INDUCTION OF CYTOTOXIC OXIDATIVE STRESS BY D-ALANINE IN BRAIN-TUMOR CELLS EXPRESSING RHODOTORULA-GRACILIS D-AMINO-ACID OXIDASE - A CANCER GENE-THERAPY STRATEGY

Hydrogen peroxide (H2O2) is a reactive oxygen species (ROS) generated in the stereoselective deamination of D-amino acids catalyzed by D-ami no acid oxidase (DAAO). H2O2 readily crosses cellular membranes and da mages DNA, proteins, and lipids, The scarcity of DAAO substrates in ma mmalian organisms and its co-localization with catalase in the peroxis omal matrix suggested that the cytotoxicity of ROS could be harnessed by administration of D-amino acids to tumor cells ectopically expressi ng DAAO in the cytoplasm, To evaluate this hypothesis, the cDNA encodi ng the highly active DAAO from the red yeast Rhodotorula gracilis was mutated to remove the carboxy-terminal peroxisomal targeting sequence, A clonal line of 9L glioma cells stably transfected with this constru ct (9Ldaao17) was found to synthesize active R. gracilis DAAO, Exposur e of 9Ldaao17 cells to D-alanine resulted in cytotoxicity at concentra tions that were nontoxic to parental 9L cells, Depletion of cellular g lutathione further sensitized 9Ldaao17 cells to D-alanine (D-Ala), Thi s result, combined with stimulation of pentose phosphate pathway activ ity and the production of extracellular H2O2 by 9Ldaao17 cells incubat ed with D-alanine implicates oxidative stress as the mediator of cytot oxicity. These results demonstrate that expression of R. gracilis DAAO in tumor cells confers chemosensitivity to D-alanine that could be ex ploited as a novel cancer gene therapy paradigm.