Comparative study of sialidase activity and G(M3) content in B16 melanoma variants with different metastatic...

We previously demonstrated that transfection of a sialidase cDNA into B16-BL6 cells, a highly metastatic and invasive cell line derived from the mouse B16 melanoma, resulted in a marked suppression of metastasis accompanied by decreased cellular content of the GM3 that is one of the target molecules of the sialidase expressed (Tokuyama et al., 1997 Int. J. Cancer, 73, 410-415). To obtain further insight into the involvement of sialidase in metastasis, we made a comparison of the levels of sialidase activity and GM3 content between B16 melanoma cell lines with low (B16-F1) and high (B16-F10 and -BL6) metastatic potential. The cells exhibited sialidase activity towards 4-methylumbelliferyl N-acetylneuraminic acid (4MU-Neu5Ac) and gangliosides at acidic pH in the particulate fractions, but not in the cytosol. The activity toward 4MU-NeuAc was significantly lower in highly metastatic cells. The activity toward gangliosides, on the other hand, varied independently of metastatic potential: B16-F10 cells with a high potential for experimental metastasis showed the lowest level and B16-BL6 cells having high invasiveness had rather a higher level of ganglioside sialidase along with a much greater GM3 synthase activity than the other two cell lines. Flow cytometric analysis with anti-GM3 antibody revealed that highly metastatic cell lines were higher in the binding affinity as compared to B16-F1 cells, B16-BL6 cells containing twice as much cellular GM3 as B16-F1 cells on thin-layer chromatography.