Analysis of cellular functions of cells transfected with sense or antisense β-1,4-galactosyltransferase cDNA

正义或反义β-1,4-半乳糖基转移酶cDNA转染细胞的细胞功能分析

基本信息

批准号：
10680696
负责人：
FURUKAWA Kiyoshi
金额：
$ 2.11万
依托单位：
Tokyo Metropolitan Institute of Gerontology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

Mammalian cells are now shown to contain several β-1、4-galactosyltransferases (β-1,4-GalTs) which are involved in the biosynthesis of glycoprotein. When cells are transformed, they express the highly branched sugar chains with tumor-associated carbohydrate antigens on proteins which are critical to tumorigenicity and metastasis of the cell. During transformation, the gene expression levels of β-1,4-GalTs I, III, IV and VI were not changed while that of β-1,4-GalT II decreased and that of β-1,4-GalT V increased. In order to investigate biological significance of the carbohydrates formed by β-1,4-GalT II and V, their genes were introduced into several cell lines. When β-1,4-GalT II-sense cDNA or β-1,4-GalT V-antisense cDNA was transfected into mouse melanoma F10 cells, altered glycosylation of the membrane glycoproteins was detected by lectin blot analysis. The cells were inoculated into C57B mouse and the ability to form tumors were examined. Mouse melanoma F10 cells formed tumors in the animals while those transfected with β-1,4-Galt II-sense cDNA or β-1,4-GalT V-antisense cDNA failed or developed tumors very slowly. These results indicated that the sugar chains synthesized by β-1,4-GalTs II and V are involved in the growth control mechanism of cells. Further studies are necessary to identify which carbohydrate structures are really engaged in this biological event.

现在显示哺乳动物细胞包含几种参与糖蛋白的生物合成的几种β-1，4-半乳糖基转移酶（β-1,4-galts）。当细胞转化时，它们会在蛋白质上表达高度分支的糖链，并在蛋白质上与肿瘤相关的碳水化合物抗原表达对肿瘤性和细胞转移至关重要的蛋白质。在转化过程中，β-1,4-GALTS I，III，IV和VI的基因表达水平没有变化，而β-1,4-GALT II的基因表达水平降低，β-1,4-GALT V的基因表达水平并增加了。为了研究由β-1,4-加升II和V形成的碳氢化物的生物学意义，它们的基因被引入了几种细胞系。当将β-1,4-GALT II-sense cDNA或β-1,4-GALT V-抗固定cDNA转化为小鼠黑色素瘤F10细胞时，通过杠杆作用印迹分析检测到膜糖蛋白的糖基化改变。将细胞接种到C57B小鼠中，并检查形成肿瘤的能力。小鼠黑色素瘤F10细胞在动物中形成肿瘤，而用β-1,4-galt II-Sensens粘液cDNA或β-1,4-galt V-抗固定症cDNA翻译的肿瘤则非常缓慢地失败或发育。这些结果表明，由β-1,4-galts II和V合成的糖链参与细胞的生长控制机理。需要进一步的研究来确定哪些碳水化合物结构真正参与了这一生物学事件。