Grigoryan Rita S, Yang Bo, Keshelava Nino, Barnhart Jerry R, Reynolds C Patrick
Developmental Therapeutics Program, USC-CHLA Institute for Pediatric Clinical Research, Los Angeles, California 90027, USA.
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.
据报道，F7-26单克隆抗体（Mab）对单链DNA损伤（ssDNA）具有特异性，并且还能识别凋亡细胞。我们开展了研究，以确定通过流式细胞术检测的F7-26结合是否能够特异性识别外源性ssDNA，而非凋亡引起的DNA损伤。用美法仑（L-PAM）、芬维A胺、4-氢过氧环磷酰胺（4-HC）+/-泛半胱天冬酶抑制剂BOC-d-fmk、拓扑替康处理神经母细胞瘤细胞，或用10Gyγ射线+/-过氧化氢（H2O2）处理，辐射后立即固定。通过DIMSCAN数字成像荧光测定法测量细胞毒性。使用Mab F7-26通过流式细胞术分析ssDNA损伤程度，用碘化丙啶复染使DNA可视化。流式细胞术用于测量通过末端脱氧核苷酸转移酶（TUNEL）测定法检测的凋亡以及通过羧基二氯荧光素二乙酸酯检测的活性氧（ROS）。经辐射并立即固定的神经母细胞瘤细胞显示ssDNA增加，但TUNEL检测未显示凋亡（TUNEL阴性）。4-HC或L-PAM+/-BOC-d-fmk增加了ssDNA（F7-26阳性），但BOC-d-fmk阻止了TUNEL染色。芬维A胺通过TUNEL增加了凋亡，但未增加F7-26检测到的ssDNA损伤。用辐射+H2O2处理的神经母细胞瘤细胞中增强的ssDNA与ROS增加有关。拓扑替康增加了4-HC处理细胞中的ssDNA和细胞毒性。这些数据表明，Mab F7-26识别的是由外源性DNA损伤而非凋亡导致的ssDNA。该检测方法对于表征抗肿瘤药物的作用机制应该是有用的。
