Radiation dose determines the method for quanti fi cation of DNA double strand breaks

Ionizing radiation induces DNA double strand breaks (DSBs) that trigger phosphorylation of the histone protein H2AX (γH2AX). Immunofl uorescent staining visualizes formation of γH2AX foci, allowing their quantifi cation. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fl uorescent signal in each single cell. In practice there are problems in quantifi cation of γH2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fl uorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to γ-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofl uorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany) microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany). Obtained results show that the level of γH2AX is time and dose dependent. Immunofl uorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of γH2AX foci.


INTRODUCTION
The most important direct damages induced by ionizing radiation (IR) are DNA double strand breaks (DSBs).As a consequence of DSBs the histone H2AX becomes phosphorylated (Mischo et al. 2005).Phosphorylated H2AX (γH2AX) foci appear at the damage sites and their number is considered as one to one DNA DSB (Schmid et al. 2012).The level of γH2AX has been used in in vitro studies as a tool for the estimation of cellular radiosensitivity and as a biomarker for individual studies in patient trials to measure DNA damage following radiotherapy and/or chemotherapy (Qvarnström et al. 2004).
In practice, the obtained results may be different when various detection systems are used (Ivar et al. 2010).Effects of cancer therapeutics are often analyzed by immunocytochemistry (ICC), fl ow cytometry and immunoblotting (Western blot) TANJA BULAT et al. in order to profile the activity of cell signaling proteins.However, the relative sensitivity and specificity of these methods has not yet been established (Lorincz and Nusser 2008).All of the above techniques are based on the particular chemical interaction with specifi c antibody.Fluorescence microscopy is frequently used in cell biology because of its sensitivity and the ability to stain and visualize specific parts of the cell, while the light microscopy is not suitable for the detection of weakly stained cellular particles (Fritschy and Härtig 2001).In fluorescence microscopy, there is no background light and the fl uorescently stained object is being contrasted to a black background.Fluorescent molecules absorb the light of one particular wavelength that produces excitation of electrons, thus emitting the light of another but higher wavelength (Radek et al. 2014).The fl uorescence microscope takes advantage of this difference, fi ltering out the excitation light.Consequently, only the emitted light is seen (Petty 2007).Immunofluoresence microscopy can be used in several microscope designs for the analysis of immunofl uorescence samples.The simplest is the epifluorescence microscope, while confocal microscope is widely used.Moreover, there are powerful super resolution microscopes capable of detecting of nanostructures.
One of the crucial steps in the preparation of samples for immunofl uorescence microscopy is fixation.The goal of fixation is to maintain cellular structure as close as possible to the native state (Bancroft and Gamble 2008).Once fi xed, it is possible to carry on with the procedure without losing the proteins of interest and the rest of the cell.There are various fi xation methods suitable for immunocytochemistry.Fixation can damage or mask antigenic sites, thereby compromising the intensity of immunostaining (Carson and Hladik 2009).Therefore, the choice of fi xative and fi xation protocol may depend on the additional processing steps and biological endpoints.Flow cytometry is the method which can rapidly detect presence or absence of the protein of interest in the large cell population.Moreover, multi-parameter analysis of the cytometric data makes it possible to correlate them with other attributes of the cells, such as the cell cycle distribution (MacPhail et al. 2003, Yoshida et al. 2003, Huang et al. 2004, Huang and Darzynkiewicz 2006).However, during sample preparation one of the requirements is serial centrifugation.This can cause important cell loss, thus reducing the quantity of sample.Based on that, it is advisable to compare data obtained by this method with results obtained by other assays.
Despite its overall simplicity, Western blot has been proven to be a powerful procedure for immunodetection of proteins, especially those that are of low concentration (Geilfus et al. 2010).This procedure is used in combination with other important antibody-based methods such as immunocytochemistry to provide confirmation of the results both in research and diagnostic trials.Specificity of antibodies used for ICC is of crucial importance and therefore Western blot is essential to address antibodies specifi city (Kurien et al. 2011).Literature data suggest that all mentioned methods have limitations in sensitivity but they do enable consistent characterization of protein phosphorylation and activation of specifi c pathways.
In this study the human HTB140 melanoma cells were chosen as a model system to disclose the formation of DNA DSBs after irradiation with γ-rays through different experimental approaches.Therefore, optimization of experimental conditions for Western blot and immunofluorescence microscopy was performed.This optimization involved two criteria: the dose and post-irradiation incubation time.Flow cytometry was not taken into account because it was not considered as the dose-time-dependent method (Yoshida et al. 2003).The results obtained by ICC and Western blot were compared.The aim was to analyze which method is more suitable for the detection of γH2AX in samples irradiated under the above mentioned conditions.Moreover, data obtained by two types of microscopes: AxioVision microscope comprising an ApoTome software and AxioImagerA1 microscope were compared in order to analyze infl uence of microscope performances on the result.

