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Growth inhibition of tumor cells by transfection with β-1,4-glanctosyltransferase gene II or V.

通过转染β-1,4-半乳糖基转移酶基因II或V抑制肿瘤细胞的生长。

基本信息

批准号：
12680708
负责人：
FURUKAWA Kiyoshi
金额：
$ 2.3万
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：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

Upon malignant transformation of cells, highly branching of N-linked oligosaccharides is induced by activation of N-acetylglucosaminyltransferase V. However, no significant change in β-1, 4-GalT activities was found during this biological event. When gene expression levels of the β-1, 4-GalT family members were examined between malignantly transformed cells and their normal counterparts, those of β-1, 4-GalT II decreased while those of β-1, 4-GalT V increased without significant changes in those of other β-1, 4-GalTs. When B16-F10 mouse melanoma cells were transfected with human β-1, 4-GalT II sense CDNA or β-1, 4-GalT V anti-sense cDNA, and transplanted subcutaneously in C57B/L6 mouse, tumor formation was suppressed in the gene-transfected cells. In order to investigate how tumor suppression was induced by them, the cells were cultured on fibronectin (FN)-coated plates without serum. The mock-transfected B16-F10 cells stayed in a round shape while both gene-transfected cells showed cell spreading aligned with actin filament as revealed with phalloidin staining. Lectin blot analysis of membrane glycoproteins from the tumors and cultured cells using RCA-I which interacts with oligosaccharides terminated with β-1, 4-linked galactose and L-PHA which binds to highly branched oligosaccharides showed that reactivities to these lectins are changed upon transfection of the β-1, 4-GalT genes. These results indicate that the altered galactosylation induced to the membrane glycoproteins including fibronectin receptor affects adhesiveness of the cells to fibronectin substrate, thus leading to the suppression of the tumor formation.

在细胞恶性转化时，N-乙酰葡糖胺转移酶V的活化诱导了N-连接寡糖的高度分支。然而，在此生物学事件期间未发现β-1，4-GalT活性的显著变化。当在恶性转化细胞和它们的正常对应物之间检测β-1，4-GalT家族成员的基因表达水平时，β-1，4-GalT II的基因表达水平降低，而β-1，4-GalT V的基因表达水平升高，而其他β-1，4-GalT的基因表达水平没有显著变化。将人β-1，4-GalT Ⅱ正义cDNA或β-1，4-GalT V反义cDNA转染B16-F10小鼠黑色素瘤细胞，并将其移植于C57 B/L 6小鼠皮下，肿瘤形成受到抑制。为了研究它们如何诱导肿瘤抑制，将细胞在无血清的纤连蛋白（FN）包被的平板上培养。模拟转染的B16-F10细胞保持圆形，而两种基因转染的细胞均显示与肌动蛋白丝对齐的细胞铺展，如鬼笔环肽染色所示。用与β-1，4-半乳糖封端的寡糖相互作用的RCA-Ⅰ和与高度分支寡糖结合的L-PHA对肿瘤和培养细胞的膜糖蛋白进行凝集素印迹分析，结果表明，转染β-1，4-GalT基因后，对这些凝集素的反应性发生了变化。这些结果表明，对包括纤连蛋白受体的膜糖蛋白诱导的改变的半乳糖基化影响细胞对纤连蛋白底物的粘附，从而导致肿瘤形成的抑制。