High throughput evaluation of gamma-H2AX
© Avondoglio et al; licensee BioMed Central Ltd. 2009
Received: 2 June 2009
Accepted: 24 August 2009
Published: 24 August 2009
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 γ-H2AX is a known marker for irradiation-induced DNA DSBs. However, the first generation assay involves the use of immunofluorescent staining of γ-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 γ-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 γ-H2AX versus irradiation dose.
Because the DSB is the critical lesion induced by ionizing radiation in terms of cell killing, their analysis provides essential insight into fundamental and translational radiobiology. However, DSBs are relatively infrequent as compared to the other radiation-induced lesions such as SSB and base damage, resulting in technical challenges in the development of specific analytical procedures. Standard techniques for quantifying DSB induction and repair have included pulsed field gel electrophoresis (PFGE) and the neutral comet assay . Over the last several years, γ-H2AX expression has been established as a sensitive indicator of DSBs . At sites of radiation-induced DNA DSBs, the histone H2AX becomes rapidly phosphorylated (the phosphorylated form is referred to as γ-H2AX) forming readily visible nuclear foci [2, 3]. Although the specific role of γ-H2AX in the repair of DSBs has not been defined, recent reports indicate that the dephosphoryation of γ-H2AX and dispersal of γ-H2AX foci in irradiated cells correlates with the repair of DNA DSBs [4–6]. Moreover, Macphail et al in their study of ten cell lines reported that the loss of γ-H2AX correlates with clonogenic survival after irradiation .
Currently, immunofluorescent staining is one method for evaluation of γ-H2AX . However, the assay typically involves the manual counting of nuclear foci, with each focus containing γ-H2AX molecules. The assay also has a limited dose range and is not amenable to high throughput screening (HTS). γ-H2AX may also be evaluated by immunoblot assay but this technique is time and labor intensive, has a fairly narrow range of detection, and also is not scalable to HTS. Lastly, flow cytometry has been used to analyze γ-H2AX; however, flow cytometry methods are not readily integrated into HTS. Although each of the aforementioned methods of evaluating γ-H2AX is effective and has provided important information, there is still a need for an analytical high throughput assay that is capable of screening radiomodifying drugs across diverse cell lines and in vivo tissue. We show that using an electrochemiluminescent detection system, γ-H2AX can be evaluated in both cultured cell lines and in vivo murine tissue with an efficient, reproducible methodology that is scalable for HTS .
Materials and methods
Cell lines and treatment
The human glioblastoma cell line (U251) and pancreatic cell line (MiaPaca) were obtained from the National Cancer Institute Frederick Tumor Repository. The breast tumor cell line variant MDA-MB-231BR was supplied by the laboratory of Patricia Steeg (National Cancer Institute, Bethesda, MD). Cells were grown in DMEM (Invitrogen) with glutamate (5 mmol/L) and 10% fetal bovine serum, and maintained at 37°C, 5% CO2. 17DMAG and perifosine, provided by the Developmental Therapeutics Program of the National Cancer Institute, were reconstituted in DMSO (100 mmol/L) and PBS (100 mmol/L) respectively, and stored at -20°C. Cells were irradiated using a Pantak X-ray source at a dose rate of 2.28 Gy/min.
Cultures were trypsinized to generate a single cell suspension and a specified number of cells was seeded into each well of a six-well tissue culture plate. After allowing cells time to attach (4 h), cultures received 17DMAG (50 nmol/L) and perifosine (9 μmol/L) or DMSO (vehicle control) for 16 h before irradiation: media was then removed and replaced with drug-free media. Ten to fourteen days after seeding, colonies were stained with crystal violet, the number of colonies containing at least 50 cells was determined, and surviving fractions were calculated.
Immunofluorescent staining for γ-H2AX
Immunofluorescent staining and counting of γ-H2AX nuclear foci was performed as previously described . Slides were examined on a Leica DMRXA fluorescent microscope. Images were captured by a Photometrics Sensys CCD camera (Roper Scientific) and imported into IP Labs image analysis software package (Scanalytics, Inc.). For each treatment condition, γ-H2AX foci were determined in at least 50 cells. Cells were classified as positive (i.e., containing radiation-induced γ-H2AX foci) when more than five foci were detected.
MSD Direct Coat Assay
Cells were grown and treated on 100 mm plates. After specified treatments and incubations, cells were harvested: scraped into PBS, washed, and frozen overnight at -80°C. Each cultured condition was resuspended in lysis buffer containing NaCL (500 mM), EDTA (2 mM), Triton X-100 (1%), sodium deoxycholate (1%), SDS (1%), Tris HCl (50 mM), NaF (10 mM), phosphatase and protease inhibitors (1×), and PMSF (2 mM). Proteins were solubilized by sonication, concentrations determined by Bradford assay, and lysates coated onto MSD high bind plates. Wells were blocked with 3% blocking solution, washed, and a sulfo-ester tag conjugated phospho-H2AX (Abcam) detection antibody was added in 1% blocking solution (1 μg/ml). Wells were washed 3× and 1× MSD Read Buffer was added before analysis in a MSD Sector Imager 2400.
All animal studies were conducted in accordance with the principles and procedures outlined in the NIH Guide for the Care and Use of Animals. Four to six week old nude mice were injected subcutaneously with U251 cells (1 × 106) on the lateral aspect of the rear leg. When tumors reached 500 mm3 mice were irradiated. Mice were sacrificed, tumors extracted, tissue homogenized, and resuspended in lysis buffer. The MSD Assay was carried out as stated above.
In vitro experiments were repeated thrice and statistical analysis was done using a Student's t test. Data are presented as mean ± SD. A probability level of a P value of < 0.05 was considered significant.
Results and Discussion
Because γ-H2AX expression is an indicator of DSB induction and repair, the development of an analytical method adaptable to a high throughout approach would appear to have a number of applications related to drug development for either radiation sensitizers or for other drugs that kill tumor cells via induction of DNA DSBs. Towards this end, we have demonstrated that the γ-H2AX MSD assay has excellent reproducibility, is quantitative, and applicable to multiple cell types from either in vitro or in vivo samples. We have also shown that the MSD assay may allow the more rapid development of radiomodifying drugs in a high throughput fashion.
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