Background
Double Strand Breaks (DSBs) are one of the most important types of DNA damage. DSBs are more difficult to repair than many other lesions and their incorrect repair (e.g., misrejoining of broken DNA strands from different chromosomes) can result in cytotoxic or genomic alterations. Defects in the DNA repair machinery may increase cell vulnerability to DNA-damaging agents and accumulation of mutations in the genome, and could lead to the development of various disorders including cancers. Epidemiological evidence supports a strong association between global DSB repair capacity and cancer risk [
1‐
3], radiation sensitivity [
4,
5] and response to cancer therapy [
6,
7]. The association between genetic defects in DNA repair and increased clinical radiosensitivity has been identified in many studies and used as a basis for the development of predictive assays for normal tissue toxicity [
8].
Over the past decade, the γ-H2AX assay has been applied to a variety of cell types and tissues to correlate γ-H2AX levels with DNA damage and repair [
9‐
13]. Following radiation exposure, histone H2AX is rapidly phosphorylated by ATM and/or DNA-PK kinases at or near the vicinity of DNA DSB sites to form γ-H2AX [
14]. Immunolabeling of γ-H2AX provides a quantitative measurement and direct visualization of DSBs as fluorescent nuclear foci. At the cellular level, the kinetics of formation or loss of γ-H2AX foci may reflect the rate or efficiency of DSB repair [
15]. The biphasic nature of DSB repair kinetics has been associated with different repair pathways that allow repair for a fast (initial few hours) and slow component (hours to days) of repair [
16,
17]. Additionally, there is evidence that the DSBs assayed several hours after the initial radiation challenge that still remain unrepaired known as residual DNA damage, may be predictive of individual susceptibility to complex DNA lesions that can be lethal [
18]. Current evidence suggests that there is a large inter-individual variation in DSB DNA repair capacity in lymphocytes from healthy individuals [
19‐
21]. Further, clinical radiosensitivity is often linked to defects in DNA repair [
5,
22,
23]. The capacity to repair DSB is therefore an important factor to consider in risk assessment, however studies to date are limited due to no large-scale prospective evidence or ability to conduct high-throughput phenotypic assays [
24].
The objective of the present study was to develop a rapid, high-throughput γ-H2AX assay based on imaging flow cytometry (IFC) using the ImageStream®
X Mk II (ISX MKII) platform to evaluate DNA DSB repair kinetics in human peripheral blood cells after exposure to ionizing irradiation. Imaging flow cytometry is a relatively new technique which combines the speed of flow cytometry with the imaging capability of conventional microscopy [
25‐
27]. It has been used to analyze cell death, apoptosis and immune response as an advanced method for fluorescence-based analysis of cellular morphology and heterogeneity [
28‐
33]. Combining the strength of flow cytometry and conventional microscopy enables high-throughput characterization of cells on a microscopic scale [
34]. This paper presents: 1) dose response curves based on γ-H2AX fluorescence intensity and foci number, 2) measurements of DNA repair kinetics up to 24 h after exposure to 4 Gy γ rays and, 3) a mathematical approach for modeling DSB rejoining kinetics using two key parameters a) rate of γ-H2AX decay, and b) yield of residual unrepaired breaks.
Methods
Blood collection and irradiation
Blood was collected by venipuncture in 5 mL lithium-heparinized Vacutainer® tubes (BD Vacutainer™, Franklin Lakes, NJ) from healthy adult donors (2 female, 2 male) with informed consent and approval by the Columbia University Medical Center Institutional Review Board (IRB protocol IRB-AAAE-2671). All donors were non-smokers in relatively good health at the time of donation with no obvious illnesses such as colds, flu, or infections and no known exposures to medical ionizing radiation within the last 12 months. Fresh blood aliquots (1 mL) were dispensed into 15 mL conical bottom tubes (Santa Cruz Biotechnology, Dallas, TX) and were irradiated with γ rays (0, 2 and 4 Gy) using a Gammacell® 40
137Cesium irradiator (Atomic Energy of Canada, Ltd., Chalk River, ON). The blood sample tubes were placed on their side in the middle of the chamber and irradiated with a dose rate of 0.73 Gy/min [
35]. The
137Cs irradiator was calibrated annually with TLDs and homogeneity of exposure across the sample volume was verified using EBT3 Gafchromic film with less than 2% variation within the sample (Ashland Advanced Materials, Gafchromic, Bridgewater, NJ).
