ARTICLE |
Correspondence to: Suguru Yonezawa, Second Dept. of Pathology, Kagoshima U. Faculty of Medicine, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan. E-mail: syoneza@m2.kufm.kagoshima-u.ac.jp
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Summary |
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Sialyl-Tn (STn), a mucin-associated disaccharide antigen carried by apomucins such as MUC2, plays an important role in tumor biology. However, little is known about the subcellular localization and compartments involved in STn synthesis. In this study we used immunoelectron microscopy to localize STn and MUC2 apomucin in human colorectal tissues. MUC2 apomucin was localized predominantly in the rough endoplasmic reticulum (RER) in normal colorectal mucosa (n=6), colorectal adenoma (n=8), and colorectal adenocarcinoma (n=10). STn, recognized by monoclonal antibody TKH2, was not readily detectable in normal colorectal mucosa but becomes manifest in both trans-Golgi apparatus and mucin droplets in colorectal adenoma. In colorectal adenocarcinoma, STn was localized not only in late but also in early Golgi compartments, and particularly in some RER lumens. Furthermore, electron microscopic in situ hybridization revealed that gold particles representing MUC2 mRNA are primarily localized over the RER. Our findings indicate that in colorectal adenoma STn sialylation takes place in the trans-Golgi apparatus, whereas in colorectal cancer STn sialylation occurs in all the Golgi compartments and in the RER.
(J Histochem Cytochem 49:15811591, 2001)
Key Words: sialyl-Tn, MUC2 mucin, immunoelectron microscopy, in situ hybridization
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Introduction |
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Sialyl-Tn (STn) is a mucin-associated disaccharide (
Biochemically, STn is formed by the sialylation of Tn antigen, i.e., GalNAc-O-Ser/Thr, which represents the common core O-glycan carbohydrate structure (2,6-sialyltransferase (STn sialyltransferase) (
2,3Galß1, 3GalNAc and NeuAc
2,6Galß1, 4GlcNAc (
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Materials and Methods |
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Tissue Specimens
Six normal colorectal mucosae were obtained from endoscopically resected tissues from patients who were verified to have no malignant colorectal diseases. Eight colorectal adenomas came from endoscopic polypectomy and were divided into two categories: low-grade atypia and high-grade atypia. Ten well- to moderately differentiated primary colorectal adenocarcinoma tissues were obtained from patients undergoing surgical resection. Tissue specimen collection was in accordance with human study guidelines and approval. The separation of adenomas into low and high grades and the diagnosis of adenocarcinoma were performed according to the definitions and explanatory notes by the World Health Organization (
Fresh tissues taken from several locations of each case were cut into fine pieces and fixed in freshly prepared 4% paraformaldehyde (Sigma Chemical; St Louis, MO) in 0.1 M phosphate buffer, pH 7.4, at 4C for 3 hr. Fixed tissues were then washed overnight in PBS, pH 7.4. On average, 10 tissue blocks from each patient were chosen and processed for embedding in LR White (Polyscience; Warrington, PA) as described by 80 nm) were collected on 300-mesh nickel grids. For EM in situ hybridization (ISH), thin sections (
100 nm) were attached onto carbon/Formvar-coated 200-mesh nickel grids.
Antibodies
The murine monoclonal antibody (MAb) TKH2 (IgG1) was obtained from Ohtsuka Pharmaceutical (Tokushima, Japan). This antibody specifically detects STn epitopes residing in the mucin glycoproteins (
MUC2 cDNA Labeling Procedure
The MUC2 probe pHAM1, a 90-bp portion of the MUC2 tandem repeat inserted at the Eco RI site of Bluescript SK-, which was isolated from a tracheal cDNA library12, was kindly provided by Dr. Carol B. Basbaum (Department of Anatomy, Cardiovascular Research Institute, University of California, San Francisco). Digoxigenin-labeled pHAM1 DNA probe and digoxigenin-labeled pHAM1 RNA probes (both antisense and sense) were prepared as described previously (
EM Immunocytochemical Labeling Procedure
For EM immunocytochemical labeling, colloidal gold was chosen on the basis of its unique properties, such as precise localization and easy differentiation without obscuring fine cell architectures (
Labeling controls were done by substituting the primary antibodies with corresponding normal sera from the same species. In addition, omission of the first and secondary antibodies was also included. To verify the specificity of silver enhancement, sections without gold labeling were allowed to react with the enhancement reagent alone. All the control experiments showed negative labeling.
EM ISH Procedure
EM ISH was performed according to published protocols (
The hybridization specificity was controlled as follows: (a) ISH without the RNA probe; (b) ISH with the sense RNA probe; (c) replacement of the anti-digoxigenin antibody with normal sheep serum; (d) treatment of the sections with RNase (50 µg/ml; Amersham Life Science) before initiation of the ISH. These control experiments yielded negative results. In addition, competition using unlabeled pHAM1 DNA probe followed by digoxigenin-labeled pHAM1 DNA probe showed significantly reduced specific binding at the probe throughout the areas showing positive reaction.
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Results |
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Normal Colorectal Mucosa
In normal colorectal mucosa, positive labeling was restricted to goblet cells. MUC2, as visualized by silver-enhanced gold particles (large), was found predominantly in the supranuclear and the perinuclear RER but was also detectable in some RER at the thin peripheral cytoplasm far away from the nucleus and in RER interspersed among the mucin droplets. The Golgi apparatus and mucin droplets were essentially devoid of MUC2 labeling except for occasional sparse background binding. STn, as visualized by non-silver-enhanced gold particles (small), could not be seen in a significant amount in any subcellular structures, such as the RER, the Golgi apparatus, and the mucin droplets (Fig 1).
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Colorectal Adenoma
In colorectal adenomas, adenoma cells resembling normal goblet cells could be found and positive labeling was seen in these adenomatous goblet cells. In both low- and high-grade adenomas, MUC2 apomucin was localized predominantly in the RER. In low-grade adenoma, STn expression was observed less often than in high-grade adenoma but, when expressed, it was either only in the trans-Golgi apparatus (Fig 2), or in both the trans-Golgi apparatus and the mucin droplets. In contrast, goblet cell maturation became progressively lost in high-grade adenomas. Although typical mucin droplet aggregates, as found in mature goblet cells, were rarely seen, mucin droplets of various sizes could be found in the cytoplasm of these dysplastic cells. In such high-grade adenomatous cells, STn was expressed more frequently in both the trans-Golgi apparatus and mucin droplets (Fig 3).
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Colorectal Adenocarcinoma
In adenocarcinoma, MUC2-STn double-positive cells were often observed. In these MUC2-expressing cancer cells, STn antigen expression showed several different subcellular localization profiles irrespective of the tumor differentiation level. Extracellularly, STn was often localized in the luminal contents, cell surface membranes, and microvilli (Fig 4). Intracellularly, STn was frequently found in mucin droplets of various sizes and electron densities (Fig 4). Another localization feature was that STn was heavily distributed over the entire Golgi apparatus, more notably in the cis-Golgi and trans-Golgi apparatus (Fig 5). Furthermore, STn was clearly detected in some RER lumens (Fig 6). In all of the above-mentioned situations, MUC2 apomucin was localized predominantly in the RER but could also be observed in some Golgi apparatus (Fig 5). Nevertheless, MUC2 apomucin was not seen in the cell surface or in the microvilli, nor in the glandular luminal contents in any appreciable amount (Fig 4).
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A considerable number of MUC2-negative tumor cells were seen interspersed among MUC2-positive cells. In these MUC2-negative cancer cells, STn was localized in similar structures to those described above, except that the intracellular mucin droplets were usually smaller compared to their counterparts seen in MUC2-positive cells (Fig 4). Malignant goblet cell and columnar cell phenotypes are the two major lineages in colorectal adenocarcinoma. Because MUC2 expression is related to the goblet cell lineage in colorectal cancer (
Although the above-mentioned localization patterns could be seen in all adenocarcinoma cases examined here, MUC2-STn double-positive cancer cells with abundant STn expression in the cell surface and in the RER/entire Golgi compartments were encountered more frequently in adenocarcinoma with lymph node and distant metastasis.
MUC2 mRNA Localization
EM ISH yielded clear hybridization signals without severely compromising the ultrastructural morphology. In all the specimens from normal colorectal mucosa, colorectal adenoma, and colorectal adenocarcinoma examined, gold particles representing MUC2 mRNA were consistently found to be closely associated with the RER (Fig 7). Other fine structures, such as the Golgi apparatus, mucin droplets, and mitochondria were not hybridized to any significant level.
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The data on EM subcellular localization patterns of STn, MUC2 apomucin, and MUC2 apomucin mRNA described above are summarized in Table 1.
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Discussion |
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Abnormal sialylation is a glycoconjugate change frequently seen in the neoplastic transformation of epithelial cells in a number of tumors, including colorectal cancer (
In this study we were able to examine the subcellular localization patterns of STn in the normal colorectal mucosa, colorectal adenoma, and adenocarcinoma by utilizing the high-resolution IEM labeling method. In agreement with previous STn light microscopic immunohistochemical reports (Golgi apparatus
secretory granules (
In relation to the subcellular localization of the Golgi enzymes in the colorectal cancer,
In recent years it has been shown that normal colorectal mucosa constitutively produces STn (
In human colorectal tissue, MUC2 has been found to be a major mucin type (
In animal models, human colon cancer cells selected for high metastatic potential produce high levels of MUC2 mucin (
Methodologically, studies on the subcellular glycosylation compartments are usually conducted through localization of the glycosylated products or the enzyme proteins of corresponding glycosyltransferases (
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Footnotes |
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1 Present address: John P. Robarts Research Institute, and Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada.
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Acknowledgments |
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Supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture, Japan (12218233, 12218234, 12470046, and 13220016) and also by a USPHS Grant from the National Cancer Institute (CA24321), the Department of Veterans Affairs Medical Research Service, and the Theodore Betz Foundation (USA).
Received for publication March 19, 2001; accepted July 11, 2001.
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