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

Cytometry A. 2007 Nov;71(11):951-60. doi: 10.1002/cyto.a.20458.


The F7-26 monoclonal antibody (Mab) has been reported to be specific for single-strand DNA damage (ssDNA) and to also identify cells in apoptosis. We carriedout studies to determine if F7-26 binding measured by flow cytometry was able to specifically identify exogenous ssDNA as opposed to DNA damage from apoptosis. 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 (H2O2) and fixed immediately postradiation. Cytotoxicity was measured by DIMSCAN digital imaging fluorescence assay. The degree of ssDNA damage was analyzed by flow cytometry using Mab F7-26, with DNA visualized by propidium iodide counterstaining. Flow cytometry was used to measure apoptosis detected by terminal deoxynucleotidyltransferase (TUNEL) assay and reactive oxygen species (ROS) by carboxy-dichlorofluorescein diacetate. Irradiated and immediately fixed neuroblastoma cells showed increased ssDNA, but not apoptosis by TUNEL (TUNEL-negative). 4-HC or L-PAM+/-BOC-d-fmk increased ssDNA (F7-26-positive), but BOC-d-fmk prevented TUNEL staining. Fenretinide increased apoptosis by TUNEL but not ssDNA damage detected with F7-26. Enhanced ssDNA in neuroblastoma cells treated with radiation+H2O2 was associated with increased ROS. Topotecan increased both ssDNA and cytotoxicity in 4-HC-treated cells. These data demonstrate that Mab F7-26 recognized ssDNA due to exogenous DNA damage, rather than apoptosis. This assay should be useful to characterize the mechanism of action of antineoplastic drugs.

MeSH terms

  • Antibodies, Monoclonal / metabolism*
  • Antineoplastic Agents / metabolism
  • Apoptosis / physiology
  • Benzyl Compounds / metabolism
  • Cell Line, Tumor / metabolism
  • Cell Line, Tumor / radiation effects
  • Child
  • DNA Damage*
  • DNA, Single-Stranded* / chemistry
  • DNA, Single-Stranded* / metabolism
  • Enzyme Inhibitors / metabolism
  • Fenretinide / metabolism
  • Flow Cytometry / methods*
  • Gamma Rays
  • Humans
  • Hydrocarbons, Fluorinated / metabolism
  • Hydrogen Peroxide / metabolism
  • In Situ Nick-End Labeling
  • Melphalan / metabolism
  • Neuroblastoma / genetics
  • Oxidants / metabolism
  • Reactive Oxygen Species / metabolism
  • Topotecan / metabolism


  • Antibodies, Monoclonal
  • Antineoplastic Agents
  • Benzyl Compounds
  • Boc-D-FMK
  • DNA, Single-Stranded
  • Enzyme Inhibitors
  • Hydrocarbons, Fluorinated
  • Oxidants
  • Reactive Oxygen Species
  • Fenretinide
  • Topotecan
  • Hydrogen Peroxide
  • Melphalan