Hiraiwa N, Tsuyuoka K, Li Y T, Tanaka M, Seno T, Okubo Y, Fukuda Y, Imura H, Kannagi R
Department of Clinical Science and Laboratory, Kyoto University, Japan.
Cancer Res. 1990 Sep 1;50(17):5497-503.
Two murine monoclonal antibodies, 2A3D2 and 2D11E2 (both IgM), which are directed to the gangliosides and sialoglycoproteins related to a rare blood group antigen, Cad, were obtained by using a ganglioside mixture prepared from human hepatocellular carcinoma cells (PLC/PRF/5) as the immunogen. These two monoclonal antibodies detected multiple ganglioside antigens present in the PLC/PRF/5 cells, and the major antigenic ganglioside was characterized as IV4GalNAc beta-GD1a, which has the carbohydrate structure GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----3GalNAc beta 1---- 4(NeuAc alpha 2----3)Gal beta 1----Cer. The two antibodies also reacted with GM2 (GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4Glc beta 1----Cer) and a Cad-active lactoseries ganglioside (IV4GalNAc beta-sialosylparagloboside, GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4GlcNAc beta 1---- 3Gal beta 1----4Glc beta 1----Cer), which have carbohydrate structures related to IV4GalNAc beta-GD1a. Beside gangliosides, both antibodies recognized the carbohydrate determinant carried by glycophorin A on very rare Cad-positive human RBC; the structure of which is GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----3(NeuAc alpha 2---- 6)GalNAc alpha 1----Ser/Thr. From these findings, it is clear that monoclonal antibodies 2A3D2 and 2D11E2 both recognize the nonreduced carbohydrate terminus composed of three sugar residues, GalNac beta 1----4(NeuAc alpha 2----3)Gal beta 1----R, and are useful for detecting the Cad-related antigen in cells and tissues. By using these monoclonal antibodies, it was revealed that many cultured human hepatocellular carcinoma cell lines and cancer tissues taken from patients with hepatocellular carcinoma contain both Cad-active glycoprotein antigens and related gangliosides, while normal liver tissues contain no appreciable amount of either species of antigen. The Cad-active glycoprotein antigens in cultured human hepatocellular carcinoma cells appeared as triplet bands having molecular weights of 92,000, 75,000, and 61,000, under either reducing or nonreducing conditions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Essentially the same triplet proteins were observed in as many as 4 of 9 cases (44%) of cancer tissue from patients with hepatocellular carcinoma, but not in neighboring cirrhotic tissues or normal livers tissues. These results suggest that the rare blood group antigen Cad is associated with human cancers, especially hepatocellular carcinoma.
通过使用从人肝癌细胞（PLC/PRF/5）制备的神经节苷脂混合物作为免疫原，获得了两种鼠单克隆抗体，2A3D2和2D11E2（均为IgM），它们针对与一种罕见血型抗原Cad相关的神经节苷脂和唾液酸糖蛋白。这两种单克隆抗体检测到PLC/PRF/5细胞中存在的多种神经节苷脂抗原，主要抗原性神经节苷脂被鉴定为IV4GalNAcβ-GD1a，其碳水化合物结构为GalNAcβ1----4（NeuAcα2----3）Galβ1----3GalNAcβ1---- 4（NeuAcα2----3）Galβ1----Cer。这两种抗体还与GM2（GalNAcβ1----4[NeuAcα2----3]Galβ1----4Glcβ1----Cer）和一种Cad活性乳糖系列神经节苷脂（IV4GalNAcβ-唾液酸基副球蛋白，GalNAcβ1----4[NeuAcα2----3]Galβ1----4GlcNAcβ1---- 3Galβ1----4Glcβ1----Cer）反应，它们具有与IV4GalNAcβ-GD1a相关的碳水化合物结构。除了神经节苷脂外，两种抗体都识别非常罕见的Cad阳性人红细胞上糖蛋白A携带的碳水化合物决定簇；其结构为GalNAcβ1----4（NeuAcα2----3）Galβ1----3（NeuAcα2---- 6）GalNAcα1----Ser/Thr。从这些发现可以清楚地看出，单克隆抗体2A3D2和2D11E2都识别由三个糖残基GalNacβ1----4（NeuAcα2----3）Galβ1----R组成的非还原碳水化合物末端，并且可用于检测细胞和组织中的Cad相关抗原。通过使用这些单克隆抗体，发现许多培养的人肝癌细胞系和取自肝癌患者的癌组织同时含有Cad活性糖蛋白抗原和相关神经节苷脂，而正常肝组织中两种抗原的含量均不明显。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中，无论是还原条件还是非还原条件下，培养的人肝癌细胞中的Cad活性糖蛋白抗原均表现为分子量分别为92,000、75,000和61,000的三条带。在多达9例肝癌患者的癌组织中有4例（44%）观察到基本相同的三条带蛋白，但在相邻的肝硬化组织或正常肝组织中未观察到。这些结果表明，罕见血型抗原Cad与人类癌症，尤其是肝癌有关。
