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Cyanide bystander effect of the linamarase/linamarin killer‐suicide gene therapy system
Author(s) -
Luisa Cortés Maria,
GarcíaEscudero Vega,
Hughes Monica,
Izquierdo Marta
Publication year - 2002
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.280
Subject(s) - bystander effect , population , biology , chemistry , biochemistry , microbiology and biotechnology , immunology , medicine , environmental health
Background The killer‐suicide system linamarase/linamarin ( lis /lin) uses the plant gene linamarase (β‐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. Methods 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, C6 lis and C6 gfp (green), to linamarin as a percentage of cell survival. Results 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% C6 lis ‐positive cells are sufficient to eliminate the entire cell culture in 96 h. Conclusions 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 . Copyright © 2002 John Wiley & Sons, Ltd.