Modulation of EGF receptor activity by changes in the GM3 content in a human epidermoid carcinoma cell line, A431.

Gangliosides have been described as modulators of growth factor receptors. For example, GM3 addition in cell culture medium inhibits epidermal growth factor (EGF)-stimulated receptor autophosphorylation. Furthermore, depletion of ganglioside by sialidase gene transfection appeared to increase EGF receptor (EGFR) autophosphorylation. These data suggested that changes in GM3 content may result in different responses to EGF. In this study, the ceramide analog d-threo-1-phenyl-2-decannoylamino-3-morpholino-1-propanol ([D]-PDMP), which inhibits UDP-glucose-ceramide glucosyltransferase, and addition of GM3 to the culture medium were used to study the effects of GM3 on the EGFR. Addition of 10 microM [D]-PDMP to A431 cells resulted in significant GM3 depletion. Additionally, EGFR autophosphorylation was increased after EGF stimulation. When exogenous GM3 was added in combination with [D]-PDMP, the enhanced EGFR autophosphorylation was returned to control levels. [D]-PDMP also increased EGF-induced cell proliferation, consistent with its effect on autophosphorylation. Once again, the addition of GM3 in combination with [D]-PDMP reversed these effects. These results indicate that growth factor receptor functions can be modulated by the level of ganglioside expression in cell lines. Addition of GM3 inhibits EGFR activity and decrease of GM3 levels using [D]-PDMP treatment enhances EGFR activity. Modulation of growth factor receptor function may provide an explanation for how transformation-dependent ganglioside changes contribute to the transformed phenotype.

Development of a new inhibitor of glucosylceramide synthase.

Analogs of the potent inhibitor of glucosylceramide (GlcCer) synthase, D-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4), based on substitutions in the palmitoyl group were made by means of a stereo-selective synthetic method in order to elucidate the role of the hydrophobic portion in both the inhibitory action toward the enzyme and the biological effects. While P4 strongly inhibited GlcCer synthase with an IC(50) of 0.5 microM in vitro, it also inhibited cell growth by 50% at the concentration of 7 microM. The shorter N-acyl chain analogs including decanoyl, octanoyl, and hexanoyl groups showed similar IC(50) values for GlcCer synthase (around 2 microM) but the hexanoyl analog exhibited only a slight inhibitory effect on cell growth, showing the dissociation between GlcCer depletion and cell growth. Several compounds which exhibit similar hydrophobicity to the hexanoyl analog of P4 were subsequently designed. We found that D-threo-1-phenyl-2-benzyloxycarbonylamino-3-pyrrolidino-1-pr opanol (PBPP) was a most potent inhibitor, showing an IC50 of 0.3 microM. In cultured cells, PBPP was able to deplete glycosphingolipids without affecting cell growth or the ceramide level.

Glycosphingolipid deficiency affects functional microdomain formation in Lewis lung carcinoma cells.

In view of the increasing evidence that gangliosides in membrane microdomains or rafts are closely associated with various signal transducing molecules including Src family kinases, we compared rafts in two subclones of 3LL mouse lung carcinoma cell line, J18 and J5, characterized by high and very low GM3 ganglioside contents, respectively. Rafts were isolated from cell lysates as low density detergent-insoluble microdomains (DIM) by sucrose density gradient centrifugation. J5 and J18 cells expressed comparable amounts of Src family kinases and the majority of Src kinases in both clones were concentrated in their DIMs, suggesting that GM3 is not necessary for DIM localization of Src kinases and there is no direct interaction between Src and GM3. However, the Src kinases were eliminated from DIMs after depletion of the major neutral GSLs of J5 cells, glucosylceramide and lactosylceramide, by an inhibitor of glucosylceramide synthase (D-PDMP), indicating that GSLs in general are required for Src kinase association to DIM. J5 and the D-PDMP-treated J5 cells had very similar DIM protein profiles and moreover cholesterol and sphingomyelin in the GSL-depleted cells were enriched in DIM similar to the untreated control cells. Interestingly, the levels of tyrosine-phosphorylated DIM proteins and cell proliferation of J5 cells were much lower than those of J18 cells, suggesting that GM3 might be involved in tyrosine phosphorylation of DIM proteins required for cell growth. Thus, our data suggest that GSLs are essential for functional raft formation.