CELL CULTURE
Human HTB140 melanoma cells were obtained from the American Tissue Culture Collection (ATCC, Rockville, MD, USA) and cultured in the RPMI 1640 cell culture medium supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) and penicillin/streptomycin (Sigma-Aldrich Chemie GmbH), in a humidifi ed atmosphere of 5% CO 2 at 37 °C (Heraeus, Hanau, Germany).

IRRADIATION CONDITIONS
Exponentially growing HTB140 cells were irradiated with γ-rays using 60 Co source at the Vinča Institute of Nuclear Sciences in Belgrade, Serbia.Applied doses ranged from 1 to 16 Gy, at the average dose rate ~ 1 Gy/min.Irradiations of cells were carried out in air at ~ 0 °C.

IMMUNOFLUORESCENCE MICROSCOPY
Cells attached to coverslips were washed once with cold PBS and fi xed with ice cold methanol-acetone (ratio 1:1) for 15 min, then blocked with 5% bovine serum albumin (fraction V, Sigma-Aldrich Chemie GmbH) in PBS for 60 min at room temperature then incubated with 1:500 dilution of the primary Alexa Fluor® 488 anti-H2AX-Phosphorylated (Ser139) Antibody (BioLegend), at 4 ºC overnight.After fi ve washings in PBST, lasting 3 min each, cells were counterstained with Dapi-antifade solution (Sigma-Aldrich Chemie GmbH).The visualization of cells was done using two microscopes: AxioVision (Carl Zeiss) comprising ApoTome software and AxioImager A1 (Carl Zeiss) with a 100 x objective.All images were captured using AxioVision software.Before the observation of foci, digital images were processed with ImageJ to adjust brightness and contrast.

AUTOMATED DIGITAL IMAGE ANALYSIS
CellProfi ler (www.cellprofiler.org) is an open source software that is used for a custom and automated approach to image analysis workfl ow (Kamentsky et al. 2011).This software enables objective and quantitative fl uorescence microscopy, based on userdefi ned thresholds for brightness, size, shape and other parameters (Carpenter et al. 2006).The image was converted from color (Aleksa Flour 488 -green, DAPI -blue) to monochrome.Foci were identifi ed with objects area which range from 6-30 pixels, while others were discarded.Data were exported to Excel for further analysis.Automated segmentation of foci was visually compared against manually counted foci.Relative fl uorescence was measured as described previously (Burgess et al. 2010).

STATISTICAL ANALYSES
Duplicate measurements were made during each experiment and all experiments were repeated at least three times.For immunofluorescence microscopy and Western blot analysis results were presented as the mean ± SEM (Standard Error of the Mean).

