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The genetic (stable overexpression of sialyltransferase I, GM3 synthase) or pharmacological (selective pressure by N-(4-hydroxyphenyl)retinamide)) manipulation of A2780 human ovarian cancer cells allowed us to obtain clones characterized by higher GM3 synthase activity compared with wild-type cells. Clones with high GM3 synthase expression had elevated ganglioside levels, reduced in vitro cell motility, and enhanced expression of the membrane adaptor protein caveolin-1 with respect to wild-type cells. In high GM3 synthase-expressing clones, both depletion of gangliosides by treatment with the glucosylceramide synthase inhibitor D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol and silencing of caveolin-1 by siRNA were able to strongly increase in vitro cell motility. The motility of wild-type, low GM3 synthase-expressing cells was reduced in the presence of a Src inhibitor, and treatment of these cells with exogenous gangliosides, able to reduce their in vitro motility, inactivated c-Src kinase. Conversely, ganglioside depletion by D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol treatment or caveolin-1 silencing in high GM3 synthase-expressing cells led to c-Src kinase activation. In high GM3 synthase-expressing cells, caveolin-1 was associated with sphingolipids, integrin receptor subunits, p130(CAS), and c-Src forming a Triton X-100-insoluble noncaveolar signaling complex. These data suggest a role for gangliosides in regulating tumor cell motility by affecting the function of a signaling complex organized by caveolin-1, responsible for Src inactivation downstream to integrin receptors, and imply that GM3 synthase is a key target for the regulation of cell motility in human ovarian carcinoma

Cultures of MDCK II and human fibroblast cells were fed radioactive sphingosine and a radioactive GM3 ganglioside derivative containing a photoactivable group. The derived cell homogenates were treated with Triton X-100 and fractionated by sucrose-gradient centrifugation to prepare a detergent-insoluble membrane fraction known to be enriched in sphingolipid and caveolin-1, i.e. of caveolae. The detergent-insoluble membrane fraction prepared after feeding [1-H-3]sphingosine to cells, was found to be highly enriched, with respect to protein content, in metabolically radiolabeled sphingomyelin and glycosphingolipids (about 18-fold). By feeding cells photoactivable radioactive GM3, after 2 h-chase, caveolin-1, CAV1, and proteins of high molecular mass became cross-linked to GM3, the cross-linking complexes being highly concentrated in the detergent-insoluble membrane fraction. The interaction between the ganglioside derivative and CAV1 was a time-dependent, transient process so that CAV1 cross-linking to GM3 was hardly detectable after a 24-h chase followed the pulse time. After a 24-h chase, only the high molecular mass proteins cross-linked to GM3 could be clearly observed. These results suggest that a portion of the GM3 administered to cells enters caveolae and moves to the glycosphingolipid domains, or enters caveolae that are then rapidly catabolized. Electron microscopy of cells in a culture immunostained with a monoclonal antibody to GM3 and a secondary gold-conjugated antibody detected several clusters of gangliosides on the plasma membranes separate from caveolae; gangliosides located inside the caveolae could not be detected. Scanning confocal microscopy of cells immunostained with anti-GM3 and anti-CAV1 Ig showed only a very small overlap with the CAV1 and GM3 signals. Thus, the biochemical and microscopic studies suggest that caveolae contain at most a low level of gangliosides and are separate from the GM3 ganglioside enriched domains

The ganglioside mixture from the cerebellum of young, 6 month old and two years old rats, was fractionated by reversed phase high performance liquid chromatography, each ganglioside homogeneous in the oligosaccharide chain as well as in the long-chain base being subsequently quantified. Two long-chain bases, LCB, were components of the five major gangliosides GM1, GD1a, GD1b, GT1b and GQ1b, these being the C18:1 LCB and C20:1 LCB. The content of C20:1 ganglioside molecular species was lower than that of the C18:1 one. In very young animals, day 8, the C20:1 ganglioside species represented about 8% of the total ganglioside content, then they progressively increased and reached, at 2 years, about 42% of the total. C18:1 GD1a and C18:1 GT1b, were the major species in young animals and reached their highest content at day 29, being 1.45 and 1.28 nmol/mg protein, respectively. The content of these two species decreased in adult and old animals and at two years it was 0.71 and 0.82 nmol/mg protein, respectively.