Cyanide bystander effect of the linamarase/linamarin killer-suicide gene therapy system.

Abstract

The killer-suicide system linamarase/linamarin (lis/lin) uses the plant gene linamarase (beta-glucosidase) to convert the cyanogenic glucoside substrate, linamarin, into glucose and cyanide. We have studied the bystander effect associated with this new system mediated by the production of the cyanide ion that diffuses freely across membranes. Immunofluorescent staining of cells treated with an anti-linamarase antibody allowed us to localize the enzyme within the cells. Flow cytometry was used to determine the sensitivity of different mixtures of cells, C6lis and C6gfp (green), to linamarin as a percentage of cell survival. We demonstrate here that rat glioblastoma C6 cells carrying the linamarase gene (lis), mixed with naive C6 cells and exposed to linamarin, induce generalized cell death. Cells expressing lis efficiently export linamarase, whereas linamarin enters cells poorly by endocytosis; as a result most of the cyanide is produced outside the cells. The study was facilitated by the presence of the green fluorescent protein (gfp) gene in the bystander population. As few as 10% C6lis-positive cells are sufficient to eliminate the entire cell culture in 96 h. This bystander mechanism does not preferentially kill toxic metabolite producer cells compared with bystander cells, thus allowing production of sufficient cyanide to cause tumor regression. In this report we confirm the potential of the lis/lin gene therapy system as a powerful tool to eliminate tumors in vivo.