The presented method combines the quantitative analysis of DNA double-strand breaks (DSBs), cell cycle distribution and apoptosis to enable cell cycle-specific evaluation of DSB induction and repair as well as the consequences of repair failure.
The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer treatments as used in clinical oncology. It enables a quantitative and longitudinal analysis of the DNA damage response after genotoxic stress, as induced by radiotherapy and a multitude of anti-cancer drugs. The method covers all stages of the DNA damage response, providing endpoints for induction and repair of DNA double-strand breaks (DSBs), cell cycle arrest and cell death by apoptosis in case of repair failure. Combining these measurements provides information about cell cycle-dependent treatment effects and thus allows an in-depth study of the interplay between cellular proliferation and coping mechanisms against DNA damage. As the effect of many cancer therapeutics including chemotherapeutic agents and ionizing radiation is limited to or strongly varies according to specific cell cycle phases, correlative analyses rely on a robust and feasible method to assess the treatment effects on the DNA in a cell cycle-specific manner. This is not possible with single-endpoint assays and an important advantage of the presented method. The method is not restricted to any particular cell line and has been thoroughly tested in a multitude of tumor and normal tissue cell lines. It can be widely applied as a comprehensive genotoxicity assay in many fields of oncology besides radio-oncology, including environmental risk factor assessment, drug screening and evaluation of genetic instability in tumor cells.
The goal of oncology is to kill or to inactivate cancer cells without harming normal cells. Many therapies either directly or indirectly induce genotoxic stress in cancer cells, but also to some extend in normal cells. Chemotherapy or targeted drugs are often combined with radiotherapy to enhance the radiosensitivity of the irradiated tumor1,2,3,4,5, which allows for a reduction of the radiation dose to minimize normal tissue damage.
Ionizing radiation and other genotoxic agents induce different kinds of DNA damage, including base modifications, strand crosslinks and single- or double-strand breaks. DNA double-strand breaks (DSBs) are the most serious DNA lesions and their induction is key to the cell killing effect of ionizing radiation and various cytostatic drugs in radiochemotherapy. DSBs do not only harm the integrity of the genome, but also promote the formation of mutations6,7. Therefore, different DSB repair pathways, and mechanisms to eliminate irreparably damaged cells like apoptosis have developed during evolution. The entire DNA damage response (DDR) is regulated by a complex network of signaling pathways that reach from DNA damage recognition and cell cycle arrest to allow for DNA repair, to programmed cell death or inactivation in case of repair failure8.
The presented flow cytometric method has been developed to investigate the DDR after genotoxic stress in one comprehensive assay that covers DSB induction and repair, as well as consequences of repair failure. It combines the measurement of the widely applied DSB marker γH2AX with analysis of the cell cycle and induction of apoptosis, using classical subG1 analysis and more specific evaluation of caspase-3 activation.
The combination of these endpoints in one assay not only reduces time, labor and cost expenses, but also enables cell cycle-specific measurement of DSB induction and repair, as well as caspase-3 activation. Such analyses would not be possible with independently conducted assays, but they are highly relevant for a comprehensive understanding of the DNA damage response after genotoxic stress. Many anti-cancer drugs, such as cytostatic compounds, are directed against dividing cells and their efficiency is strongly dependent on the cell cycle stage. The availability of different DSB repair processes is also dependent on the cell cycle stage and pathway choice which is critical for the repair accuracy, and in turn determines the fate of the cell9,10,11,12. In addition, cell cycle-specific measurement of DSB levels is more accurate than pooled analysis, because DSB levels are not only dependent on the dose of a genotoxic compound or radiation, but also on the DNA content of the cell.
The method has been used to compare the efficacy of different radiotherapies to overcome resistance mechanisms in glioblastoma13 and to dissect the interplay between ionizing radiation and targeted drugs in osteosarcoma14,15 and atypical teratoid rhabdoid cancers16. Additionally, the described method has been widely used to analyze side effects of radio- and chemotherapy on mesenchymal stem cells17,18,19,20,21,22,23,24, which are essential for the repair of treatment-induced normal tissue damage and have a potential application in regenerative medicine.
