Tatsuro Irimura

MUC1 mucin expression as a marker of progression and metastasis of human colorectal carcinoma.

BACKGROUND/AIMS The MUC1 mucin distributes among a variety of epithelial tissues (except the intestinal epithelia) and is often detectable in colorectal carcinoma tissues and cell lines. This study aimed to elucidate whether MUC1 mucin expression correlated to the progression of colorectal carcinomas. METHODS We collected 113 tissue specimens, including primary colorectal carcinoma, normal mucosa, liver metastases, lymph node metastases, and normal livers from 58 patients with colorectal carcinoma. Immunohistochemical staining and Western blotting analysis with mature MUC1 mucin-specific monoclonal antibodies were performed. RESULTS The levels of mature MUC1 mucins were significantly higher in carcinoma tissues than those in normal colonic mucosa (P < 0.001). Furthermore, the levels of mature MUC1 mucins were significantly higher in primary tumors from patients having metastasis or metastatic tumors than in primary tumors from patients without metastasis (P < 0.001). In the primary sites, mature MUC1 mucin levels apparently increased according to progression of the stages (P < 0.001). CONCLUSIONS These results strongly suggest that mature MUC1 mucins become ectopically expressed in colorectal carcinoma progressed to the metastatic stages and that mature MUC1 mucins may be a useful marker for advanced colorectal carcinoma.

Molecular cloning of a novel actin-binding protein, p57, with a WD repeat and a leucine zipper motif

A 57 kDa protein (p57) was obtained during the study on phosphatidylinositol‐specific phospholipase C. Its cDNA was isolated from calf spleen and human leukemia cell line HL60 libraries and cloned. In the primary structures of p57, they have two unique amino acid sequence motifs, a WD repeat and a leucine zipper motif. Furthermore, p57 shared sequence similarity (40%) with coronin, an actin‐binding protein responsible for chemotaxis, cell motility, and cytokinesis of Dictyostelium discoideum, which has only the WD repeat. p57 also showed an actin‐binding activity and was mainly expressed in immune tissues. From these results, we conclude that p57 is a coronin‐like novel actin‐binding protein in mammalian cells but may also have a different function from coronin.

Human colon carcinoma cells with increased invasive capacity obtained by selection for sialyl-dimeric Lex antigen

Sialyl-dimeric LeX antigen (NeuAc alpha 2-3Gal beta 1-4[Fuc-alpha 1-3] GlcNAc beta 1-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc beta 1-R; SLX) is an oncodevelopmental carbohydrate antigen expressed both on glycolipids and on mucin-like glycoproteins in human colorectal carcinomas. The levels are higher in primary tumors at advanced stages than in tumors at early stages, and metastatic lesions contain a greater quantity of this antigen than corresponding primary tumors. To study whether this antigen influences the metastatic behavior of tumor cells, we selected SLX variant cells from the human colon carcinoma cell line KM12C using the specific monoclonal antibody FH6. We obtained two stable cell lines: a high expresser (KM12-HX) and a low expresser (KM12-LX) of this antigen. The growth rate of these cells are similar. A mucin-like glycoprotein reactive with monoclonal antibody FH6 was detected after electrophoretic separation of KM12-HX cell lysates but not of KM12-LX lysates. The degree of invasion was compared in assays in vitro using matrigel-coated filter membranes. The number of KM12-HX cells that invaded the membranes was significantly higher than KM12-LX cells.

