To investigate the significance of sialylation and sulfation of
lactosylceramide in transformed cells, we established
ganglioside GM3- and lactosylsulfatide (SM3)-reconstituted cells by transfecting cDNAs of
GM3 synthase and
cerebroside sulfotransferase into the J5 subclone of 3LL
Lewis lung carcinoma cells. The J5 clone was selected for the transfection of these genes because it lacks GM3 and SM3 but accumulates
lactosylceramide. The anchorage-dependent growth of both GM3- and SM3-reconstituted cells was similar. However, anchorage-independent growth (as measured by colony-forming ability in soft
agar) of the SM3- reconstituted cells was almost completely lost, which supports our previous observation showing the suppression of tumorigenic potential in vivo and
beta1 integrin gene expression induced by the introduction of
cerebroside sulfotransferase gene (Kabayama et al. [2001] J. Biol. Chem., 276, 26777-26783). The GM3-reconstituted cells formed a significantly higher number of colonies in soft
agar compared to mock-transfected cells and began to proliferate and become resistant to apoptosis when serum was depleted, indicating that endogenous GM3 is essential for maintaining these fundamental properties of malignant cells. We also found that serum-induced ERK1/2 activation was suppressed in the GM3-reconstituted cells, suggesting that anchorage-independent cell cycle initiation by endogenous GM3 is elicited through pathway(s) independent of ERK1/2 activation. The selective down-regulation of
platelet-derived growth factor (PDGF)-dependent ERK1/2 activation in the GM3-reconstituted cells was due to the substantial decreases of
PDGF alpha receptor mRNA and
protein, but in the SM3-reconstituted cells
PDGF alpha receptor expression was similar to mock cells. Thus, endogenously produced GM3 and SM3 differentially and distinctly regulate
tumor-progression ability, that is, GM3 leads the transformed phenotype of J5 cells to promotion and SM3 to abrogation.