Flow cytometry analysis of single-strand DNA damage in neuroblastoma cell lines using the F7-26 monoclonal antibody
Rita S Grigoryan1, Bo Yang, Nino Keshelava
1Developmental Therapeutics Program, USC-CHLA Institute for Pediatric Clinical Research, Los Angeles, California 90027, USA.
Abstract:
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.
Insights
The F7-26 monoclonal antibody (Mab) specifically detects single-strand DNA (ssDNA) damage from external sources, not apoptosis. This assay is valuable for understanding how anti-cancer drugs work.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- The F7-26 monoclonal antibody (Mab) is known to identify single-strand DNA (ssDNA) damage and apoptosis.
- Distinguishing ssDNA damage from apoptosis is crucial for understanding drug mechanisms.
Purpose of the Study:
- To determine if F7-26 Mab binding via flow cytometry can specifically identify exogenous ssDNA damage versus DNA damage resulting from apoptosis.
- To validate the utility of F7-26 Mab as a tool for characterizing antineoplastic drug mechanisms.
Main Methods:
- Neuroblastoma cells were treated with various agents (melphalan, fenretinide, 4-hydroperoxycyclophosphamide, topotecan, gamma radiation +/- hydrogen peroxide).
- Single-strand DNA damage was assessed using F7-26 Mab and flow cytometry.
- Apoptosis was measured by terminal deoxynucleotidyltransferase (TUNEL) assay.
- Reactive oxygen species (ROS) were detected using carboxy-dichlorofluorescein diacetate.
Main Results:
- Irradiated cells showed increased ssDNA but were TUNEL-negative.
- 4-hydroperoxycyclophosphamide or melphalan increased ssDNA, with BOC-d-fmk inhibiting apoptosis.
- Fenretinide induced apoptosis (TUNEL-positive) without increasing ssDNA damage.
- Radiation plus hydrogen peroxide led to increased ssDNA and ROS.
- Topotecan enhanced ssDNA and cytotoxicity in 4-HC treated cells.
Conclusions:
- Mab F7-26 reliably detects ssDNA damage caused by exogenous agents, distinct from apoptosis.
- This assay provides a valuable method for elucidating the mechanisms of action for anti-cancer drugs.
