Detection of DNA damage in individual cells by flow cytometric analysis using anti-DNA monoclonal antibody |
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| Authors: | Oskar S Frankfurt |
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| Institution: | 1. Department of Biomedical Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran;2. Mechanical Engineering Program, School of Science, Engineering and Technology, Pennsylvania State University, Harrisburg, Middletown, PA 17057, USA;3. Department of Mechanical Engineering, Texas A&M University, College Station, TX 77843-3123, USA;1. Department of Molecular Toxicology, German Institute of Human Nutrition (DIfE) Potsdam-Rehbrücke, 14558 Nuthetal, Germany;2. Institute of Organic Chemistry, University of Tuebingen, 72076 Tuebingen, Germany;3. Department of Food Safety, Federal Institute for Risk Assessment (BfR), 10589 Berlin, Germany;1. Dipartimento di Medicina Clinica e Chirurgia, Unità di Fisiologia, Università degli Studi di Napoli Federico II, Italy;2. Dipartimento di Scienze della Vita, Seconda Università di Napoli, Italy;3. Dipartimento di Scienze, Università della Basilicata, Italy;4. Dipartimento di Neuroscienze e Scienze Riproduttive ed Odontostomatologiche, Università degli Studi di Napoli Federico II, Italy;1. Department of Biochemical Sciences, “Sapienza” University of Rome, Italy;2. Plants Biology Department, Faculty of Biology, University of Havana, Cuba |
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| Abstract: | A new method for the measurement of DNA damage in individual cells treated with alkylating agents is described. The method is based on the binding of anti-DNA monoclonal antibody to DNA in situ. Monoclonal antibody F7-26 was obtained by fusion of mouse myeloma cells with spleen cells isolated from a mouse immunized with DNA treated by nitrogen mustard (HN2). Binding of antibody was evaluated by flow cytometry with indirect immunofluorescence. No binding of antibody to DNA in non-treated HeLa S3 cells was detected. Treatment of cells with HN2 or L-phenylalanine mustard induced binding of antibody to DNA in situ. Binding of antibody was observed after treating cells with doses of drugs which reduced the surviving fraction below 20%. Intensity of binding increased in proportion to the drug dose. Two-parameter analysis for the antibody binding and DNA content showed no binding of antibody to replicating DNA in control cells. In HN2-treated cells a cell subset with the lowest antibody binding was observed among cells in G1 phase. Binding of antibody to DNA in HN2-treated cells was eliminated by single-strand (ss) specific S1 nuclease. In competition assay, antibody was inhibited by thermally denatured DNA, but not by native double-stranded (ds) DNA, RNA, nucleosides and deoxyribohomopolymers. Binding of monoclonal antibody specific for the determinants expressed on ssDNA to the cells treated with alkylating agents may be attributed to local DNA denaturation. Potentiation of L-phenylalanine mustard cytotoxicity by buthionine sulfoximine or hyperthermia was accompanied by increased antibody binding to cellular DNA. Immunoreactivity of cells with the monoclonal antibody F7-26 may be a useful probe for the assessment of cell damage induced by alkylating agents, especially in heterogeneous cell populations. |
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