γ-H2AX assay immunolabeling protocol
Immediately after irradiation, 100 μl blood aliquots were transferred to 1.4 mL 2D Matrix™ microtubes (Thermo Scientific™, Waltham, MA) containing 900 μL RPMI 1640 culture medium (Gibco, Waltham, MA) supplemented with 15% FBS and 2% Penicillin and Streptomycin (all reagents from Invitrogen, Eugene, OR). The rack containing microtubes was placed into an incubator at 37 °C, 5% CO2 up to 24 h. At specific time points after irradiation (0.5, 1, 3, 6 and, 24 h), cultured blood samples were lysed and fixed with 1X Lyse/fix solution (BD Phosflow™,; BD Biosciences, San Jose, CA), washed with 1X phosphate buffered saline (PBS, Gibco, Gaithersburg, MD), suspended in 50% cold methanol, and stored at 4 °C for 24 h. Fixed cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) at room temperature for 10 min and then incubated with Alexa Fluor® 488 Mouse anti-H2AX (pS139) antibody (clone N1–431, BD Pharmingen™, Franklin Lakes, NJ), diluted 1:1000 with 1% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO) at room temperature for 1 h, after which the samples were washed with 1X PBS and stained with 5 μM DRAQ5™ (Thermo Scientific™) at RT for a minimum of 5 min. All solution transferring or mixing in microtubes was performed using a 1.2-ml multichannel electronic pipet (Eppendorf Inc., Westbury, NY). All steps in the procedure were performed at room temperature (RT) and microtubes in racks were spun at 250×g for 3 min.
Data acquisition and analysis on the ISX and IDEAS®
The 96-well plate of samples were transferred to the ImageStream®X Mk II (ISX MKII) imaging flow cytometer (LUMINEX Corporation, Austin, Texas) for automated sample acquisition and captured using the ISX INSPIRE™ data acquisition software. Images of 5000–12,000 cells were acquired at 40x magnification using the 488 nm excitation laser at 200 mW: Bright field (BF) images were captured on channel 1, γ-H2AX immunostaining on channel 2, DRAQ5 images on channel 5 and side scatter on channel 6. Data was collected with only the Area feature applied in the BF channel, such that events with areas less than 60 pixels (15 μm2) were gated out in order to minimize the collection of small debris. For the compensation, irradiated blood cells were stained with γ-H2AX antibody or DRAQ5 only and captured using the 488 nm laser without brightfield illumination. The compensation coefficients were calculated automatically using the compensation wizard in the Image Data Exploration and Analysis Software (IDEAS) package (v6.2). To quantify the γ-H2AX expression levels, the viable lymphocytes population was gated for foci quantification and total γ-H2AX fluorescence intensity. Nuclear foci formation was identified using the spot counting wizard in IDEAS which automated identification and enumeration of foci. The geometric mean of the γ-H2AX fluorescence intensity of individual cells from each sample was analyzed. For the dose response curve, γ-H2AX foci and intensity levels were measured at 1 h post irradiation. All curves were generated using GraphPad Prism 7 (GraphPad software Inc., La Jolla, CA), and R2 value was calculated to assess goodness of fit of curves from linear regression analysis.
