Flow cytometry analysis of single-strand DNA damage in neuroblastoma cell lines using the F7-26 monoclonal antibody.
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.
Grigoryan
Rita S
RS
Developmental Therapeutics Program, USC-CHLA Institute for Pediatric Clinical Research, Los Angeles, California 90027, USA.
Yang
Bo
B
Keshelava
Nino
N
Barnhart
Jerry R
JR
Reynolds
C Patrick
CP
D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000378:metabolism; D017209:Apoptosis / Q000502:physiology; D001593:Benzyl Compounds / Q000378:metabolism; D045744:Cell Line, Tumor / Q000378:metabolism / Q000528:radiation effects; D002648:Child; D004249:DNA Damage; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D017313:Fenretinide / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D005720:Gamma Rays; D006801:Humans; D006845:Hydrocarbons, Fluorinated / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D008558:Melphalan / Q000378:metabolism; D009447:Neuroblastoma / Q000235:genetics; D016877:Oxidants / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D019772:Topotecan / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D001593:Benzyl Compounds; C476029:Boc-D-FMK; D004277:DNA, Single-Stranded; D004791:Enzyme Inhibitors; D006845:Hydrocarbons, Fluorinated; D016877:Oxidants; D017382:Reactive Oxygen Species; D017313:Fenretinide; D019772:Topotecan; D006861:Hydrogen Peroxide; D008558:Melphalan