Expression of Sulfomucins in Normal Mucosae, Colorectal Adenocarcinomas, and Metastases

We have examined the expression of specific mucin antigens in tissue sections from 92 cases of colorectal carcinoma, using sulfomucin‐specific monoclonal antibody (MAb) 91.9H. The expression of sulfomucins was high in normal mucosae and much lower in primary colorectal carcinoma, in metastatic lesions in lymph nodes or in liver. The intracellular localization of sulfomucins was also different among these tissues. In normal mucosae, MAb 91.9H binding was seen in the supranuclear area, presumably Golgi complexes, the luminal surface, and secretory products. In primary colorectal carcinomas and in their metastatic lesions, MAb 91.9H was preferentially localized in the cell surface and substances attached to the luminal surface of glandular structures. Analysis of the lysates of normal and tumor tissues showed that very‐high‐molecular‐weight components contained the antigenic epitopes. The intensity of MAb 91.9H binding was lower in tumors at advanced stages than in tumors at early stages. These high‐molecular‐weight components were apparently reactive with MAb FH6 specific for sialyl‐Lex (s‐Lex) structures. Histological specimens with low levels of MAb 91.9H reactivity often exhibited relatively high levels of MAb FH6 reactivity. These two mucins may have reversed expression during carcinogenesis and carcinoma progression, and this change may be related to metastatic potential.

Metastatic behavior and cell surface properties of HT-29 human colon carcinoma variant cells selected for their differential expression of sialyl-dimeric Lex antigen

Immunochemical studies of human colorectal carcinoma with various monoclonal antibodies against Lex-related carbohydrate antigens previously revealed that the amount of sialyl-dimeric Lex antigen (NeuAcα2–3Gal/⋏1–4(Fucαr1–3)GlcNAcβ1–3Galβ1–4 (Fucα1–3)GlcNAcβ1-R: SLX) associated with metastatic lesions was greater than in the primary tumors. To assess whether an experimental model can be used to study the direct relationship between this carbohydrate antigen and the tumor cells metastatic behavior, we selected variant cells with increased surface SLX from established human colon carcinoma cell line HT-29. The cells in the upper 5% or lower 5% population in fluorescence intensity after reacting with a monoclonal antibody, FH6, were retrieved separately by a fluorescence-activated cell sorter and propagated. After three- or four-times selection, we obtained stable cell lines with low and high cell surface SLX antigens (HT-29 M1 and HT-29 M2, respectively). Binding of monoclonal antibody FH6 was detected to glycolipids extracted from HT-29 M2 cells but not from HT-29 Ml cells. Glycoprotein components having reactivity with monoclonal antibody FH6 were below the detectable level. HT-29 M2 cells injected intrasplenically into nude mice showed a slightly reduced incidence of metastatis to lung, liver and lymph nodes than did HT-29 M1 cells. Subsequently we found that SLX antigen was not detectable by immunohistochemical examination of these tumor cells grown in nude mice. Re-established cell line from nude mice xenografts expressed SLX antigenin vitro.

Expression of Sulfated Carbohydrate Chains Detected by Monoclonal Antibody 91.9H in Human Gastric Cancer Tissues

Expression of sulfated carbohydrate chains in intestinal metaplasia (IM) and gastric cancer tissues was immunohistochemically evaluated by using a monoclonal antibody (MAb) 91.9H. While normal gastric tissues were negative for MAb 91.9H, IM and cancer tissues were positively stained with high frequency. The incidence was 67% for IM with chronic gastritis (n = 12), 95% for IM with gastric cancer (n=21), 77% for well and moderately differentiated adenocarcinomas (n = 13), 48% for poorly differentiated adenocarcinoma (n = 23), 89% for signet‐ring cell carcinoma (n = 9) and 100% for mucinous adenocarcinoma (n = 7). When poorly differentiated adenocarcinoma cases were divided into two groups, solid (n = 13) and non‐solid types (n = 10), the incidences were 8% and 100%, respectively. These data suggest that MAb 91.9H could be of practical use as a new marker for IM and gastric cancer, and may be valuable for subgrouping poorly differentiated adenocarcinomas. Analyses of the core proteins for 91.9H epitope were then carried out. Comparison of immunostaining of ten poorly differentiated adenocarcinoma cases by MAb MUSE11 against MUC1 gene product with that by MAb 91.9H suggested that 91.9H epitope is not expressed on MUC1. Northern blot analysis of 10 pairs of gastric cancer and adjacent normal tissues with a MUC2 cDNA probe showed that the expression level of MUC2 mRNA was below the limit of detection. Thus, 91.9H epitope may be expressed on other proteins than MUC1 or MUC2 core proteins in gastric cancer.

