A high throughput fluorescence assay to quantify DNA double strand breaks in cells

1024 Introduction: Radiotherapy and radioimmunotherapy exploit the use of α, β and γ-irradiation to induce DNA double strand breaks (DSBs) which may lead to cell death if not repaired. These DNA DSBs are always followed by phosphorylation of the histone protein component H2AX to form γ-H2AX. The phosphorylated γ-H2AX protein is an established marker for DNA DSBs as it has been shown to represent DSBs in a 1:1 manner. Quantifying γ-H2AX as an indicator of DNA DSBs could potentially be used to tailor radiation therapy specifically to match the aberrant cell type and assess impacts on surrounding normal tissue. Conventional methods to assess γ-H2AX expression involve fluorescence microscopy to count individual foci which is tedious, time-consuming and subject to operator induced variability. Alternatively, flow cytometry has been used to quantify DSBs in a sensitive and reliable manner, however this method requires an expensive instrumentation and highly trained users. The ability to assess DSBs using a standard plate reader makes this technique more widely accessible and offers the potential for high-throughput screening. Methods: The A549 lung epithelial carcinoma cell line was used in this study and DNA DSBs were induced by treating the cells with different doses of etoposide (10 nM - 100 μM) for 1.5 h. Immunofluorescence was performed using an anti-γ-H2AX antibody to stain cells and γ-H2AX foci were counted under an epifluorescence microscope to detect and quantify DSBs. For comparison a 96-well plate assay was employed as a high-throughput method to quantify DNA DSBs using a plate reader to measure the total γ-H2AX fluorescence intensity per well. This assay utilized an unlabeled primary anti-γ-H2AX antibody followed by either a secondary detection antibody labeled with Alexa Fluor-488 for a direct fluorescence measurement or incubation with a lanthanide-labeled secondary antibody for time-resolved fluorescence measurement. Standard staining with phalloidin (F-actin) and DAPI (nuclei) was employed to confirm the uniformity of the cells within the 96-well plate and to eliminate well-to-well variability. Detection of DSBs after radiation exposure was also assessed using this procedure. Results: Employing a lanthanide-coupled secondary antibody and using time resolved fluorescence enabled significant detection and differentiation of low numbers of DNA DSBs. When using the traditional Alexa-488 fluorophore to stain cells for γ-H2AX expression, the fluorescence intensity of cells increased with the number of DNA DSBs and was comparable to counting foci at the time interval tested. This increase was significant with high doses of etoposide which induced higher number of DSBs. Conclusion: It is feasible to use conventional immunofluorescence and plate readers to assess DNA DSBs with comparable sensitivity to foci counting using fluorescent microscopy. Additional sensitivity was enabled by using a lanthanide-coupled secondary antibody and time-resolved fluorescence. Adapting this assay to a 96-well plate format makes it more amenable to high-throughput screening applications. The sensitivity and ease of use of this assay has the potential to aid in the development of improved radiotherapy, assess the protective effect of compounds against ionizing radiation, and measure DNA damage induced by chemotherapy and or combined radiation and chemotherapies. Acknowledgements: This work was supported by the ORNL Laboratory Directed Research and Development program.