Assessing the level of DNA damage present in cells is integral to understanding the molecular mechanisms involved in the genome repair process and consequences for cell fate. Double-strand breaks (DSBs) in DNA can occur from exposure to exogenous genotoxic agents such as ultraviolet light, oxidative stress and chemical mutagens, as well as from normal cellular processes and discrete recombination reactions.1‒3 Defects in DNA damage pathways can result in a number of genomic instability disorders that can lead to heightened predisposition to neoplasia, making the ability to accurately measure DNA DSB critical to cancer and aging research.
Regardless of the cause of DNA damage, normal cell responses trigger either cell cycle checkpoint arrest and apoptosis or DNA repair. Phosphorylation of histone H2A.X is required for checkpoint-mediated cell cycle arrest and DNA repair following double-strand DNA breaks. During cellular response to DSBs, histone H2A.X is rapidly phosphorylated at Ser139 by ataxia telangiectasia mutated protein kinase (ATM), which is itself activated through phosphorylation on Ser1981.2 Although there are other kinases that regulate the checkpoint pathways in the DNA damage response, ATM is the primary mediator of the response to DNA DSBs that result from exposure to ionizing radiation. Cells lacking functional ATM have major problems in repairing DSBs and sustain major chromosome instability.4,5 Antibodies specific for H2A.X and ATM can therefore be used to detect DNA DSB at the cellular level.
Traditionally, DNA damage has been measured by Western blotting (WB) techniques using phospho-sensitive antibodies.6 WB requires the production of cell lysates, during which population heterogeneity is lost. Markers of DSB induction may vary on individual cells depending on such factors as cell cycle status, rendering WB and other methods that require homogenization of cell populations insensitive to characterization of individual cell status. Moreover, WB readouts are qualitative in that they do not provide the ability to evaluate cell damage responses with statistical power.
In contrast, cytometric methods for interrogating DNA damage provide multiparameter data with single-cell resolution.6,7 The Muse cell analyzer (EMD Millipore, Billerica, Mass.) is a benchtop instrument designed for three-parameter analysis of fluorescent populations. The integrated computer and software package includes a multicolor DNA damage module for rapid assessment of phosphorylated histone H2A.X and ATM on a cell-by-cell basis via capillary-based flow cytometry.
To assess the capacity for the system to quantitatively assess downstream markers of DNA DSB, phosphorylation of both H2A.X and ATM was evaluated in cells treated with the topoisomerase inhibitor etoposide. Analysis at the cellular level demonstrated the ability to reveal a time-dependent activation for each target as the exposure time to etoposide was prolonged. Similarly, ATM and H2A.X activation trends were assessed in HeLa cells exposed to UV irradiation, and results established the capacity of the system to quantitatively measure DNA damage secondary to physical as well as chemical triggers.
Antibodies with the ability to discriminate phosphorylated targets are useful for demonstrating whether cell populations display markers indicative of DNA damage, but WB is significantly limited by the capacity to return only a “yes or no” result based on a homogenized population. Results shown here demonstrate an enhanced capacity for a cell-based immunodetection approach to return quantitative DNA damage data following genotoxin exposure. A single multiplexed experiment was sufficient to provide multiparametric, quantitative data from two DNA damage markers.
Methods
Cell culture and treatment
HeLa cells were propagated in EBSS/MEM SH30244.02 (HyClone, GE Healthcare Life Sciences, Pittsburgh, Penn.) supplemented with 10% fetal bovine serum (FBS), 1% nonessential amino acids, 1% penicillin/streptomycin, 1% L-glutamine, 1% sodium pyruvate and 1% HEPES, and cultured in a humidified tissue culture incubator at 37 °C, 5% CO2. To model radiation exposure, cells were exposed to ultraviolet irradiation from a Stratagene Stratalinker 2400 UV crosslinker (Agilent Technologies, Santa Clara, Calif.) ranging from 25 J/m2 to 500 J/m2 to induce DNA DSBs. Chemically induced DSB damage was tested by treating cells with a 10-μM solution of the cytotoxic anticancer drug etoposide for 0, 1, 2, 4, 6 or 24 hours in standard tissue culture conditions.
Sample preparation
For cellular analysis, both treated cultures and untreated controls were detached using Accutase solution. Cells were washed, centrifuged to pellet and fixed in ice-cold fixation buffer, followed by washing and permeabilization in ice-cold buffer. Washed cell pellets were then incubated in darkness for 30 minutes at room temperature, with anti-phospho-specific H2A.X-PECy5 and anti-ATM-PE diluted 1:10 in assay buffer. All buffers were from EMD Millipore. WB was performed on lysates of etoposide-treated cells.
Data acquisition and analysis
For cellular analysis, test and control samples of 2 × 105 cells each were acquired using the Muse cell analyzer, and results were analyzed using the multicolor DNA damage module of the standard software analysis package.
Results
Multiparametric evaluation of DSB
Data obtained via traditional Western blot confirmed that this method was sensitive to DSB-induced phosphorylation of histone H2A.X in cells treated with the topoisomerase inhibitor etoposide (Figure 1a). However, this Western blot data is limited to confirming the presence or absence of phospho-H2A.X in a homogenized population, and the ability to quantitate protein based on a band image is not considered reliable.
