Increased tumorigenicity and invasiveness of C6 rat glioma cells transfected with the human alpha-2,8 sialyltransferase cDNA.

E Sottocornola, I Colombo, V Vergani, G Taraboletti, B Berra
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引用次数: 27

Abstract

Gangliosides are thought to be involved in tumor cell proliferation, migration and invasiveness as so far demonstrated by the addition of exogenous gangliosides to the culture medium. To better understand the direct influence that alterations in ganglioside synthesis can exert on these functional aspects of cell biology, in the present study, we investigated the behaviour of C6 rat glioma cells after stable transfection with the human CMP-NeuAc:NeuAcalpha2-3Galbeta1-4GlcCer alpha2,8-sialyltransferase (SAT-II, EC 2.4.99.8) gene. The enzyme synthesizes ganglioside GD(3) by adding a sialic acid residue to ganglioside GM(3). Stable transfection of the constructs into C6 cells and expression of the human SAT-II gene were evaluated using PCR and RT-PCR amplification, respectively. Qualitative and quantitative analysis of the ganglioside profile was performed by conventional HP-TLC and identity of de novo synthesized species was assessed by TLC immunostaining. Results show that whereas C6 parental cells and C6 cells transfected with the empty expression vector synthesize, almost exclusively, ganglioside GM(3), de novo synthesis of GD(3) is clearly observed in clones expressing the alpha2,8-sialyltransferase. Subcutaneous grafting in athymic nude mice of cells expressing high levels of GD(3) induces tumors growing faster and more aggressively than controls. In in vitro assays, the same cells demonstrate increased proliferation rate, motility and invasiveness. Chemotaxis and chemoinvasion were assayed using the modified Boyden chamber. Data obtained suggest that endogenously neosynthesized GD(3) is able to modify proliferation rate, motility and invasion of C6 rat glioma cells, enhancing the features of malignancy of this tumor cell line.

转染人α -2,8唾液基转移酶cDNA的C6大鼠胶质瘤细胞的致瘤性和侵袭性增强
神经节苷脂被认为参与肿瘤细胞的增殖、迁移和侵袭,迄今为止已通过向培养基中添加外源性神经节苷脂证明了这一点。为了更好地了解神经节苷脂合成的改变对细胞生物学这些功能方面的直接影响,在本研究中,我们研究了用人CMP-NeuAc: neuacalpha2 - 3galbeta1 - 4glcer alpha2,8-唾液基转移酶(SAT-II, EC 2.4.99.8)基因稳定转染C6大鼠胶质瘤细胞后的行为。该酶通过在神经节苷脂GM(3)中加入唾液酸残基合成神经节苷脂GD(3)。分别用PCR和RT-PCR扩增检测构建体转染C6细胞的稳定性和人SAT-II基因的表达情况。采用常规的HP-TLC对神经节苷脂进行定性和定量分析,并通过TLC免疫染色对新合成的物种进行鉴定。结果表明,C6亲代细胞和转染空表达载体的C6细胞几乎完全合成神经节苷脂GM(3),而在表达α 2,8-唾液基转移酶的克隆中可以明显地观察到GD(3)的从头合成。在胸腺裸小鼠中皮下移植表达高水平GD(3)的细胞,诱导肿瘤比对照更快、更有攻击性地生长。在体外实验中,同样的细胞表现出增加的增殖率,运动性和侵袭性。采用改良的Boyden室检测趋化性和化学侵袭性。结果表明,内源性新合成GD(3)能够改变C6大鼠胶质瘤细胞的增殖速率、活力和侵袭性,增强该肿瘤细胞系的恶性特征。
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