Isolation of variants of BALB/c 3T3 cells defective in complex ganglioside biosynthesis

In an attempt to clarify the relationship between altered glycosphingolipid metabolism and other aspects of the transformed phenotype, we have isolated variants of BALB/c 3T3 cells (clone A31) which are defective in synthesis of the more complex gangliosides (GM2, GM1 and GD1a). The selection protocol was based on the specificity of cholera toxin for ganglioside GM1, and the ability to lyse cells which bound toxin using anti-toxin and complement. Following treatment of cells with ethane methane sulfonate (EMS), populations resistant to lysis were obtained after 5-6 rounds of selection, despite a low and rather variable killing efficiency (75-95%). Five out of six clones isolated from such populations showed reduced toxin-binding capacity and loss of gangliosides more complex than GM3, as determined by metabolic labelling with [1-14C] palmitate. An identical phenotype was displayed by a variant isolated from a non-mutagenized population of cells. The phenotype remained stable for several months in culture and for over at least 40 cell doublings. Ganglioside nomenclature is according to Svennerholm [24]. GM3 = NeuAc α 2 → 3 Gal β 1 → 4 Glc → Cer; GM2 = GalNac β 1 → 4 (NeuAc α 2 → 3) Gal β 1 → 4 Glc → Cer; GM1 = Gal β 1 → 3 GalNac β 1 → 4 (NeuAc α 2 → 3) Gal β 1 → 4 Glc → Cer; GD1a = NeuAc α 2 → 3 Gal β 1 → 3 GalNac β 1 → 4 (NeuAc α 2 → 3) Gal β 1 → 4 Glc → Cer; GD1b = Galβ 1 → 3 GalNac β 1 → 4 (NeuAc α 2 → 8 NeuAc α 2 → 3) Gal β 1 → 4 Glc → Cer); GT1b = NeuAc α 2 → 3 Gal β 1 → 3 GalNac β 1 → 4 (NeuAc α 2 → 8 Neu Ac α 2 → 3) Gal β 1 → 4 Glc → Cer. © 1985.