REAL-TIME PET ANALYSIS OF METASTATIC TUMOR CELL TRAFFICKING IN VIVO AND ITS RELATION TO ADHESION PROPERTIES

Although a number of studies have indicated that highly metastatic cells tend to adhere more to target endothelium in vitro than low or non-metastatic cells, direct evidence about the correlation between cellular adhesiveness and organ disposition of the cells has not been obtained. Using positron emission tomography (PET), we have developed a novel technique that enables the non-invasive detection of the real-time tumor cell trafficking. The present study shows the correlation between trafficking of murine large cell lymphoma RAW117 and the adhesion properties of the cells in vitro. Cells accumulated in the liver time-dependently, and accumulation of RAW117-H10, liver metastatic subline cells, was more intense than that of RAW117-P, the parental cells, indicating that the metastatic potential is correlated with the in vivo accumulation of the cells in the target tissue. To examine whether the adhesion properties of the cell membrane determine the cell trafficking, we performed PET analysis after altering the adhesion properties on the cell membrane by means of cellular protein kinase C modulation, since the modulation of this enzyme is known to alter the surface adhesion molecules, i.e., those of the integrin superfamily. The treatment of RAW117-P with 12-O-tetradecanoylphorbol 13-acetate, which caused augmentation of adhesion to hepatic sinusoidal microvessel endothelial cells (HSE) in vitro, enhanced the hepatic accumulation of the cells in vivo. On the contrary, treatment of RAW117-H10 with the protein kinase C inhibitor H-7, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, which reduced the adhesion activity of the cells to HSE, suppressed their accumulation in the liver, although the suppression was observed only during the first 30 min after administration of the cells. These data suggest that the adhesion properties of metastatic lymphoma cells are critical for the accumulation of these cells in the target tissue.

Quantitative measurement of carbohydrate binding activity of mouse macrophage lectin

A simple ELISA assay measuring lectin activity of a mouse macrophage galactose/N-acetylgalactosamine-specific C-type lectin (MMGL) was developed. The binding of galactosylated poly-lysine (termed a ligand) to the immobilized soluble form of MMGL (rML) was measured quantitatively. Consistent with the characteristics of MMGL, the binding was calcium dependent and inhibited by galactose and N-acetylgalactosamine. An antiserum against rML inhibited the ligand binding, demonstrating the usefulness of this method for the screening of blocking antibodies. Using this assay, we found a significant interaction between MMGL and carrageenans, a group of sulfated polygalactans. The inhibitory effect of carrageenans was not attributable to a nonspecific interaction because other types of sulfated polysaccharides, such as glycosaminoglycans and fucoidin, did not interfere with the ligand binding. The relevance of the present finding to the biological activities of carrageenans is discussed.

Cancer Metastasis Determined by Carbohydrate-Mediated Cell Adhesion

During the progression of cancer to advanced stages, highly malignant and metastatic tumor cells are thought to arise within primary tumors and become predominant. On the basis of this hypothesis, surgical specimens from patients at various clinical stages of colorectal carcinomas were compared for a variety of molecular phenotypes possibly related to metastasis. As a result, colorectal carcinomas with increased metastatic potential and with poor prognosis have been characterized by the increased expression of sialyl-LeX antigens. Human colon carcinoma cell lines were selected for their high or low expression of sialyl-LeX antigens on the cell surfaces. These cells differ in their metastatic behavior in nude mice. The high expresser cells strongly adhere to activated endothelial cells in vitro. Retrospective studies with immunohistochemical technique have revealed that these molecules could be used as a predictive marker for colorectal cancer metastasis.