1. Preparation
2. Sample Collection
3. Washing and Staining
4. Measurement
5. Data Evaluation
Human U87 or LN229 glioblastoma cells were irradiated with 4 Gy of photon or carbon ion radiation. Cell cycle-specific γH2AX levels and apoptosis were measured at different time points up to 48 h after irradiation using the flow cytometric method presented here (Figure 3). In both cell lines, carbon ions induced higher γH2AX peak levels that declined slower and remained significantly elevated at 24 to 48 h compared to photon radiation at the same physical dose (Figure 4A). This indicated that carbon ions induced higher peak levels of DNA double-strand breaks (DSBs) than photons that were repaired less efficiently. For both radiation types, the γH2AX levels were highest in G1 cells, probably because DSB repair is limited to the pathway of non-homologous end-joining at this stage of the cell cycle.
In line with the higher DSB induction rate and slower repair kinetics, carbon ions induced a stronger and longer-lasting cell cycle arrest in G2 phase (Figure 3B) and a higher rate of apoptosis than photons (Figure 4C). Carbon ion radiation may therefore help to overcome radioresistance mechanisms observed in glioblastoma upon classical radiotherapy with photons (see Lopez Perez, et al.1 for detailed discussion).
The results shown in Figure 4 are examples of an optimal outcome of this method, showing clear differences between untreated and irradiated cells and statistically significant differential effects between different treatments. In cases where induction of DSBs and apoptosis is less clear, it is important to include positive controls. As shown here, cells fixed 1 h after photon irradiation are good positive controls for γH2AX induction. Photon radiation is also known to efficiently induce apoptosis in lymphocytes, which makes them useful as positive controls for apoptosis 2-4 days after irradiation. Another possibility is to use drugs for apoptosis induction, such as the proteasome inhibitor MG132 or a death receptor ligand (Figure 5).
Another important aspect for an optimal outcome of the assay is the quality of the cell cycle profiles. In Figure 4B the G1 and G2 peaks were narrow and clearly separated, making it easy to apply cell cycle modelling and to define the gates for cell-cycle-specific measurement of γH2AX. Depending on the cell type and the strength of the treatment effect (Figure 6A,B), the resolution of the different cell cycle phases can be significantly worse. In some cases, the DAPI concentration has a big impact on the quality of the cell cycle profile and needs to be adjusted (Figure 6C). In cases where no clear G1 peak is detectable due to the treatment, the cell cycle modelling tool cannot be applied accurately. It is advisable then to only use manual gating in the DAPI-A versus DAPI-W plot based on controls with a well-defined cell cycle profile, as described in the protocol.
The analysis of a cell cycle profile without a clear G1 peak can be further complicated if the treatment leads to low cell numbers that result in large shifts in the overall DAPI signal strength. In this situation, it can be difficult to judge, if a peak is the G1 or the G2 peak. To avoid this problem, the cells can be counted with a Neubauer chamber or by other means after the last washing step (step 3.3) and the cell numbers can be adjusted. The peak positions in the treated cells will then match with the controls.
Figure 1: Worksheet setup for sample acquisition. Plots and gates for signal display in the Worksheet window of the flow cytometer software (see the Table of Materials). Please click here to view a larger version of this figure.
Figure 2: Gating strategy for data evaluation. Population tree and diagrams showing how the gates are set to define the different sub-populations in the flow cytometric analysis software. DJF refers to the cell cycle modelling tool using the Dean-Jett-Fox method25,26. Please click here to view a larger version of this figure.
Figure 3: Raw data export table. Setup of the 'Table Editor' for raw data export in the flow cytometric analysis software. Please click here to view a larger version of this figure.
