleling the tumour's dedifferentiation

Oestrogen Receptor β (ERβ) may protect against prostate and mammary cell proliferation and malignant transformation. Epidemiological studies indicate that oestrogens may reduce colon cancer risk. Since ERα is minimally expressed in normal and malignant colon, the aim of this study was to investigate the expression of ERβ in both normal colonic wall and colon cancer. ERβ expression was evaluated by immunohistochemistry in 90 cases of colon adenocarcinoma and nearby (>30-cm away) normal colonic wall, using a monoclonal antibody. Moderate or strong nuclear immunostaining was detected in superficial and crypt epithelium, endothelial cells, vascular smooth muscle cells, lymphocytes, enteric neurons and smooth muscular cells of the normal colonic wall. Superficial epithelial cells in normal colon demonstrated a significantly higher ERβ expression than colon adenocarcinoma cells in both genders. The decline in ERβ expression paralleled the loss of differentiation of malignant colon cells, regardless of the tumour's localisation. These findings suggest a protective role for ERβ against colon carcinogenesis.

Subtractive immunization using highly metastatic human tumor cells identifies SIMA135/CDCP1, a 135 kDa cell surface phosphorylated glycoprotein antigen.

We have previously used a subtractive immunization (SI) approach to generate monoclonal antibodies (mAbs) against proteins preferentially expressed by the highly metastatic human epidermoid carcinoma cell line, M(+)HEp3. Here we report the immunopurification, identification and characterization of SIMA135/CDCP1 (subtractive immunization M(+)HEp3 associated 135 kDa protein/CUB domain containing protein 1) using one of these mAbs designated 41-2. Protein expression levels of SIMA135/CDCP1 correlated with the metastatic ability of variant HEp3 cell lines. Protein sequence analysis predicted a cell surface location and type I orientation of SIMA135/CDCP1, which was confirmed directly by immunocytochemistry. Analysis of deglycosylated cell lysates indicated that up to 40 kDa of the apparent molecular weight of SIMA135/CDCP1 is because of N-glycosylation. Western blot analysis using a antiphosphotyrosine antibody demonstrated that SIMA135/CDCP1 from HEp3 cells is tyrosine phosphorylated. Selective inhibitor studies indicated that an Src kinase family member is involved in the tyrosine phosphorylation of the protein. In addition to high expression in M(+)HEp3 cells, the SIMA135/CDCP1 protein is expressed to varying levels in 13 other human tumor cell lines, manifesting only a weak correlation with the reported metastatic ability of these tumor cell lines. The protein is not detected in normal human fibroblasts and endothelial cells. Northern blot analysis indicated that SIMA135/CDCP1 mRNA has a restricted expression pattern in normal human tissues with highest levels of expression in skeletal muscle and colon. Immunohistochemical analysis indicated apical and basal plasma membrane expression of SIMA135/CDCP1 in epithelial cells in normal colon. In colon tumor, SIMA135/CDCP1 expression appeared dysregulated showing extensive cell surface as well as cytoplasmic expression. Consistent with in vitro shedding experiments on HEp3 cells, SIMA135/CDCP1 was also detected within the lumen of normal and cancerous colon crypts, suggesting that protein shedding may occur in vivo. Thus, specific immunodetection followed by proteomic analysis allows for the identification and partial characterization of a heretofore uncharacterized human cell surface antigen.

Novel isoglobo-neolacto-series hybrid glycolipid detected by a monoclonal antibody is a rat colon tumor-associated antigen

Isoglobotetraosylceramide (GalNAc(beta 1-3)Gal(alpha 1-3)Gal(beta 1-4)Glc (beta 1-1)Cer), the major glycolipid species in dimethylhydrazine-induced rat tumors of colorectal origin, was not detected in epithelial cells of normal colon but was present in the non-epithelial stroma and could be extracted from each of nine tumors studied. Monoclonal antibodies produced against isoglobotetraosylceramide detected this and another novel rat tumor-associated glycolipid not present in epithelial cells nor in non-epithelial stroma of normal rat colon (Brodin, T., Thurin, J., Strömberg, N., Karlsson, K.-A. and Sjögren, H.O. (1985) Eur. J. Immunol. 16, 951-956). This novel glycolipid was present in 8/9 of the studied tumors and was also present in two in vitro cell clones. These were originally obtained from a W49/T4 colon tumor isograft. The novel glycolipid was characterized by mass spectrometry, 1H-NMR, and methylation analysis as a hybrid between the isoglobo- and neolacto-series, with the structure GalNAc(beta 1-3)Gal(alpha 1-3)Gal(beta 1-4)GlcNA(beta 1-3)Gal (beta 1-4)Glc(beta 1-1)Cer.

Cell population kinetics of 1,2-dimethylhydrazine-induced colonic neoplasms and their adjacent colonic mucosa in the mouse.

The parameters of cell population kinetics of symmetrical 1,2-dimethylhydrazine-induced colonic neoplasms and their adjacent colonic mucosa in the mouse were analyzed using the fraction labeled-mitoses curve method and compared with those of three groups of epithelial cells in the crypt of the descending colon of normal mouse. The analysis of three groups of epithelial cells in the crypt of normal mouse indicates that differentiation of epithelial cells was associated not only with a smaller proliferative pool of cells but also with a shortening of the duration of G2 phase and a prolongation of mitotic time. Other parameters of cell cycle did not change significantly. The mean cell cycle time of neoplastic cells in chemically induced colonic neoplasms was similar to that of epithelial cells in normal colon, but the variance was much greater in neoplastic cells. In neoplastic cells, the proliferative pool was greater, the G1 phase prlonged, and the S phase and the mitotic time became shorter as compared to epithelial cells in normal colon. The duration of G2 phase of neoplastic cells fell between the values of presumptive stem cells and differentiating cells in normal colon, compatible with the hypothesis that neoplastic cells are transformed stem cells defective in cellular differentiation. In the colonic mucosa immediately adjacent to neoplasms, the fraction-labeled-mitoses curve showed a flat second wave, indicating that the group of cells initially labeled by the pulse became a mixture of cells, some continuing the proliferative cycle normally, some going out of cycle, some slowing down in their passage from S through G2 to M, and some being arrested in mitotic phase. Such heterogeneous behavior of cells may be closely related to expansion of neoplasms. With some assumptions, however, cell cycle parameters of those normally cycling cells were estimated: the cell cycle time and the duration of G1 phase and mitotic phase were prolonged as compared to neoplastic cells and epithelial cells of normal colon.