Transfection of the DAAO gene and subsequent induction of cytotoxic oxidative stress by D-alanine in 9L cells

Abstract

D-amino acid oxidase (DAAO) can catalyze the dehydrogenation of D-amino acids, such as D-alanine, to the corresponding amino acids and is then reoxidized by molecular oxygen to yield hydrogen peroxide, a reactive oxygen species, which reacts with DNA, lipids and protein, inducing cell death. This study investigated whether rat glioma 9L cells infected with the recombinant retrovirus containing the DAAO cDNA fragment can be induced in order to undergo cytotoxic oxidative stress by D-alanine. The recombinant retroviral vector, plzrus-DAAO-FLAG-GFP (pl-Dfg), was constructed and used to transfect packaged phoenix cells. The supernatant containing recombinant retroviral particles from the transfected phoenix cells was harvested and utilized to infect target 9L cells. The cytotoxic oxidative stress of infected 9L cells was induced by the DAAO substrate, D-alanine. The plasmid pl-Dfg was successfully constructed. The high titer retroviral supernatant was obtained from the transfected phoenix cells. Infected 9L cells were less viable after exposure to D-alanine compared to the control group. Anti-apoptotic proteins significantly inhibited cell death. The DAAO/D-alanine system has a potential utility for gene therapy and may be an effective strategy for the treatment of brain cancer and other malignant tumors.