Functional analysis of carbohydrate antigens in chemically induced tumor cells derived from beta-1,4-galactosyltransferase-I knockout mice

β-1,4-半乳糖基转移酶-I 敲除小鼠化学诱导肿瘤细胞中碳水化合物抗原的功能分析

• 批准号: 15500298
• 负责人: HASHIMOTO Noriyoshi
• 金额: $ 2.37万
• 依托单位: Kanazawa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15500298/
• 关键词: Carbohydrate; Knockout mice; Galactosyltransferase; Tumor antigen; Galactose; Chemical carcinogen; Skin tumor; Malignancy; Carbohydrate; Knockout mice; Galactosyltransferase; Tumor antigen; Galactose; Chemical carcinogen; Skin tumor; Malignancy

Beta-1,4-galactosyltransferase I (beta4GalT-I) is an essential glycosyltransferase to synthesize some kinds of type 2 N-glycans and core 2 O-glycans. We have generated beta4GalT-I knockout (KO) mice to study the multiple in vivo function of these carbohydrates. First, we have generated embryonic fibroblast cell lines from both beta4GalT-I homozygously mutated and heterozygously mutated, phenotypically normal, mice. Both cell lines were immortalized by repetitive passages. Transfection of mouse beta4GalT-I expression vector to beta4GalT-I null fibroblast cell line have no effect on immortalization of cells. In order to evaluate tumor specific carbohydrate antigens such as sialyl Lewis antigens in tumorigenesis, we have conducted functional analysis of carbohydrate antigens using chemically induced skin tumor cell lines derived from beta4GalT-I KO mice. No significant differences in cellular growth and adherent abilities to fibronectin were observed between beta4GalT-I null cells and those which were transfected with mouse beta4GalT-I expression vector. When motility and invasiveness of beta4GalT-I null cell lines through fibronectin-coated and/or Matrigel-coated transwells were assessed, significant migrations and invasions were observed. Moreover, the cell motility and invasiveness were declined by the transfection of mouse beta4GalT-I expression vector in an expression level dependent manner. Though sialyl Lewis antigens have not be detected after beta4GalT-I transfections to beta4GalT-I null cell lines, significant expressions of galactose residuedetected by RCA 120 lectin in the beta1,4-linkage was observed. These results suggest that carbohydrates synthesized by beta4GalT-I regulate the malignancy of tumor cells.

β-1,4-半乳糖基转移酶I（beta4galt-i）是合成某些类型2型N-聚糖和核心2 O-聚糖的必不可少的糖基转移酶。我们已经产生了beta4galt-i基因敲除（KO）小鼠，以研究这些碳水化合物的多重体内功能。首先，我们从beta4galt-i纯合突变和杂合突变的，表型正常的小鼠中产生了胚胎成纤维细胞系。两种细胞系都通过重复通道永生。将小鼠β4GALT-1表达载体转染至beta4galt-i NULL成纤维细胞系对细胞的永生化没有影响。为了评估肿瘤特异性碳水化合物抗原（例如siAllyl刘易斯抗原）在肿瘤发生中，我们使用源自beta4galt-I KO小鼠的化学诱导的皮肤肿瘤细胞系对碳水化合物抗原进行了功能分析。在β4GALT-I NULL细胞与用小鼠beta4galt-I表达载体转染的细胞之间，细胞生长和依从能力在纤连蛋白之间没有显着差异。当评估了通过纤连蛋白包被和/或基质涂层的transwells的β4GALT-1无效细胞系的运动性和侵入性时，观察到显着的迁移和入侵。此外，通过以表达水平依赖性方式转染小鼠beta4galt-i表达载体的细胞运动和侵袭性下降。尽管在β4galt-i转染向β4Galt-i null细胞系后未检测到siAlyl刘易斯抗原，但观察到在beta1,4-链接中，RCA 120凝集素残留的半乳糖表达的显着表达。这些结果表明，由β4Galt-I合成的碳水化合物调节肿瘤细胞的恶性肿瘤。