title: Quantification of specific DNA O-alkylation products in individual cells by monoclonal antibodies and digital imaging of intensified nuclear fluorescence.
abstract: We report the establishment of a standardized, monoclonal antibody (Mab)-based immunocytological assay (quantitative ICA) for the visualization and quantification of low levels of specific DNA O-alkylation products in individual cells by electronically intensified, indirect or direct immunofluorescence. In terms of specific binding to alkali-denatured nuclear DNA and low background noise, 10 Mabs from a collection of 154 Mabs specific for O6-methyl-2'-deoxyguanosine (O6-MedGuo), O6-ethyl-2'-deoxyguanosine (O6-EtdGuo), O6-n-butyl-2'-deoxyguanosine (O6-BudGuo) and O4-ethyl-2'-deoxythymidine (O4-EtdThd) with antibody affinity constants ranging between 1.0 x 10(6)-3.0 x 10(10) l/mol, were found to be best suited for ICA. At present, > or = 200 O6-EtdGuo residues (corresponding to an O6-EtdGuo/dGuo molar ratio in DNA of > or = 8.4 x 10(-8)), > or = 400 O6-BudGuo residues (O6-BudGuo/dGuo, > or = 1.7 x 10(-7)), > or = 1800 O4-EtdThd residues (O4-EtdThd/dThd, > or = 7.5 x 10(-7)) and > or = 4800 O6-MedGuo residues (O6-MedGuo/dGuo, > or = 2.0 x 10(-6)), can be quantified per diploid genome. Using a SIT video camera in combination with multiparameter image digital analysis, DNA adduct-specific rhodamine fluorescence signals are measured relative to nuclear DNA content (DAPI fluorescence). Adduct-specific fluorescence recordings in three different rat cell lines (BT3Ca, Fao and NO) were in excellent agreement with the data obtained by competitive radioimmunoassay (RIA) for hydrolysates of DNA isolated from the respective cells exposed in parallel to the same alkylating carcinogens (N-methyl-, N-ethyl- and N-[n-butyl]-N-nitrosourea). Accordingly, the kinetics of O6-EtdGuo repair, as determined by ICA and RIA, respectively, were superimposable. Cell-specific, quantitative ICA can, therefore, be used for the quantification of specific, stable DNA adducts induced by alkylating carcinogens or chemotherapeutic agents and for DNA repair measurements in individual (e.g. human) cells. Work is currently underway to extend the spectrum of carcinogen--DNA adduct-specific Mabs suited for quantitative ICA.
authors.affiliation: Institute of Cell Biology (Cancer Research), West German Cancer Center Essen, University of Essen Medical School.
authors.forename: F
authors.initials: F
authors.lastname: Seiler
authors.forename: U
authors.initials: U
authors.lastname: Kirstein
authors.forename: G
authors.initials: G
authors.lastname: Eberle
authors.forename: K
authors.initials: K
authors.lastname: Hochleitner
authors.forename: M F
authors.initials: MF
authors.lastname: Rajewsky
mesh_terms: D000478:Alkylation; D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D051381:Rats; D013936:Thymidine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D004247:DNA; D003849:Deoxyguanosine; D013936:Thymidine