Dahiya R, Kwak K S, Byrd J C, Ho S, Yoon W H, Kim Y S
Department of Medicine, University of California, San Francisco/Veterans Administration Medical Center 94121.
Cancer Res. 1993 Mar 15;53(6):1437-43.
Previous studies have suggested that mucin gene expression is tissue-specific; however, the relationship between unique mucin gene products and the biochemical properties of mucins is unknown. The purpose of this study was to determine the biochemical and molecular characteristics of mucin synthesized by adenocarcinoma cell lines derived from breast (ZR-75-1), stomach (MGC-803), pancreas (Capan-2), and lung (Chago K-1). Mucin was quantitated by [3H]glucosamine labeling and Sepharose CL-4B chromatography. The mucinous nature of the labeled high molecular weight glycoproteins (HMG) was verified by alkaline borohydride treatment, cesium chloride density gradient ultracentrifugation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Specific mucin gene expression was determined using cDNA probes for 2 distinct intestinal mucins (MUC-2 and MUC-3) and one breast cancer mucin (MUC-1). Specific core mucin proteins were confirmed by immunoblots using antibodies that recognize MUC-1, MUC-2, and MUC-3 core peptides. These experiments demonstrate that all cell lines contained HMG in the medium, cytosol, and membrane fractions. The HMG was mucinous in breast, pancreatic, and lung cell lines. In contrast, most of the HMG secreted by the gastric cell line was proteoglycan-like, due to its susceptibility to hyaluronidase, heparinase, and chondroitinase avidin-biotin complex. Ion-exchange (DEAE-Sephacel) chromatography of [3H]glucosamine-labeled HMG demonstrated that the acidic or basic nature of the mucin was different in all cancer cell lines tested. Despite these differences, mRNA and immunoblot analysis suggest that all cell lines predominantly express MUC-1 apomucin, small amounts of MUC-2 apomucin, and no MUC-3. Immunoprecipitation of MUC-1-type mucin using the 139H2 monoclonal antibody demonstrated that different sizes of mucin peptides were present in all cell lines, corresponding to the known length polymorphism of this mucin. The amount and nature of carbohydrate epitopes were analyzed by immunoblots using anti-T (peanut lectin), anti-Tn (91S8 monoclonal antibody), and anti-sialosyl Tn (JT10e monoclonal antibody). T and Tn antigens were significantly higher in breast and pancreatic cells as compared with lung and gastric cell lines. These findings correlated with increased activities of polypeptidyl N-acetylgalactosaminyl transferase and beta-1,3-galactosyltransferase.(ABSTRACT TRUNCATED AT 400 WORDS)
以往研究表明黏蛋白基因表达具有组织特异性；然而，独特的黏蛋白基因产物与黏蛋白生化特性之间的关系尚不清楚。本研究的目的是确定源自乳腺（ZR-75-1）、胃（MGC-803）、胰腺（Capan-2）和肺（Chago K-1）的腺癌细胞系合成的黏蛋白的生化和分子特征。通过[3H]葡糖胺标记和琼脂糖CL-4B柱层析对黏蛋白进行定量。通过碱性硼氢化钠处理、氯化铯密度梯度超速离心和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳验证标记的高分子量糖蛋白（HMG）的黏液性质。使用针对2种不同肠黏蛋白（MUC-2和MUC-3）和一种乳腺癌黏蛋白（MUC-1）的cDNA探针确定特异性黏蛋白基因表达。使用识别MUC-1、MUC-2和MUC-3核心肽的抗体通过免疫印迹法确认特异性核心黏蛋白蛋白。这些实验表明所有细胞系在培养基、胞质溶胶和膜组分中均含有HMG。乳腺、胰腺和肺细胞系中的HMG具有黏液性质。相比之下，胃细胞系分泌的大多数HMG类似蛋白聚糖，因为它对透明质酸酶、肝素酶和软骨素酶敏感。[3H]葡糖胺标记的HMG的离子交换（DEAE-琼脂糖凝胶）柱层析表明，在所有测试的癌细胞系中，黏蛋白的酸性或碱性性质不同。尽管存在这些差异，但mRNA和免疫印迹分析表明所有细胞系主要表达MUC-1脱辅基黏蛋白，少量表达MUC-2脱辅基黏蛋白，不表达MUC-3。使用139H2单克隆抗体对MUC-1型黏蛋白进行免疫沉淀表明，所有细胞系中均存在不同大小的黏蛋白肽，这与该黏蛋白已知的长度多态性相对应。通过使用抗-T（花生凝集素）、抗-Tn（91S8单克隆抗体）和抗唾液酸化Tn（JT10e单克隆抗体）的免疫印迹分析碳水化合物表位的数量和性质。与肺和胃细胞系相比，乳腺和胰腺细胞中的T和Tn抗原明显更高。这些发现与多肽基N-乙酰半乳糖胺基转移酶和β-1,3-半乳糖基转移酶活性增加相关。（摘要截断于400字）
