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

Abstract

The F7-26 monoclonal antibody (Mab) has been reported to be specific for singlestrand DNA damage (ssDNA) and to also identify cells in apoptosis. We carried out 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) 6 pan-caspase inhibitor BOC-d-fmk, topotecan or with 10Gy c radiation 6 hydrogen peroxide (H 2 O 2 ) 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 6 BOC-d-fmk increased ssDNA (F7-26-positive), but BOC-dfmk prevented TUNEL staining. Fenretinide increased apoptosis by TUNEL but not ssDNA damage detected with F7-26. Enhanced ssDNA in neuroblastoma cells treated with radiation 1 H 2 O 2 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. ' 2007 International Society for Analytical Cytology

Application of anti‐ssDNA monoclonal antibody to study exogenous and apoptosis‐associated DNA damage

“…In contrast to the observations reported above, data published by Grigoryan et al (1) show that detection of the exogenous DNA damage in their study was not performed under optimal conditions. For sensitive detection of nonapoptotic DNA breaks, the denaturation medium should be different and temperature should be higher than that they used (2,9-13).…”

“…For sensitive detection of nonapoptotic DNA breaks, the denaturation medium should be different and temperature should be higher than that they used (2,9-13). Indeed, the intensity of staining in irradiated and H 2 O 2 -treated cells is very low (1). Only under conditions designed specifically for the measurement of exogenous DNA damage can the effect of pharmacologically relevant drug concentrations on DNA be detected.…”

“…Grigoryan et al (1) concluded that the formamide-MAb F7-26 procedure does not recognize apoptotic cells. However, their own data does not support this conclusion.…”

The glutathione synthesis inhibitor buthionine sulfoximine synergistically enhanced melphalan activity against preclinical models of multiple myeloma

“…F7-26 (mAb) was from Millipore (Billerica, MA, USA). 23 The JC-1 probe, vitamin C, vitamin E, N -acetylcysteine (NAC) and sodium thiosulfate (STS) were from Sigma-Aldrich. The anti-CD38 phycoerythrin (PE) and anti-CD138 fluorescein isothiocyanate (FITC) antibodies, and APO-DIRECT kit (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)) were purchased from BD Biosciences (San Jose, CA, USA).…”

“…The anti-CD38 phycoerythrin (PE) and anti-CD138 fluorescein isothiocyanate (FITC) antibodies, and APO-DIRECT kit (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)) were purchased from BD Biosciences (San Jose, CA, USA). 23, 24 Caspase-3, caspase-9, poly ADP ribose polymerase and anti-rabbit immunoglobulin G horseradish peroxidase antibodies were from Cell Signaling (Danvers, MA, USA); anti-β-actin was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).…”