Quantitative modeling of DNA repair kinetics
For the kinetic curves, γ-H2AX levels were measured at 0.5, 1, 3, 6 and, 24 h after 4 Gy irradiation. The data on γ-H2AX foci (F) at different time points (T) after irradiation were quantitatively modeled by the following equation, where F
bac is the background value prior to irradiation, F
res is the residual value remaining at long times (e.g. 24 h) after irradiation, K
prod is the constant for induction of foci by radiation, and K
dec is the constant for decay of foci after irradiation [
20]:
$$ \mathrm{F}={\mathrm{F}}_{\mathrm{bac}}+{\mathrm{F}}_{\mathrm{res}}+{\mathrm{K}}_{\mathrm{prod}}\ T\ \mathit{\exp}\left(-{\mathrm{K}}_{\mathrm{dec}}\ T\ \right) $$
(1)
We used least squares fitting in Maple 2017 software (
https://www.maplesoft.com/) as a practical approach for estimating K
dec and F
res, involving curve fitting of each sample data set to Eq. (
1). Thus, as we propose below we will use both the decay constant (K
dec) and residual excess fluorescence intensity (F
res) to describe each individual’s DNA DSB Repair Capacity.
Discussion
Since it was first demonstrated by Rogakou, Bonner and colleagues that histone H2AX is rapidly phosphorylated on residue serine 139 in cells when DSBs are introduced into the DNA by ionizing radiation [
37], the γ-H2AX assay has been widely used as a sensitive molecular marker of DNA damage and DSB repair capacity in a variety of human tissue and cell types [
38,
39]. In recent years, the γ-H2AX biomarker has become a powerful tool to monitor DNA DSBs in translational cancer research with the potential to assess the radiosensitivity of prospective radiotherapy patients [
5,
40]. The goal of the present work was to develop and optimize the γ-H2AX immunocytofluorescence protocol for high-content screening of double-stranded DNA breaks in finger-stick sized blood samples using IFC. The IFC technique allows fast and accurate analysis of γ-H2AX yields in several thousand cells per sample which would be extremely time consuming using conventional manual immunocytofluorescence protocols. In the present work, we have used our high-throughput IFC-based γ-H2AX assay to measure dose-dependent response and DSB repair kinetics in irradiated human blood samples.
To assess individual DSB repair capacity, radiation-induced γ-H2AX yields were measured for dose/time response in ex-vivo irradiated blood samples taken from four healthy donors (2 male, 2 female). Measurements of γ-H2AX fluorescence intensity and foci number at specific time points up to 24 h after exposure with 0, 2 and 4 Gy gamma rays showed a linear dose-dependent response and pattern of DNA repair, consistent with previous studies [
10,
17,
20,
41]. The results highlight that the fluorescence intensity endpoint showed a better dose response compared to foci number given the small difference in foci number between 2 and 4 Gy. The reduced dose-response is likely attributed to the current configuration of our ISX IFC platform which only contains a 40x objective lens for image acquisition. The lower resolution of the 40x lens in comparison to a 60x objective is therefore likely to be responsible for the underestimation of γ-H2AX foci in the irradiated blood lymphocytes. Particularly in cells exposed to higher doses of radiation, there will be many γ-H2AX foci in close proximity to each other, leading to poor differentiation in smaller images with low spatial resolution. Recent studies by Durdik et al. [
42] and Parris et al. [
43] have shown that increasing the magnification from 40x to 60x together with extended depth of field (EDF) focus stacking option provided a more accurate assessment of foci number throughout the complete nuclear region in human lymphocytes exposed to low-dose ionizing radiation [
42] and 2 Gy-irradiated immortalized fibroblasts [
43]. Thus, these studies suggest that the 60x + EDF ISX configuration would permit enhanced foci identification thereby allowing better differentiation between the 2 and 4 Gy dose points, and identification of lower doses between 0 and 2 Gy. Further studies are necessary to address the assay dose limits for the sensitivity of the γ-H2AX foci and fluorescence intensity endpoints after ionizing radiation exposure and to expand this work to evaluate individual DNA repair capacity within a larger population.
Quantitative modeling of DNA repair kinetics based on fluorescence intensity showed that the decay constant of γ-H2AX foci after irradiation (K
dec) was not markedly different among donors tested, whereas residual γ-H2AX fluorescence intensity (F
res) was apparently higher in M2 and F2 than in the other two donors (M1 and F1), suggesting that M2 and F2 may have more unrepaired DSB 24 h after irradiation (Fig.
