Flow cytometry analysis of single strand DNA damage in neuroblastoma cell lines using the F7-26 monoclonal antibody

1806 Most chemotherapeutic agents act via DNA damage, and drugs such as alkylating agents and topoisomerase inhibitors act primarily by inducing single-strand DNA (ssDNA) breaks. We developed a flow cytometry method to measure the proportion of cells with ssDNA using the F7-26 monoclonal antibody (Mab) specific for ssDNA. Neuroblastoma cells were treated with melphalan (L-PAM), fenretinide, 4-hydroperoxycyclophosphamide (4-HC) ± pan-caspase inhibitor BOC-d-fmk, topotecan or with 10Gy gamma radiation ± hydrogen peroxide (H 2 O 2 ). Cytotoxicity was measured by DIMSCAN digital imaging fluorescence assay. Apoptosis by TUNEL, reactive oxygen species (ROS) by carboxy-dichlorofluorescein diacetate, and ssDNA by Mab F7-26 were measured with a BD LSRII flow cytometer, with DNA visualized by propidium iodide counterstaining. Irradiated and immediately fixed neuroblastoma cells showed increased ssDNA, but not apoptosis by TUNEL. 4-HC or L-PAM ± BOC-d-fmk increased ssDNA (F7-26-positive), but BOC-d-fmk prevented TUNEL staining in the same cells. Fenretinide increased apoptosis by TUNEL but not ssDNA damage detected with F7-26. Enhanced ssDNA in neuroblastoma cells treated with radiation + H 2 O 2 was associated with increased ROS. Topotecan increased both ssDNA and cytotoxicity in 4-HC-treated cells. Table shows mean percentages (%) of ssDNA damage assayed by Mab F7-26 or apoptosis assayed by TUNEL assay in neuroblastoma cell lines (n = 3). Thus, Mab F7-26 recognized ssDNA due to exogenous DNA damage, rather than apoptosis. This 2-color assay can be used to accurately and sensitively quantify alkylator- and irradiation-induced ssDNA damage, and also to exclude DNA damage as a mechanism of drug action. This assay should be useful not only for studying the mechanism of drug action in cell culture but also as a pharmacodynamic assay with patient specimens.