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（beta4 GalT-I）是合成某些2型N-聚糖和核心2型O-聚糖所必需的糖基转移酶。我们已经产生了β 4GalT-I敲除（KO）小鼠，以研究这些碳水化合物的多种体内功能。首先，我们已经从β 4GalT-I同源突变和异源突变的表型正常小鼠中产生了胚胎成纤维细胞系。两种细胞系均通过重复传代而永生化。将小鼠β 4GalT-I表达载体转染到β 4GalT-I缺失的成纤维细胞系中，对细胞的永生化没有影响。为了评价肿瘤特异性碳水化合物抗原如唾液酸刘易斯抗原在肿瘤发生中的作用，我们使用化学诱导的β 4 GalT-I KO小鼠皮肤肿瘤细胞系对碳水化合物抗原进行了功能分析。转染小鼠β 4GalT-I表达载体的细胞和β 4GalT-I敲除细胞的生长和对纤连蛋白的粘附能力无显著差异。当评估β 4GalT-I无效细胞系通过纤连蛋白包被和/或Matrigel包被的transwell的运动性和侵袭性时，观察到显著的迁移和侵袭。此外，转染小鼠β 4 GalT-I表达载体以表达水平依赖的方式降低细胞的运动性和侵袭性。虽然在β 4 GalT-I转染到β 4 GalT-I缺失细胞系后未检测到唾液酸刘易斯抗原，但观察到在β 1，4-连接中RCA 120凝集素检测到显著的半乳糖残基表达。这些结果表明，β 4 GalT-I合成的碳水化合物调节肿瘤细胞的恶性程度。

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.