RESULTS
In order to follow the formation of γH2AX foci, the HTB140 melanoma cell monolayers were irradiated with γ-rays.Applied dose range was from 1 to 16 Gy.Cells were maintained under standard conditions from 30 min to 6 h after irradiation.
The dose-and time-depended phosphorylation of H2AX was followed for all time points and doses.The samples were visualized and then subjected to the automated digital image analysis with CellProfi ler software.Software parameters were optimized according to the properties of the cell line (size and shape of nuclei), size and shape of foci, characteristics of micrographs, etc. (Carpenter et al. 2006).In order to optimize software parameters, manual counting of γH2AX foci in sample irradiated with 2 Gy was used as the control measurement for computer counting.All time points were taken into consideration.Data obtained by both ways of counting are shown in Figure 1.doses of 12 and 16 Gy.For these two doses values obtained by AxioImager A1 microscope were low, thus are not given in the fi gure.
It has been noticed that higher radiation doses have weaker effects on DNA, as observed through the number of foci per nucleus.In order to perform further analysis of the results, the relative fluorescence was measured for threshold doses.According to the results presented in Figure 2, threshold doses are 12 and 8 Gy for AxioVision containing ApoTome software and AxioImager A1, respectively.From the results presented in Figure 3, representing relative fluorescence that was evaluated for threshold doses, it can be seen that 12 and 8 Gy have present the expected higher effect on the formation of the DSBs than 1 Gy of γ-rays.
For the Western blot analysis the cells were incubated for 30 min to 24 h after irradiation.The increase of the γH2AX started 30 min after irradiation and was detected throughout of doses applied (Fig. 4).The acquired data for other post irradiation incubation times exhibited similar dose dependence (data not presented).foci was attained 30 min after irradiation (Fig. 5).Again, the results obtained from AxioImager A1 resulted in lower values.
Nevertheless, for the same dose (2 Gy) Western blot analysis did not show time dependence of γH2AX formation (appearance / disappearance).In the samples irradiated with 12 Gy the highest induction of γH2AX was reached 2 h after irradiation, while it was signifi cantly reduced 6 h post-irradiation (Fig. 6).
In order to investigate the unexpected weak effect for higher doses (12 and 16 Gy, Fig. 2), despite high immunofl uorescence (Fig. 3), further analyses of images were performed.Among analyzed micrographs the one that was chosen is shown in Figure 7, presenting the sample irradiated with 12 Gy γ-rays.Lots of overlapping signals are most likely the reason of underestimation of the results presented in Figure 2.
CellProfiler software outlines foci and provides data that characterize each of them.The outlined γH2AX foci in the HTB140 cell sample irradiated with 2 Gy γ-rays obtained by AxioVision having ApoTome software (a) and AxioImager A1 (b) microscopes, shown in Figure 8. Due to the features of ApoTome software the real number of foci per nucleus was acquired and is presented in Figure 8a.However, on the micrograph obtained by AxioImager A1 presented in Figure 8b, only the foci that are in the focused object plane were recorded, while the others were detected as blurred structures.

DISCUSSION
The detection of γH2AX foci that refl ect dynamics of the DNA DSB repair was analyzed after irradiation with low and high doses of γ-rays.The human HTB140 melanoma cells were used as a model system.Experimental conditions and data analyses were optimized according to the methods that were used.
Immunofl uorescence microscopy enabled in situ visualization of each DNA DSB separately, as well as the distribution of breaks inside the nucleus of a single cell (Hu et al. 2005).Before counting, CellProfiler image analysis software was optimized.The software establishes whether the object that is analyzed is an individual one or if there are two or more clumped ones.This is a very important initial parameter which determines appropriate module by recognizing primary (nuclei) and secondary objects (foci).Most options within the module use this estimation of the size range of the objects in order to distinguish them from the noise in the image.Since pixels are used as units in CellProfiler, it is easy to zoom in on objects and determine typical diameters (Carpenter et al. 2006).When the software parameters are correctly set the results obtained by software and by manual counting of foci are compatible (Fig. 1).
During capture of images for the analysis with AxioImager A1 microscope each image consists of signal contributions coming from the focused object plane.However it also includes contributions coming from the structures located above and below it.The blurred structures from above or below are either recognized to be out of focus or, if they are clearly outside the focal plane, they give the effect of brightening the image background (Ju et al. 2009).With the AxioVision comprising ApoTome software, a grid structure is inserted into the light fi eld diaphragm plane of the refl ected-light beam path.Advantage of this system, as compared to AxioImager A1, is its possibility to precisely make an image of the object plane.This is done by a high precision scanning mechanism that moves the grid pattern in defi ned steps across the sample plane.The result is a precise optical section through the specimen with no blurring and with high contrast (Conchello and Lichtman 2005).Different performances of the microscopes explains some discrepancy in the number of foci detected with AxioVision having ApoTome software and AxioImager A1, presented in Figures 2 and 5.For higher doses, clumped objects that are shown in Figure 7 most probably cause underestimation of the number of foci.This can be misinterpreted as a weaker effect of irradiation.Image analyses through relative fl uorescence (Fig. 3) indicate the need for parallel analysis on at least two different approaches for the same method, in this case for ICC.In combination with Western blot analysis, ICC confi rms the assumption that results for higher doses presented in Figure 2 are underestimated, thus the doses of 8 and 12 Gy have stronger effects on the DNA level than the dose of 1 Gy of γ rays.
For analyzed cell system AxioVision with ApoTome software and AxioImager A1 are valid for up to 8 and 6 Gy, respectively.Differences in counting of foci by the two microscopes are illustrated in Figure 8 where the outlined γH2AX foci correspond to the previously defi ned parameters in CellProfi ler.In images captured with the AxioVision with ApoTome software there is negligible number of discarded γH2AX foci, due to the ability of this software to make precise optical sections, thus giving higher number of foci per nucleus.
Quantifi cation of phosphorylation of H2AX protein as a function of dose is also possible by Western blot analysis (Fig. 4), using actin for normalization purpose (Gaba et al. 2010).However, the analysis could not be fully attained with this method when using lower radiation doses at different post-irradiation incubation times (Fig. 6).In the Western blot assay, the protein to be quantifi ed is coming from a large number of cells.Therefore, this method is not appropriate to provide the precise information about the level of γH2AX per single cell.On the other hand, since the irradiation with doses of 1 or 2 Gy caused lower level of phosphorylation of H2AX, the intensity of corresponding bands obtained by the Western blot do not follow the real changes in the γH2AX kinetic (Fig. 6).Yet, the Western blot analysis was very useful for the analysis of phosphorylation of H2AX after irradiation with higher doses when the overlapping of signals disabled reliable quantification using immunofluorescence microscopy.Moreover, Western blot is a favorable method when following different cellular pathways triggered by radiation or other treatments (Wang et al. 2013).
In summary, since γH2AX is considered to be a sensitive indicator of DNA DSBs, the development of different γH2AX focus formation assays and the corresponding data analyses are of great experimental and even clinical importance.The described experimental techniques are becoming powerful tools for further studies of the cellular response to DNA damage.The combined use of ICC and Western blot analysis with respect to the dose level and other properties of a toxic agent, provide data about cellular processes important for the maintenance of genome stability.

