Abstract
We describe a sensitive, rapid, and simple assay for mammalian O 6-alkylguanine DNA alkyltransferase ( O 6-AGT) utilizing solid-phase DNA as the substrate and a monoclonal antibody (Mab)-based immuno-slotblot (ISB) for quantitation of O 6-ethylguanine ( O 6-EtG). γ-phage DNA was treated with N-ethyl- N-nitrosourea and immobilized on newly developed hydrophilic latex beads. After incubation with cell extracts to be assayed for O 6-AGT activity, the substrate DNA could be isolated easily by a brief centrifugation through 50% glycerol. The amount of O 6-EtG retained in the substrate DNA was determined by ISB using the anti-( O 6-ethyl-2′-deoxyguanosine) Mab ER-6. As little as 2 fmol of O 6-AGT per reaction tube can be reproducibly measured by this procedure, which is suitable for handling large numbers of samples within a short time (e.g., 80 samples within 2 days). In normal and malignant cells, respectively, O 6-AGT activity protects against O 6-alkylguanine-mediated mutagenesis and oncogenesis following exposure to N-nitroso carcinogens or confers resistance against cytocidal anti-cancer drugs such as chloroethylnitrosoureas and related compounds. The analysis of cellular O 6-AGT activity by a highly sensitive, routinely applicable method is, therefore, of particular interest in studies related to carcinogenesis, molecular epidemiology, and clinical oncology.
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