Flow Cytometric Analysis of Phosphorylated Histone H2AX Following Exposure to Ionizing Radiation in Human Microvascular Endothelial Cells

Flow cytometry/ γγγγ H2AX/Ionizing radiation/DNA damage/N-acetyl-cysteine. We applied a flow cytometric method to quantify IR-induced histone H2AX phosphorylation at serine 139 ( γ H2AX) and compared those values to those obtained using a standard microscopy based foci counting method. After PFA fixation, methanol permeabilization was suitable for both FITC- or Alexa647- γ H2AX. In contrast, Alexa647- γ H2AX was not suitable for ethanol permeabilization. Antibody concentrations at 1–2 µ g/ml yielded the highest γ H2AX positive percentage for both antibodies. Without DAPI staining, γ H2AX formation can be measured as a relative fold increase. Values determined by bivariant flow cytometric analysis and those obtained using microscopic foci formation exhibited a good quantitative correlation. Values obtained by both methods could vary according to the gating or threshold setting used. γ H2AX positive cells increased as a function of radiation dose (2–16 Gy) followed by a dose-dependent decay. The free radical scavenger N-acetyl-L-cysteine (NAC), if administered at a concentration of 4 mM 30 min before IR, was effective in reducing IR-induced γ H2AX formation in all phases of the cell cycle. We have developed a simplified and quantitative flow cytometry based method to measure IR-induced γ H2AX in cells and demonstrated strong correlation to values obtained by a standard automated digital microscopic foci analysis along with NIH ImageJ


INTRODUCTION
DNA double-strand breaks (DSBs) are potentially damaging events in cells that are highly mutagenic when misrepaired and lethal if left unrepaired. Ionizing radiation (IR) induces a broad spectrum of different types of DNA lesions including a variety of base damages, single-strand breaks (SSBs) and DSBs in cells. 1) Since DSBs are a major damaging lesion to the cells following IR, the ability to quantitatively measure them in a rapid manner would be extremely important for both biodosimetry and risk assessment applications. The measurement of γ H2AX as a marker of DSB damage offers several advantages over the other analytical methods such as neutral elutriation, pulsed field gel electrophoresis and neutral comet assays that are described in detail elsewhere. 2) Recently developed γ H2AX based analysis as a marker for DSBs is both relatively robust and practical to assess IRinduced damage in cells. Exposure to IR results in the rapid phosphorylation of a minor nucleosomal histone protein, H2AX, at the sites of DSB damage that precedes the involvement of repair enzymes involved in the processes of homologous recombination (HR) and non-homologous endjoining (NHEJ) DNA repair. [3][4][5] An important modification that occurs specifically at the site of DSB damage is the rapid phosphorylation of H2AX 5) covering about 2 Mb of DNA that surrounds the break. 6) This can be detected using a specific antibody. The number of γ H2AX foci in a nucleus is known to be directly proportional to the number of DSBs formed, and dephosphorylation coincides with DSB repair. 7) Thus, γ H2AX foci formation is a sensitive and specific marker for the detection of DSBs. [7][8][9] γ H2AX can be detected visually as microscopic foci. Other effective techniques include: immunoprecipitation, Western blotting, flow cytometry and laser scanning cytometry. Thus, there are a number of methods utilizing γ H2AX as a marker of DSBs available with each method having for its own merits and drawbacks. Although direct visualization of γ H2AX is probably the most specific and efficient technique for spotting DSBs in cells it is very time consuming. The measurement of γ H2AX within cells have here-to-fore largely relied on immunocytochemical approaches to detect microscopic foci formation to study the DNA repair mechanism(s), replication and recombination. [10][11][12] The intensity of γ H2AX immunofluorescence (IF) measured by cytometry has been reported to correlate with the dose of IR. 2,13,14) However, the frequency of γ H2AX detected varies as a function of the phase of the cell cycle. 15) This observation highlights the potential usefulness of flow cytometry in detecting γ H2AX. When combined with DAPI nuclear staining, not only can the frequencies of γ H2AX be integrated across the cell cycle, but specificity in quantization as a function of the specific phase of the cell cycle can be determined. Flow cytometric methods have been employed to assess drug-or UV-induced DNA damage related to apoptosis. [16][17][18][19][20] At present, however, assessment of IRinduced γ H2AX using flow cytometry is quite limited and usually expressed only in a relatively semi-quantitative manner. 2,13,15,21) A further improvement and refinement is needed to develop a more rapid and quantitative technique to measure IR-induced DNA DSBs.
To assess DNA DSB damage following IR, we applied the flow cytometric analysis for the detection of γ H2AX quantification and provided automated digital microscopic foci analysis using NIH ImageJ macro software (Appendix). We then compared these data with data obtained using flow cytometric analysis for IR-induced γ H2AX measurement combined with DAPI staining. Because about two thirds of the biologic damage induced by low linear energy transfer (LET) IR, such as X-rays, is through the generation of highly reactive free radicals, i.e., the indirect effect of IR on DNA, 22) we also investigated the role of a thiol containing free radical scavenger, N-acetyl-L-cysteine (NAC), on mediating the formation of IR-induced γ H2AX across all phases of the cell cycle.

