The mechanisms of cytotoxic activity of Griffonia simplicifolia 1-B4 (GS1B4) and wheat germ agglutinin (WGA) lectins against various murine tumour cell lines were studied. Tumour cells that lack lectin-binding carbohydrates were resistant to lysis by these lectins. However, YAC-1 cells that expressed GS1B4 lectin-binding sites showed low sensitivity to lysis. To further analyse the relative importance of cell surface carbohydrates in lectin cytotoxicity, BL6-8 melanoma cells, which do not express the alpha1,3 galactosyltransferase (alpha1,3GT) gene and cell surface alpha-galactosyl epitopes reacting with GS1B4 lectin, were transfected with cDNA encoding alpha1,3GT. After transfection, BL6-8 cells expressed high levels of GS1B4-binding alpha-galactosyl epitopes, but remained resistant to lysis by GS1B4 lectin, suggesting that the presence of lectin-binding epitopes, while essential, is not sufficient for tumour cell lysis and probably some intracellular mechanisms are involved in the regulation of lectin-mediated cytotoxicity. We found that the GS1B4 and WGA lectins induced apoptosis with DNA fragmentation of sensitive, but not resistant, tumour cell lines. DNA fragmentation, as well as tumour cell lysis, was blocked in the presence of the specific inhibitory sugar. To determine whether binding of the lectin to cell surface carbohydrates is sufficient to trigger tumour cell lysis, lectin-sensitive CL8-1 melanoma cells were incubated with GS1B4 lectin immobilized on agarose beads. Although these tumour cells bind to the immobilized lectin, it failed to trigger tumour cell death, suggesting that only soluble lectin is capable of tumour cell lysis and lectin internalization is probably required for their lysis. The haptenic sugar (methyl alpha-D-galactopyranoside) added simultaneously with the soluble GS1B4 lectin was capable of complete protection of tumour cells from lysis. However, this sugar had little or no protection when added 1-4 h after cell preincubation with GS1B4 lectin. Failure of the haptenic sugar to reverse lectin cytotoxicity after 1-4 h of incubation with tumour cells indicates that at this time point lectin was internalized and triggered irreversible lytic signal. Thus, lectin cytotoxicity is mediated by soluble lectins that bind to the specific receptor, internalize and trigger programmed cell death (apoptosis).
