Growth suppression of tumor cells by controlling the galactosylation of cell surface N-glycans

我们先前的研究表明，逆转肿瘤细胞中β-1，4-半乳糖基转移酶(β-1，4-GalT)II或V基因的表达可以抑制肿瘤的生长，提示表面碳水化合物在细胞生长中的重要性。由于正常细胞在较高的细胞密度下相互作用后停止增殖，我们研究了细胞表面糖基化，特别是N-糖基化是否会因不同的细胞密度而改变。用刮刀收集密度分别为20%、50%和100%的小鼠NIH3T3细胞，并进行凝集素印迹分析。CBB染色显示，除50K和250K蛋白的含量增加外，大多数组成蛋白的表达在生长过程中没有变化。当与β-1，4-半乳糖化低聚糖结合的Rca-I与印迹杂交时，凝集素在80K、110K、120K和160K带的反应性增加，尤其是在230K带的反应性随细胞密度的增加而增加。使用L-PHA也观察到了类似但弱得多的对这些蛋白条带的反应。Northern印迹分析表明，β-1，4-GalT II基因仅在100%细胞密度时表达显著增加，提示β-1，4-GalT II编码的半乳糖残基对正常细胞的生长抑制起重要作用。用asialo转铁蛋白-Sepharose柱从高密度细胞中获得了半乳糖结合蛋白，表明该蛋白参与了高密度细胞上表达的半乳糖残基的结合。

Our previous study showed that reverting the expression of the β-1,4-galactosyltransferase (β-1,4-GalT) II or V gene in cancer cells results in the suppression of tumor growth, suggesting the importance of the surface carbohydrates in cell growth. Since normal cells cease to proliferate upon interaction of cells at higher cell density, we investigated whether or not cell surface glycosylation, particularly N-glycosylation, change by different cell density. Mouse NIH3T3 cells grown at 20%, 50% and 100% in density were harvested by scraper, and membrane preparations were subjected to lectin blot analysis. CBB-staining showed that the expression of most constitutional proteins unchanges during growth except for 50 K and 250 K proteins whose amounts increase. When the blot was incubated with RCA-I, that binds to β-1,4-galactosylated oligosaccharides, the reactivity of lectin increased in 80 K,110 K,120 K and 160 K bands and particularly in 230 K band in proportion to the cell density. A similar but much weaker reactivity towards these protein bands was observed using L-PHA. Northern blot analysis showed that the expression of the β-1,4-GalT II gene only increases significantly at 100% cell density, suggesting that the galactose residues coded by β-1,4-GalT II is important for growth inhibition of normal cells. By using an asialo-transferrin-Sepharose column, 31 K protein was obtained as galactose-binding protein from highly dense cells, suggesting the involvement of this protein in binding to galactose residues expressed on cells at higher density.