Irradiation and a drug treatment conditions
Confluent HMECs were irradiated at room temperature by an X-ray generator (RT250, Phillips, Holland) operating at 250 kVp and 15 mA at a dose rate of 1.65 Gy/min. Radiation doses ranged from 0 to 16 Gy. NAC (Sigma) was dissolved in phosphate buffered saline (PBS; 8.1 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , 140 mM NaCl; Gibco/BRL) at a final concentration of 4 mM and was added to cells 30 minutes before irradiation and maintained throughout the procedure up to 1 hour after irradiation at which time the cells were harvested.

Cell preparation for γ H2AX analysis
HMECs were grown to confluency in 60 mm dish. The medium was changed at 24 hours before IR and/or NAC treatment. One hour after IR, cells were trypsinized with 1 ml EDTA/Trypsin, stopped with 1 ml complete medium and collected in 15 ml tubes. Paraformaldehyde (PFA, Sigma) was added at a final concentration of 2% and cells were fixed at 37 ° C for 10 min. Cells were then immediately chilled on ice, centrifuged, washed three times with 0.5% bovine serum albumin (BSA; EMD Bioscience, La Jolla, CA)-PBS, permeabilized with 70% ethanol in distilled water or 90% methanol in 0.5% BSA-PBS, and kept at 4 ° C for ethanol samples and at -20 ° C for methanol samples until further staining. Cells (1-2 × 10 6 ) were washed three times with 0.5% BSA-PBS, stained with FITC-γ H2AX antibody (Upstate, Charlottesville, VA) or Alexa647 (A647; Invitrogen, Carlsbad, CA)conjugated γ H2AX antibody (Upstate) for 1 hour at 4 ° C in the dark, and then washed three times with 0.5% BSA-PBS. Cells were counterstained with DAPI (1-2 µ g/ml 4',6-diamidino-2-phenylindole dihydrochloride hydrate (Sigma) in TBS (14.04 g tris HCl, 1.34 g tris base, 0.4 g MgCl 2 , 1 ml triton X 100, 6.5 g NaCl in 1 liter, pH7.4). Stained samples were analyzed either by flow cytometry or digital microscopic observation as described below.