Figure 1 -
Figure 1 -Mean number of γH2AX foci per HTB140 cell nucleus, counted by computer software (full square) or manually (full triangle), as a function of time elapsed after irradiation with 2 Gy γ-rays (C-control).Data obtained from 3 experiments are presented as mean ± SEM.

Figure 2 -
Figure 2 -Mean number of γH2AX foci per HTB140 cell nucleus as a function of dose, 30 min after irradiation with γ-rays, measured by two types of microscopes: AxioVision comprising ApoTome software and AxioImager A1 (C-control).Data obtained from 3 experiments are presented as mean ± SEM.

Figure 3 -
Figure 3 -Relative fluorescence, given as percentage of control, estimated with the microscopes AxioImager A1 and AxioVision comprising ApoTome software for the dose of 1 Gy each and the threshold doses of 8 Gy and 12 Gy, respectively.Data obtained from 3 experiments are presented as mean ± SEM.

Figure 4 -
Figure 4 -Dose dependent phosphorylation of H2AX protein in HTB140 cells, given as percentage of control and analyzed by Western blot, 30 min after irradiation with γ-rays (C-control).Data obtained from 3 experiments are presented as mean ± SEM.

Figure 5 -
Figure 5 -Kinetics of appearance and disappearance of γH2AX foci (time dependent mean number of foci) per HTB140 cell nucleus after irradiation with 2 Gy γ-rays (C-control).Quantifi cation was performed with the microscopes AxioVision comprising ApoTome software and AxioImager A1.Data obtained from 3 experiments are presented as mean ± SEM.

Figure 6 -
Figure 6 -Time dependent phosphorylation and dephosphorylation of H2AX protein in HTB140 cells, given as percentage of control and analyzed by Western blot, after irradiation with 2 and 12 Gy γ-rays (C-control).Data obtained from 3 experiments are presented as mean ± SEM.

Figure 8 -
Figure 8 -Illustration of differences in counting of γH2AX foci per HTB140 cell nucleus irradiated with 8 Gy γ-rays, obtained by the microscopes AxioVision comprising ApoTome software (a) and AxioImagerA1 (b).Objects of interest were outlined using CellProfi ler image analysis software that was optimized for the analyses.