Figure 4: DNA damage response of human U87 and LN229 glioblastoma cells after irradiation with 4 Gy of photon versus carbon ion radiation. (A) DSB induction and repair measured by γH2AX levels. The median fluorescence intensity was normalized to the relative DNA content in each cell cycle phase (G1 = 1.0, S = 1.5, G2 = 2.0) and control levels were subtracted. Symbols indicate mean and error bars indicate SD values. Solid lines represent fits according to the function I(t) = (p1*t² + p2*t) / (t² + q1*t + q2) | t ≥ 0, p1 < 0, p1, q1, q2 > 0. (B) Cell cycle distribution (mean and SD) and representative histograms of the DNA content marker DAPI at 24 h after photon or carbon ion irradiation. (C) Apoptosis induction, as measured by caspase-3 and subG1 positive cells (mean and SD). *P < 0.05, **P < 0.01, ***P < 0.001 (two-sided Student's t test). This figure has been modified from Lopez, et al.1 with permission. Please click here to view a larger version of this figure.
Figure 5: Drug-mediated apoptosis-induction in U87 glioblastoma cells. U87 cells were treated with 5 ng/mL of a modified TNF-related apoptosis-inducing ligand (see Table of Materials) plus 2.5 µM MG132 for 20 h before fixation to validate the apoptosis measurement by active caspase-3 and subG1 analysis. (A) Histogram of the caspase-3 signal of treated versus untreated cells. (B) DAPI histogram of treated cells with a subG1 population, indicating DNA degradation. The histogram of caspase-3-positive events is overlaid in red, demonstrating that most of the apoptotic cells had not yet degraded their DNA at this time point. Please click here to view a larger version of this figure.
Figure 6: Factors influencing the quality of cell cycle profiles. Too harsh treatments complicate cell cycle analysis. (A) No G1 peak is detectable 48 h after treatment of LLC cells (Lewis lung carcinoma) with 20 Gy of photon radiation or (B) 96 h after treatment of HS68 fibroblasts with 100 mJ of UV radiation. (C) DAPI concentration in different cell lines: In HS68 fibroblasts (upper panel) the G2/M peak (see arrows) was equally well-separated from the G1 peak for DAPI concentrations between 0.01 and 1.0 µg/mL. In contrast, DAPI concentrations below 0.75 µg/mL resulted in poor separation and shape definition of the G2/M peak (see arrows) in MRC-5 fibroblasts (lower panel). Please click here to view a larger version of this figure.
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Parameter | Significance | Excitation laser | Detector | Signal | |||||
λ (nm) | P (mW) | Filter | U (V) | log | -A | -H | -W | ||
FSC | forward scatter (size) | 488 | 100 | 488/10 | 230 | ● | ● | ||
SSC | side scatter (granularity) | 488 | 100 | 488/10 | 210 | ● | ● | ||
Alexa488 | γH2AX (double-strand breaks) | 488 | 100 | 525/50 | 275 | ● | ● | ● | |
Alexa555 | phospho-histone H3 (M phase) | 561 | 150 | 586/15 | 360 | ● | ● | ● | |
Alexa647 | active caspase-3 (apoptosis) | 640 | 40 | 670/14 | 410 | ● | ● | ● | |
DAPI | DNA (cell cycle, apoptosis) | 350 | 20 | 450/50 | 350 | ● | ● | ● |
Table 1: Typical flow cytometer configuration. λ = wavelength of the excitation laser, U = detector voltage, log = logarithmic scale (otherwise linear), -A = signal area, -H = maximum signal height, -W = signal width (duration), FSC = front scatter, SSC = side scatter. Optical filter specifications are given as central wavelength/bandwidth in nanometers. Settings may vary for different flow cytometers and detector voltages need to be adjusted for different cell lines. Please click here to view this table (Right click to download).
Supplementary Figure 1: Microscopic images of γH2AX foci. U87 cells were irradiated with different doses of photon radiation, fixed and stained after 30 min according to the presented protocol and prepared for microscopy as described in the discussion. At 2 Gy, individual foci could be easily distinguished, while at 8 Gy foci counting was biased due to considerable overlap. Please click here to view a larger version of this figure.
Supplementary File 1: Please click here to download this file.