γ H2AX measurement by flow cytometer
Cells stained with FITC-or A647-γ H2AX and DAPI were analyzed with a 3 laser LSRII (Becton Dickinson, San Jose, CA) using 488 nm excitation for FITC (530/30 BP filter), 633 nm excitation for Alexa647 (680/40 BP filter) and 355 nm excitation for DAPI (450/65 BP filter). Debris was excluded from the analysis by gating a forward scatter versus side scatter plot. Cell aggregates were excluded by gating a time of flight versus forward scatter plot. Integrated fluorescence measurements for FITC or Alexa647 and DAPI were recorded for 10,000 single non-debris events. Logarithmic FITC or Alexa647 fluorescence was plotted versus linear DAPI fluorescence using FlowJo analysis software (TreeStar, Ashland, OR). Unirradiated controls were used to set the threshold gating to determine the percentage of γ H2AX positive cells. Briefly, DNA content (DAPI) was plotted on the x-axis, placing the G1 peak at channel 100 for the background autofluorescence of FITC or Alexa647 was plotted on the y-axis and an intensity level of 100. Contour or dot plots were used to set the gating strategy. At least three independent gating measurements were performed using contour patterns and the data were presented as a mean with standard deviation (SD).

Quantification of microscopic γ H2AX foci formation
Microscopic γ H2AX foci were immunolocalized and imaged using an automated multiparameter digital image microscope using the same samples that were prepared for flow cytometry. Cells were counterstained with 1 µ g/ml DAPI and placed on a cover slip at the bottom of 35 mm dish. Cellular morphology and fluorescence were examined under an automated multiparameter digital image microscope (Olympus IX81), utilizing immersion oil with a 60x (N.A. 1.45) objective lens. The FITC and DAPI digital imagers were captured serially (MetaMorph, Universal Imaging Corp.), and more than 10 fields containing 100 cells were stored. Data were analyzed on a per nucleus basis using an NIH ImageJ software (v.2.34l) with a custom macro function designed to count particles. Nuclear boundaries and foci were automatically identified in images by a threshold algorithm on digitally processed images resulting in nuclear outlines along with the number of foci per cell and details of intensities and morphometry. The custom macro program for NIH ImageJ is available as an Appendix.

γ H2AX staining conditions for flow cytometric analysis
We first examined the staining procedure to determine the conditions that might affect the relative intensity of γ H2AX fluorescence. Confluent HMECs were irradiated with 8 Gy, harvested 1 hour after IR and fixed with 2% PFA. To assess the importance of the alcohol permeabilization step on fluorescence intensity, fixed cells were divided into two groups and separately permeabilized with either 70% ethanol or 90% methanol and then kept at 4 ° C or -20 ° C, respectively. Cells were stained with either FITC-or A647-γ H2AX antibodies, each at 4 µ g/ml concentration. Fig. 1a shows single stained FITC histograms derived under the various conditions. In general, histograms shifted to the right reflecting a higher γ H2AX intensity in 8 Gy irradiated as compared to control groups with the exception of the ethanol permeabilized A647-γ H2AX stained group. Under this experimental condition the histogram describing the irradiated cell population did not clearly separate from the untreated control background (Fig. 1a), making quantification of the γ H2AX cell population induced by IR difficult. The resulting histograms are characterized as monophasic. Therefore, the mean, geometric mean and/or median fluorescence ratio can be calculated using relative γ H2AX fluorescence intensities. This is done by taking the ratio of the histograms representing the irradiated to that of the con-trol cells (Fig. 1b). Although use of these three parameters appears to yield similar ratios, the use of the median values represents the best choice due to the monophasic characteristic of the peaks.
From these results, the permeabilization step using methanol as compared to ethanol is superior for both FITC or A647-γ H2AX staining. Both ethanol and methanol were equally effective in FITC-γ H2AX staining. The reason for this discrepancy in the use of the two alcohols is not apparent at present. It may be due to differences related to quenching of Alexa647, the antibody concentration used, or differential effects on PFA in the fixation process.

Bivariant flow cytometric analysis of γ H2AX with DAPIcombined staining
The use of single staining procedures to generate histograms reflecting γ H2AX lacked the precision to effectively generate and analyze sufficiently quantitative data. The addition of DAPI staining offered the advantage of assessing γ H2AX frequencies as a function of the phase of the cell cycle. This is important since it is known that background γ H2AX intensity also varies as a function of the cell cycle phase. 2,15) To analyze γ H2AX frequencies under conditions of mixed staining that included with DAPI, we used contour plotting to gate γ H2AX positive cells from the remainder of the population expressing background γ H2AX intensities. The intensity of FITC positive fluorescing cells was evaluated at 10% increments using contour plotting with the aid of Flow-Jo software. The resulting data are presented in both contour and the pseudo-color dot plots for comparison (Fig. 1c). A representative cell cycle distribution pattern is show in Fig.  1d. The results using this gating approach on the same samples are shown in Fig. 1e and 1f.
These data demonstrate the advantage of combining both γ H2AX and DAPI staining on the same population of cells to obtain quantitative data on γ H2AX formation as a function of the cell cycle as well as for better discrimination against variable cell cycle related background γ H2AX intensities. It is important to note that in this experiment we used HMECs grown to full confluence and assessed γ H2AX formation within 1 hour after IR exposure. Under these conditions no change in cell cycle distribution was observed.

γ H2AX antibody concentrations for bivariant flow cytometric analysis
Although the antibody concentration of 4 µ g/ml used in the above experiments was useful to discriminate the γ H2AX positive population of cells from the negative/background population as shown in Fig. 1, we wanted to determine if there is a more optimal antibody concentration for γ H2AX detection when DAPI is used in the combined staining method. First, we applied FITC-or A647-γ H2AX antibody concentrations ranging from 4 to 100 µ g/ml to conflu- ent HMECs irradiated with 8 Gy and assessed 1 hour after irradiation. The result was that a 4 µg/ml concentration of antibody was the most effective concentration with respect to measuring γH2AX frequencies for both of the fluorescent dye-conjugated antibodies used (data not shown). Next we evaluated both antibodies using confluent HMECs irradiated with 16 Gy. Antibody concentrations ranged from 1 to 8 µg/ ml. As shown in Fig. 2a and 2b, both antibodies were most effective in the 1-2 µg/ml concentration range. These concentrations yielded the highest values of γH2AX frequencies detected. The relative efficiency of detection decreased as the antibody concentration was increased from 4 to 8 µg/ml. These results also reflect a higher frequency of γH2AX positive cells following 16 Gy as compared to 8 Gy exposures (see Fig. 1). From these results we chose to stain for γH2AX using antibody concentrations in the 1 to 2 µg/ml range for subsequent bivariant flow cytometric analysis.

Automated digital microscopic foci analysis for γH2AX formation and its relationship to bivariant flow cytometric analysis
To determine whether values of γH2AX frequency asse-ssed using our flow cytometric method would correlate with those determined using a visual microscopy based method of detection, we utilized a standard cell/nucleus counting method. Cells were stained with FITC-γH2AX and DAPI. Identification and counting of positive cells containing γH2AX foci was made using the NIH ImageJ macro program which is provided as an Appendix. For this purpose we used the same cell samples scored by flow cytometry analysis to compare values obtained with the visual microscopy foci scoring method. Representative images obtained using an automated multiparameter digital image microscope are shown in Fig.  3a. The digitally captured images were further analyzed using the NIH ImageJ macro software. Unirradiated control HMEC images are shown at the top panel and images of cells irradiated with 8 Gy are shown in the lower panel. γH2AX foci and DAPI stained nuclei are shown in the left panel and the images used in automatic cell counting are shown in the right panel. The numbers reported represent positive cells for γH2AX as judged by numbers of foci counted representing values greater than the mean + 2SD as derived from the results presented in Fig. 3b. The distribution of background γH2AX foci/nucleus in untreated cells seems to fall below 10 with the average number being 3.6 with an SD of 3.9. If we take the mean + 2SD as a cutoff point, the upper limit of γH2AX in untreated cells as background number for future analysis is 11.4 foci per nucleus (Fig. 3b).
A comparison of the frequencies of γH2AX determined by the two methods for cells irradiated with 8 Gy is presented in Fig. 3c. γH2AX foci formation frequencies determined using the visual microscopy method with cutoffs set at two different SD (Mean + 2SD and +3SD) were compared with those obtained using flow cytometric analysis under two different gating conditions, i.e., 10% and 15% increments, respectively. The two methods yield substantially similar data with correlations of data sets being dependent on the respective threshold and gating values chosen.

IR-induced γH2AX and phase of cell cycle
A bivariant flow cytometric method was used to quantify the values of IR-induced γH2AX formation in confluent HMEC populations 1 hour following IR with doses ranging from 2 to 16 Gy (Fig. 4). Alterations in γH2AX frequency histograms (Fig. 4a), relative γH2AX frequencies (Fig. 4b), γH2AX single stained histograms (Fig. 4c), bivariant cell clustering distributions (Fig. 4d), radiation dose response of γH2AX formation (4e), and cell cycle distribution of γH2AX formation as a function of radiation dose (4f) are presented for comparative purposes and to describe the utility of this flow cytometric method in measuring γH2AX formation as a biomarker in irradiated cells. IR-induced γH2AX frequencies increased as a function of radiation dose up to 16  flattened suggesting a saturation of the system in this very high dose range. IR induced γH2AX formation in all phases of the cell cycle with the induction in G1 and G2 higher than that observed in S phase cells (Fig. 4f).

Kinetics of IR-induced γH2AX and its decay analyzed by bivariant flow cytometry
A bivariant flow cytometric method was further utilized to analyze both IR-induced γH2AX formation and its kinetics of decrease in confluent HMEC at 1, 4 and 24 hours following 2-16 Gy (Fig. 5). A radiation dose-dependent γH2AX increase followed by a decrease as a function of time were evident as shown in Fig. 5. Induced γH2AX returned to normal levels at 24 hours following IR exposure at every radiation dose used.

Inhibition of IR-induced γH2AX by a free radical scavenger NAC
The bivariant flow cytometric method was applied to determine the effects of a free radical scavenging agent on the induction of γH2AX in HMECs by IR. The free radical scavenger NAC was added to cultures of HMECs grown to confluence at a concentration of 4 mM for 30 min just prior to exposure to 8 Gy of IR. γH2AX formation was assessed 1 hour after irradiation. As shown in Fig. 5, NAC reduced not only the γH2AX formation in the population as a whole (Fig. 6a) but also in all phases of the cell cycle (Fig. 6b). Of considerable importance, the bivariant flow cytometric method also allowed for the determination that NAC exhibited a cell cycle phase-dependent radioprotective response (G1>G2>S) in an asynchronous population of cells.

