title: Monoclonal antibody-mediated solid-phase assay for mammalian O6-alkylguanine DNA alkyltransferase activity.
abstract: We describe a sensitive, rapid, and simple assay for mammalian O6-alkylguanine DNA alkyltransferase (O6-AGT) utilizing solid-phase DNA as the substrate and a monoclonal antibody (Mab)-based immuno-slotblot (ISB) for quantitation of O6-ethylguanine (O6-EtG). lambda-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 O6-AGT activity, the substrate DNA could be isolated easily by a brief centrifugation through 50% glycerol. The amount of O6-EtG retained in the substrate DNA was determined by ISB using the anti-(O6-ethyl-2'-deoxyguanosine) Mab ER-6. As little as 2 fmol of O6-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, O6-AGT activity protects against O6-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 O6-AGT activity by a highly sensitive, routinely applicable method is, therefore, of particular interest in studies related to carcinogenesis, molecular epidemiology, and clinical oncology.
authors.affiliation: Department of Cancer Cell Research, University of Tokyo, Japan.
authors.forename: M S
authors.initials: MS
authors.lastname: Satoh
authors.forename: C
authors.initials: C
authors.lastname: Moriyama
authors.forename: A
authors.initials: A
authors.lastname: Asai
authors.forename: H
authors.initials: H
authors.lastname: Handa
authors.forename: M F
authors.initials: MF
authors.lastname: Rajewsky
authors.forename: T
authors.initials: T
authors.lastname: Kuroki
authors.forename: N H
authors.initials: NH
authors.lastname: Huh
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D015151:Immunoblotting; D008770:Methylnitrosourea / Q000378:metabolism; D008780:Methyltransferases / Q000378:metabolism*; D019853:O(6)-Methylguanine-DNA Methyltransferase; D014316:Tritium
chemical_list: D000911:Antibodies, Monoclonal; D014316:Tritium; C007673:6-ethylguanine; D006147:Guanine; D008770:Methylnitrosourea; D004247:DNA; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase