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

Gangliosides are thought to be involved in tumor cell proliferation, migrat ion and invasiveness as so far demonstrated by the addition of exogenous ga ngliosides to the culture medium. To better understand the direct influence that alterations in ganglioside synthesis can exert on these functional as pects of cell biology, in the present study, we investigated the behaviour of C6 rat glioma cells after stable transfection with the human CMP-NeuAc:N euAc alpha 2-3Gal beta 1-4GlcCer alpha 2,8-sialyltransferase (SAT-II, EC 2. 4.99.8) gene. The enzyme synthesizes ganglioside GD(3) by adding a sialic a cid residue to ganglioside GM(3). Stable transfection of the constructs int o C6 cells and expression of the human SAT-II gene were evaluated using PCR and RT-PCR amplification, respectively. Qualitative and quantitative analy sis of the ganglioside profile was performed by conventional HP-TLC and ide ntity of de novo synthesized species was assessed by TLC immunostaining. Re sults show that whereas C6 parental cells and C6 cells transfected with the empty expression vector synthesize, almost exclusively, ganglioside GM3, d e novo synthesis of GD(3) is clearly observed in clones expressing the alph a 2,8-sialyltransferase. Subcutaneous grafting in athymic nude mice of cell s expressing high levels of GD3 induces tumors growing faster and more aggr essively than controls. In in vitro assays, the same cells demonstrate incr eased proliferation rate, motility and invasiveness. Chemotaxis and chemoin vasion were assayed using the modified Boyden chamber. Data obtained sugges t that endogenously neosynthesized GD(3) is able to modify proliferation ra te, motility and invasion of C6 rat glioma cells, enhancing the features of malignancy of this tumor cell line.