DISCUSSION
Mammalian histone H2AX is a variant of H2A and accounts for approximately 2-10% of total histone H2A. 5,24) The kinases that phosphorylate H2AX are nuclear PIKKs (phosphatidylinositol 3-OH serine/threonine protein kinaselike kinases) that include ATM (ataxia telangiectasia mutated), 5,25,26) ATR (ATM and Rad3-related) 27) and DNA-PK (DNA-dependent protein kinase). 28,29) ATM and DNA-PKcs have been shown to phosphorylate histone H2AX in response to IR. 26,28,30,31) During DNA synthesis the stalled replication forks as well as UV-induced lesions are regulated prominently by ATR. 26,32,33) Under each of these conditions, the high concentration of γH2AX protein localized in chromatin adjacent to DSBs can be detected by IF giving rise to easily identifiable distinct foci. 4,7) It has been proposed that γH2AX may function as an anchor to hold broken DNA ends in close proximity in chromatin, facilitating the DNA repair. 34) As such, it has been reported that cells exhibiting enhanced radiosensitivity exhibit a relatively slower rate of γH2AX loss and a higher retention of γH2AX foci. 7,13,35) Because γH2AX foci appear within minutes after IR exposure, γH2AX focus formation is considered to be a sensitive and selective signal for identifying the existence of DSBs in cells. 24,36) However, γH2AX appears to be implicat- ed in a number of a cellular responses that include mitotic DSBs repair in yeast, 11) V(D)J recombination in lymphoid cells 10) and meiotic recombination in mice. 12) During DNA replication arrest 33) scheduled H2AX phosphorylation can occur as a result of intrinsic enzymatic processes. That can lead to a background frequency that is observed in untreated control cells. 17,21) Thus, not all γH2AX foci that are detected are due solely to exogenously induced DNA damage but may also reflect the byproduct of normal cellular processes.
There are a number of methods now in use to detect γH2AX foci in cells. These include Western blotting, immunoprecipitation, flow cytometry, laser scanning cytometry and visual microscopy based methods. Of these methods, measurement of γH2AX IF by multiparameter flow cytometry 2,20) is particularly advantageous. Large numbers of cells can be rapidly analyzed and the data can provide information relating to γH2AX incidence as a function of cell cycle distribution, the identification of rare cell subpopulations that might otherwise go undetectable by techniques such as Western blotting or microscopy. However, there are several important factors that must be addressed if cytometric techniques are to be used to quantitatively detect and analyze γH2AX incidence in IR exposed populations.
A potential confounding factor is the induction of the apoptotic process in radiation exposed cells. A number of papers have reported that the relative intensity of γH2AX IF measured by flow cytometer increases with the dose of IR or toxic chemicals used. 2,13,20,21) Following exposure to these deleterious agents both damaged mitotic cells and cells undergoing apoptosis can express very high levels of γH2AX. 4,37) The degree of H2AX phosphorylation in response to DSBs generated during apoptotic process has been reported to be many-fold higher compared with that representing initial or primary DSBs induced by damaging agent. 19) The appearance of early apoptotic cells are easily identified in flow cytometric generated histograms and appear as a subpopulation cluster expressing 5 to 10 fold higher levels of γH2AX than are observed in non-apoptotic cells. 15) This is due to the extensive DNA fragmentation that occurs during an apoptotic process. Thus, the challenge is to identify and dissociate the damage component occurring from the apoptotic process from that induced directly by the genotoxic agent such as IR. 20) As demonstrated in the histograms presented in Figs. 1a and 4a, cells exhibiting γH2AX formation following IR clearly overlap regions known to represent untreated control cells. Regardless, it is clear that the frequency of γH2AX foci increased with increasing doses of radiation. 13,15) However, when we used the geometric mean, mean or median of these histograms we were able to calculate values representing a fraction of induced the γH2AX as the fold increase over the untreated population ( Fig. 1a and Fig. 4a) resulting in a clear IR-dose response pattern (Fig. 4b). The relative level of γH2AX is defined as the ratio of the detectable γH2AX IF signal measured from the entire histogram from the irradiated populations to that of the unirradiated control. Ratios were obtained using comparisons of the geometric means, means or medians of the respective histograms. These parameters are easily calculated using FlowJo software as shown in Fig. 1b and Fig. 4b. Although each of these three parameters gave rise to qualitatively similar results, a comparison of median values was chosen as the most appropriate way to analyze these distribution histogram analyses. The median value approach has the advantage of not being heavily weighted by outliers as the mean value is. This is especially important when the frequency of positive γH2AX cells at a relatively low percentage as might be expected following exposure to low doses of IR.
