CELLULAR UPTAKE AND RELEASE OF THE IMMUNOMODULATING FUNGAL TOXIN GLIOTOXIN

Uptake of the immunomodulating agent gliotoxin into a panel of cells u sing biosynthetically radiolabelled S-35 toxin showed rapid associatio n of the toxin with all cell types studied with 70-85% of the total co unts in the media becoming cell associated. A difference in kinetics w as observed for cell lines when compared to the primary cells thymocyt es, activated T-cells and macrophages. In the latter uptake was maxima l after 10-15 min and radiolabel was lost from the cells as early as 1 00 min. In the cell lines studied, uptake was complete in less than 1 min with no loss of label after 100 min. The exception to this was a W ilms tumour line. Analysis of the fate of gliotoxin taken up into sens itive (activated T-cells) and resistant (human fibroblast) cells by HP LC showed: (a) up to 30% of the original gliotoxin taken up by sensiti ve cells was released as free gliotoxin over a 22 hr period. The remai nder was metabolized to inorganic sulphate; (b) in T-cells gliotoxin i s reduced to the dithiol form in significant amounts and this reductio n may be modulated by glutathione; and (c) no reduced gliotoxin could be detected in the resistant fibroblast cell line 27Sk even though up to 50% of the original gliotoxin was still present in the free form in these cells at 22 hr. Gliotoxin became covalently associated with mac romolecules in both cell types studied. Very little free gliotoxin is released into extracellular medium by the fibroblast cell line. Glioto xin at 500 nM was found to induce apoptosis or programmed cell death i n the Wilms tumour cell line but not in any other cell line studied, a nd this may account for the different kinetics of release of the toxin from the Wilms tumour cell line.