6b). The differences in DSB repair capacity between the 4 healthy donors tested here, show the potential of our high-throughput γ-H2AX assay to measure DNA repair kinetics on an individual-by-individual basis. Quantitative modeling of DNA repair kinetics based on foci number did not show any difference in DSB repair capacity between the four individuals (Additional file
1). This result was probably influenced by the visibly larger “scatter” in the foci data at 24 h, compared with the fluorescence intensity data at 24 h, widening the confidence intervals for F
res based on the foci data. Efforts to improve foci quantification with higher magnification and the use of EDF mentioned above, could enhance the quantification of DSB rejoining kinetics and assess the DSB repair capacity of specific individuals. Recent work by Kroeber et al. [
23] showed the capability of the γ-H2AX assay to identify distinct outliers among a large cohort of 136 rectal cancer patients. They suggested that these patients are most probably radiosensitive and may have the highest risk of suffering radiotherapy-related late sequelae [
23]. Interestingly, Yin et al. [
8] recently reported enhanced DNA repair capacity in the peripheral blood mononuclear cells from a small cohort lung cancer patients tended to be associated with a poor response to radiation therapy, implicating a modulation of DNA repair [
8].
It is known that the presence of γ-H2AX is not always linked specifically to DNA damage, but also to other cellular stages such as senescence, cell division or apoptosis [
44]. In this case, the multi-spectral nature of IFC technology for γ-H2AX analysis would allow for the expansion to a quantitative multiplexed assay to analyze multiple radiation responsive biomarkers on a single cell. Also, the ability to target specific cell populations as well as eliminate interfering cells or debris will increase the number of cells that can be analyzed and potentially improve the sensitivity of the assay. In the current study, we measured γ-H2AX yields in focused DNA positive lymphocytes population instead of the total leukocytes. It is known that the sensitivity of lymphocytes and granulocytes to radiation are different whereby γ-H2AX levels in lymphocytes increased in a dose-dependent manner after 0–10 Gy γ-ray exposure, whereas levels in granulocytes were unaffected [
36]. Further, residual levels of apoptosis in the irradiated samples are a potential confounding factor for the γ-H2AX total fluorescence analysis [
45]. IFC image analysis using the IDEAS® software allowed us to automatically detect and eliminate pan-nuclear γ-H2AX stained lymphocytes based on fluorescence intensity and morphology. Pan-nuclear γ-H2AX response has been suggested as a biomarker to distinguish apoptotic cells from DNA damaged cells [
46,
47]. We have shown here that the percentage of pan-nuclear γ-H2AX stained lymphocytes increased over time, up to 24 h after 4 Gy exposure (Fig.
3). These observations are consistent with other studies which show the apoptotic response of human lymphocytes upon radiation exposure [
48‐
50].
Another advantage of our IFC-based γ-H2AX assay is both reduced assay time and time-to-result. First, our immunolabeling protocol presented here can be completed within 2 h, eliminating the need to prepare peripheral blood mononuclear cells which requires Ficoll gradient purification, an approach that is laborious and time-consuming, and will hamper large-scale population studies [
51]. The IFC system is capable of acquiring cellular imagery at high flow rates from samples in suspension, reaching up to 1000 cells/s, making it faster than the automated microscopy systems and avoiding the need to create high quality slides [
52].
Overall, further development and validation of the IFC-based γ-H2AX assay system presented in this work will allow for evaluation of DNA damage and DSB repair capacity with increased resolution, sensitivity, accuracy and high-speed image acquisition as compared to traditional flow cytometry and traditional microscope immunohistochemical methods [
28,
30]. End-to-end automation of the IFC-based γ-H2AX assay can be achieved with the integration of our RABiT (Rapid Automated Biodosimetry Technology) platform for automated sample preparation from small volumes of blood [
35]. Measurements of individual DSB repair capacity within a large population could offer valuable information to advance this high-throughput assay for translational research such as monitoring risk and response among radiotherapy patients.
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