High throughput evaluation of gamma-H2AX

Abstract

The DNA double-strand break (DSB) is the primary lethal lesion after therapeutic radiation. Thus, the development of assays to detect and to quantitate these lesions could have broad preclinical and clinical impact. Phosphorylation of histone H2AX to form gamma-H2AX is a known marker for irradiation-induced DNA DSBs. However, the first generation assay involves the use of immunofluorescent staining of gamma-H2AX foci. This assay is time consuming, operator dependent and is not scalable for high throughput assay development. Thus, we sought to develop a new assay using a high throughput electrochemiluminescent platform from Mesoscale Discovery Systems to quantify gamma-H2AX levels. The results show that our assay utilizes significantly less time and labor, has greater intra-assay reproducibility and has a greater dynamic range of gamma-H2AX versus irradiation dose.

Figure 1

γ-H2AX evaluation post-irradiation. (A) U251 cells were plated onto chamber slides, irradiated at the specific doses, and fixed for immunocytochemical analysis. Foci were evaluated three times in 30 nuclei per treatment per experiment. (B) Plot of the linear dynamic range of the immunofluorescent staining assay. (C) Plot of the linear dynamic range of the MSD Assay.

Figure 2

Comparison of foci versus MSD for the evaluation of DNA-DSB repair. The U251 cell line was plated out onto chamber slides for the immunofluorescent assay in which foci counting was utilized. U251 cells were plated onto 100 mm plates for the MSD assay. Irradiation was carried out and the respective assays were performed at designated time points.

Figure 3

The effect of drugs on tumor cell radiosensitivity. (A) U251 Cells were seeded as a single-cell suspension and with a specified number of cells. After allowing cells time to attach (4 h), 17 DMAG or the vehicle control was added (50 nmol/L) and the plates were irradiated 16 h later. Ten to twelve days after seeding, survival curves were generated after normalizing for the cytotoxicity generated by 17 DMAG alone. Data presented are the mean ± SE from at least three independent experiments. (B) Identical experimental conditions as (A) followed by the MSD assay. (C) The non-radiosensitizing effect of perifosine carried out by the MSD assay. U251 cells were treated with perifosine (9 μmol/L) alone and the combination of perifosine and IR.

Figure 4

Evaluation of γ-H2AX In Vivo. Tumors were grown in mice injected with U251 cells subcutaneously in the flank. Mice were irradiated, tissue homogenized, protein isolated and the MSD assay was performed.