Figure 1 ‒ Immunodetection by Western blot and flow cytometric analysis for markers of DNA double-strand breaks. a) Traditional WB of etoposide-treated cells with anti-phospho H2A.X can discriminate between the unphosphorylated and phospho-histone, but is limited to a single data parameter from a population aggregate. b) HeLa cells were exposed to 10 μM
etoposide for 1, 2, 6 or 24 hours to induce DNA damage, and were then stained with both anti-phospho-histone H2A.X (Ser139) and anti-phospho-ATM (Ser1981) antibodies in multiplex. Samples were acquired using the Muse cell analyzer and statistical results obtained using the multicolor DNA damage module of the built-in Muse software.In contrast, and based on gated controls, the flow cytometer could, at the single-cell level, differentiate cells expressing the target from those that do not. Statistical analysis of scatter plots on target phosphorylation versus cell size or phospho-ATM versus phospho-H2A.X demonstrated the quantifiable multiparametric evaluation of DSB in cells exposed to agents of DNA damage (Figure 1b).
Phosphorylation of H2A.X
Exogenous DNA damage can occur secondary to both genotoxic chemical and physical agents. According to Tanaka et al., the increase in intensity of phospho-H2A.X after cell irradiation is strongly correlated with the dose of radiation.6 To test this assertion, cell cultures were exposed to a gradient of UV irradiation doses ranging from 25 J/m2 to 500 J/m2.
Data obtained from the multicolor DNA damage software demonstrated that H2A.X phosphorylation increases proportionally with increased UV irradiation (Figure 2a). The degree of DSB as measured by phospho-H2A.X signal approximately doubled with a fourfold increase in irradiation, while phosphorylation of the histone approximately tripled with a 20-fold increase in exposure.
Figure 2 ‒ Phosphorylation of H2A.X quantified by cellular analysis of cultures exposed to UV irradiation and etoposide, a therapeutic cytotoxin. a) HeLa cells were exposed to UV irradiation of 25 J/m2, 50 J/m2, 100 J/m2 and 500 J/m2 and stained with anti-histone H2A.X and anti-phospho-histone H2A.X (Ser139) antibodies in multiplex. Using statistical analysis of events shown in the scatter plot (left panels), the degree of DSB as measured by phospho-H2A.X signal approximately doubles with a fourfold increase in irradiation, and phosphorylation of the histone is approximately tripled with a 20-fold increase in exposure. b) Signal from cells expressing H2A.X increases with exposure to etoposide over time, and nearly 100% of the target is phosphorylated after 24-hour exposure. Samples were acquired using the Muse cell analyzer and statistical results are shown alongside corresponding scatter plots.A similar quantifiable increase in phospho-H2A.X signal was observed with prolonged exposure to etoposide (Figure 2b). The observed kinetics of H2A.X phosphorylation were generally consistent with previously reported observations that exposure of cultured cells to UV-B light resulted in significantly increased H2A.X phosphorylation one hour after exposure.8 After 24 hours of exposure, nearly 100% of the target was phosphorylated.
Conclusion
DNA damage can occur by environmental exposure to physical means such as irradiation with UV or other wavelengths, or may be caused by chemical agents including select anticancer drugs.1,3 In addition, DNA is continuously subject to DSBs in the chromosome from by-products of normal cellular metabolism such as reactive oxygen species.1 Checkpoint systems have therefore evolved that promote DNA repair, the activation of which can be evaluated by assessing the degree of phosphorylation of histone H2A.X and the kinase ATM.2
Although Western blot is a viable method for H2A.X analysis, it is limited by the capacity to provide bulk population data and is not quantitative. The Muse cell analyzer provides real-time quantitative assessment of protein phosphorylation by laser-based detection of each cell event, and facilitates data interpretation with built-in software and a touchscreen interface that enables rapid acquisition and analysis. Because the system interrogates fluorescent signal from every cell in a sample of several thousand, it can simultaneously return statistically powerful quantitative data from each of two critical DNA damage markers at the single-cell level.
References
- Kaufmann, W.K. and Paules, R.S. DNA damage and cell cycle checkpoints. FASEB J. 1996 Feb, 10(2), 238‒47.
- Bakkenist, C.J. and Kastan, M.B. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 2003 Jan 30, 421(6922), 499‒506.
- Ewald, B.; Sampath, D. et al. H2A.X phosphorylation marks gemcitabine-induced stalled replication forks and their collapse upon S-phase checkpoint abrogation. Mol. Cancer Ther. 2007 Apr, 6(4), 1239‒48.
- Khalil, H.S.; Tummala, H. et al. Targeting ATM pathway for therapeutic intervention in cancer. BioDiscovery 2012 July 29, 1(3).
- Burma, S.; Chen, B.P. et al. ATM phosphorylates histone H2AX in response to DNA double-strand breaks. J. Biol. Chem. 2001 Nov 9, 276(45), 42,462‒7.
- Tanaka, T.; Huang, X. et al. Cytometry of ATM activation and histone H2A.X phosphorylation to estimate extent of DNA damage induced by exogenous agents. Cytometry A 2007 Sep, 71(9), 648‒61.
- Muslimovic, A.; Ismail, I.H. et al. An optimized method for measurement of gamma-H2AX in blood mononuclear and cultured cells. Nat. Protoc. 2008 Jun 26, 3(7), 1187‒93.
- Zhao, H.; Traganos, F. et al. Kinetics of the UV-induced DNA damage response in relation to cell cycle phase. Cytometry A 2010 Mar, 77(3), 285‒93.
Mark Santos and Wenying Zhang are part of the R&D Cell Analysis Group at EMD Millipore, 290 Concord Rd., Billerica, Mass. 01821, U.S.A.; tel.: 951-514-4538; [email protected]; www.emdmillipore.com