The featured method is easy to use and offers a fast, accurate and reproducible measurement of the DNA damage response including double-strand break (DSB) induction and repair, cell cycle effects and apoptotic cell death. The combination of these endpoints provides a more complete picture of their interrelations than individual assays. The method can be widely applied as a comprehensive genotoxicity assay in the fields of radiation biology, therapy and protection, and more generally in oncology (e.g., for environmental risk factor assessment, drug screening and evaluation of genetic instability in tumor cells).
The most critical step in this protocol is the fixation of the cells. To obtain a clean sample with a clearly defined cell population and optimal resolution of different cell cycle phases, it is important to give adherent cells enough time to detach from the culture vessel and form a completely round shape. The cells should be transferred into the fixation solution as a single cell suspension without cell clumps. To separate them, the cells must be pipetted up and down or passed through a fine needle several times in difficult cases. The fixation time should be kept constant between all samples and should not exceed 20 min including the time for centrifugation before resuspension of the cells in 70% ethanol. Prolonged fixation will lead to loss of accessible epitopes for antibody binding and will decrease the signal strength and sensitivity of the method.
The main limitation of the technique is that it does not provide information on the quality of the γH2AX foci other than the intensity in the whole nucleus. Particularly large γH2AX foci as well as the occurrence of a pan-nuclear γH2AX staining have been linked to clustered DSBs1,27,28,29, which are highly complex lesions that are very difficult to repair30. Such clustered lesions seem to be characteristic for particle radiation such as clinically applied carbon ion radiation and may be the reason for the superior biological effectiveness of heavy ion radiation compared to photons31,32,33. Analysis of the γH2AX foci size may therefore be of interest as an indicator of the damage complexity. Although it is possible to use the present protocol with an image stream cytometer to visualize the γH2AX foci, the achievable resolution cannot compete with a classical microscopic approach. However, we have successfully prepared microscopic slides from the remaining samples after flow cytometric measurement (Supplementary Figure 1) and evaluated several features including γH2AX foci count, size and pan-nuclear intensity using a semi-automated imaging and analysis system. To prepare the samples for microscopy, 30-50 µL of the stained cells were spread over the surface of a 24 mm x 24 mm cover glass, air dried overnight at room temperature in the dark and then embedded with mounting medium on a glass slide.
In comparison with microscopic evaluation of DSB levels by γH2AX foci counting, the flow cytometric measurement is superior in throughput, sampling rate, dynamic range and accuracy. The dynamic range of microscopic foci counting is limited by the optical resolution, leading to the inability to distinguish overlapping foci29,34. In practice, we have observed a linear relationship between radiation dose and γH2AX foci numbers below 2 Gy, and a strong saturation effect above 2 Gy in U87 glioblastoma cells1. In contrast, the intensity of the γH2AX signal measured by flow cytometry increased linearly with dose for the whole dose range tested (0-8 Gy).
The accuracy of microscopic foci counting is limited by the inability to distinguish between different cell cycle phases without additional stainings35. The number of DSBs induced in a cell is not only dependent on the radiation dose, but also on the DNA content, and hence the cell cycle stage. G2 cells for example have twice as much DNA as G1 cells and the average number of DSBs induced at a certain radiation dose is twice as high for G2 cells compared to G1. This is clearly reflected in the γH2AX signal intensity of G2 versus G1 cells at early time points after radiation measured by flow cytometry. Therefore, it is important to evaluate DSB levels in a cell cycle-specific manner to obtain accurate results. A pooled analysis of cells in different cell cycle phases, like usual in microscopic approaches, can be biased by shifts in the cell cycle distribution particularly due to radiation-induced G2 arrest. To account for this effect and to compare DSB repair characteristics between different cell cycle stages, the γH2AX levels can easily be normalized to the DNA content in the flow cytometry method as indicated in the protocol.