In addition to the technical difficulty of gating due to the overlapping histograms, other biologically based considerations should be addressed. Cells in each phase of cell cycle will express fluorescence from γH2AX foci, with the intensity varying as a function of position within the cell cycle phase. 15) Adjustment must be made to address this phenomenon to maximize the quantization of the flow cytometric method for γH2AX detection. 13,15) Not only that, the level of the substrate H2AX is reported to be variable between different cell types and is dependent on total DNA content. 5,15) Thus, not only the DNA content doubles during cell cycle progression, but histone content also doubles throughout the cell cycle. Therefore cells in S and G2/M have 1.5-and 2fold higher levels of H2AX than G1 cells 15) while the ratio of histone to DNA content remains invariable in cells. 38) In the case of stalled replication forks, unlike IR, H2AX phosphorylation is regulated prominently by ATR. 32) This also occurs following exposure of cells to UV. 26,33) Stalled replication forks and UV-induced DNA lesions can result in DSBs and γH2AX formation predominantly in S phase cells. 20) However, the intrinsic or background γH2AX are generally smaller in size and less distinct as compared with foci that are induced by genotoxic agents. 2,17) UV-induced γH2AX in S phase cells are reported as resembling those induced by DNA topoisomerase inhibitors that can trigger apoptosis. 19) These examples illustrate the importance of including cell cycle analysis to maximize the quantitative power of flow cytometric assays in the detection of γH2AX formation frequencies following IR exposure of cells.
We quantitatively evaluated and compared the methods of automated digital microscopy and flow cytometry to detect γH2AX frequencies in cells treated under the same conditions. The automated digital microscopy method is generally well accepted for the detection of γH2AX foci because cells expressing this damage can be readily identified and thresholds for determining positive cells are easily set. As demonstrated in Fig. 3a, b, c both methods gave similar results. The automated digital imaging method we employed also included the use of NIH ImageJ macro software to assist in setting the appropriate thresholds. It is important to note that corre-lation between the two methods is highly dependent upon the settings of the respective threshold or gating values used (Fig. 3c).
The frequency of γH2AX formation as reflected by the intensity of IF increased linearly with the amount of damage induced 7) but the accuracy of measuring discrete foci (size, number and intensity) in whole nuclei could be compromised somewhat when doses higher than 2 Gy (50 or so breaks per diploid cell) are used. 2,13) This can be a limiting factor in the use of visual based microscopy based techniques to quantitatively measure γH2AX frequencies at higher radiation doses.
While flow cytometry based approaches may lack some of the specificity in γH2AX detection that can be attained with visual/digital based microscopy methods, they offer an advantage for rapid and mass screening of DNA damage in a quantitative manner. Not only can these approaches be used to routinely measure and evaluate in a rapid manner drug effects or radiation induction of γH2AX formation, but cell cycle dependent effects can also be measured at the same time. It is known that DNA repair processes involving HR and NHEJ are dependent upon cell cycle phase. 39) If these are involved in the removal of γH2AX, flow cytometric approaches will be highly useful in assessing the kinetics of damage removal as a function of position in the cell cycle especially since most of these studies are performed using asynchronously growing populations of cells.
We have applied the present method routinely at radiation doses ranging from 1-8 Gy using other cell lines such as Chinese hamster AA8, several human head-and-neck cancer cell lines and human colorectal cancer cell lines, each exhibiting a strong radiation dose response. Fig. 5 is a representative plot of γH2AX kinetics.
The background level of γH2AX is both cell-type-dependent and cell-phase-dependent; levels of γH2AX in G0/G1 phase cells are about three times lower than cells in S and G2 phases. 15) Although γH2AX foci formation occurs both in non-replicating 5,24) and replicating DNA, 27) histone H2AX phosphorylation has been reported to be more robust in cycling cells as compared to the cells arrested in G1. 15,40) For these reasons we limited our current study to assessing γH2AX under a confluent condition. γH2AX formation induced by IR was observed as expected in all phases of the cell cycle ( Fig. 4d and 4f). S phase cells exhibited a lower frequency of γH2AX formation as compared to G1 or G2/M phase cells (Fig. 4f). Furthermore, the magnitude of γH2AX formation induced by IR could be reduced with the use of a radioprotective drug. One such drug, NAC, if added to cells prior to IR, resulted in a reduced frequency of γH2AX formation in all phases of the cell cycle (Fig. 6b). NAC is known to protect cells by scavenging ROS. 41) We have developed and contrasted to a well established digital microscopy based technique and a flow cytometric based method. Using bivariant flow cytometric method it was possible to detect and quantify in a rapid manner the frequency of γH2AX formation induced by IR. Quantification of γH2AX formation could be determined not only as a function of radiation dose but also as a function of cell cycle position.