Induction of Cytotoxic Oxidative Stress byd-Alanine in Brain Tumor Cells ExpressingRhodotorula gracilisd-Amino Acid Oxidase: A Cancer Gene Therapy Strategy

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Abstract
Hydrogen peroxide (H2O2) is a reactive oxygen species (ROS) generated in the stereoselective deamination of d-amino acids catalyzed by d-amino acid oxidase (DAAO). H2O2 readily crosses cellular membranes and damages DNA, proteins, and lipids. The scarcity of DAAO substrates in mammalian organisms and its co-localization with catalase in the peroxisomal matrix suggested that the cytotoxicity of ROS could be harnessed by administration of d-amino acids to tumor cells ectopically expressing DAAO in the cytoplasm. To evaluate this hypothesis, the cDNA encoding 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 construct (9Ldaao17) was found to synthesize active R. gracilis DAAO. Exposure of 9Ldaao17 cells to d-alanine resulted in cytotoxicity at concentrations that were nontoxic to parental 9L cells. Depletion of cellular glutathione further sensitized 9Ldaao17 cells to d-alanine (d-Ala). This result, combined with stimulation of pentose phosphate pathway activity and the production of extracellular H2O2 by 9Ldaao17 cells incubated with d-alanine implicates oxidative stress as the mediator of cytotoxicity. These results demonstrate that expression of R. gracilis DAAO in tumor cells confers chemosensitivity to d-alanine that could be exploited as a novel cancer gene therapy paradigm. Gene-directed enzyme prodrug therapy (GDEPT) is an antineoplastic treatment strategy designed to overcome the systemic toxicity of chemotherapy by specifically expressing a foreign enzyme in malignant cells that converts a nontoxic prodrug into a cytotoxic metabolite. The relative inefficiency of current in situ gene transfer methodology suggests that enzyme/prodrug combinations that produce membrane permeable metabolites will elicit a more favorable therapeutic response. Ideally, the agent produced by the transduced cell “factories” would be cytotoxic toward both proliferating and quiescent cells. We describe a novel GDEPT approach using d-amino acid oxidase from the red yeast Rhodotorula gracilis and d-alanine as a substrate that generates hydrogen peroxide, a reactive metabolite of oxygen that has both these characteristics. We also demonstrate the ability to sensitize tumor cells to this GDEPT protocol by manipulating cellular antioxidant pathways.