Extensions of the present protocol are possible with relatively little extra effort. Additional antibodies with compatible fluorophores labels can be included in step 3.4 without the need for any extra steps during sample preparation. If a more detailed analysis of apoptosis is desired, caspase-7, -9 and -8 antibodies could be included. As another extension, we have recently included a live and dead cell dye, a troponin T antibody and counting beads in the protocol to specifically analyze cardiomyocytes co-cultured with fibroblasts after irradiation. The addition of the live/dead cell stain and the counting beads expands the capability of the method to include fibroblast proliferation and non-apoptotic cell death of the cardiomyocytes as further endpoints that provide useful additional information on the cell fate after genotoxic stress. The extended protocol is available upon request.
The authors have nothing to disclose.
We thank the Flow Cytometry Facility team at the German Cancer Research Center (DKFZ) for their support.
1000 µL filter tips | Nerbe plus | 07-693-8300 | |
100-1000 µL pipette | Eppendorf | 3123000063 | |
12 x 75 mm Tubes with Cell Strainer Cap, 35 µm mesh pore size | BD Falcon | 352235 | |
15 mL tubes | BD Falcon | 352096 | |
200 µL filter tips | Nerbe plus | 07-662-8300 | |
20-200 µL pipette | Eppendorf | 3123000055 | |
4’,6-Diamidin-2-phenylindol (DAPI) | Sigma-Aldrich | D9542 | Dissolve in water at 200 µg/ml and store aliquots at -20 °C |
Alexa Fluor 488 anti-H2A.X Phospho (Ser139) Antibody, RRID: AB_2248011 | BioLegend | 613406 | Dilute 1:20 |
Alexa Fluor 647 Rabbit Anti-Active Caspase-3 Antibody, AB_1727414 | BD Pharmingen | 560626 | Dilute 1:20 |
BD FACSClean solution | BD Biosciences | 340345 | For cytometer cleaning routine after measurement |
BD FACSRinse solution | BD Biosciences | 340346 | For cytometer cleaning routine after measurement |
Dulbecco’s Phosphate Buffered Saline (PBS) | Biochrom | L 182 | Dissolve in water to 1x concentration |
Dulbecco's Modified Eagle's Medium with stable glutamin | Biochrom | FG 0415 | Routine cell culture material for the example cell line used in the protocol |
Ethanol absolute | VWR | 20821.330 | |
Excel software | Microsoft | ||
FBS Superior (fetal bovine serum) | Biochrom | S 0615 | Routine cell culture material for the example cell line used in the protocol |
FlowJo v10 software | LLC | online order | |
Fluoromount-G | SouthernBiotech | 0100-01 | Embedding medium for optional preparation of microscopic slides from stained samples |
folded cellulose filters, grade 3hw | NeoLab | 11416 | |
LSRII or LSRFortessa cytometer | BD Biosciences | ||
MG132 | Calbiochem | 474787 | optional drug for apoptosis positive control |
Multifuge 3SR+ | Heraeus | ||
Paraformaldehyde | AppliChem | A3813 | Prepare 4.5% solution fresh. Dilute in PBS by heating to 80 °C with slow stirring under the fume hood. Cover the flask with aluminium foil to prevent heat loss. Let the solution cool to room temperature and adjust the final volume. Pass the solution through a cellulose filter. |
Phospho-Histone H3 (Ser10) (D2C8) XP Rabbit mAb (Alexa Fluor® 555 Conjugate) RRID: AB_10694639 | Cell Signaling Technology | #3475 | Dilute 3:200 |
PIPETBOY acu 2 | Integra Biosciences | 155 016 | |
Serological pipettes, 10 mL | Corning | 4488 | |
Serological pipettes, 25 mL | Corning | 4489 | |
Serological pipettes, 5 mL | Corning | 4487 | |
SuperKillerTRAIL (modified TNF-related apoptosis-inducing ligand) | Biomol | AG-40T-0002-C020 | optional drug for apoptosis positive control |
T25 cell culture flasks | Greiner bio-one | 690160 | Routine cell culture material for the example cell line used in the protocol |
Trypsin/EDTA | PAN Biotech | P10-025500 | Routine cell culture material for the example cell line used in the protocol |
U87 MG glioblastoma cells | ATCC | ATCC-HTB-14 | Example cell line used in the protocol |