Flow cytometric analysis of the cell cycle phase specificity of DNA damage induced by radiation, hydrogen peroxide and doxorubicin
Alan J. Potter1,
Katherine A. Gollahon1,
Ben J.A. Palanca1,3,
Mary J. Harbert1,
Young M. Choi1,4,
Alexander H. Moskovitz1,5,
John D. Potter2 and
Peter S. Rabinovitch1,2,6
1 Department of Pathology, University of Washington, Seattle, WA 98195,
2 Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
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Abstract
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We have optimized a flow cytometric DNA alkaline unwinding assay to increase the sensitivity in detecting low levels of DNA damage (strand breaks and alkali-labile sites) and to permit the measurement of the extent of DNA damage within each cell cycle compartment. The lowest
radiation dose that induced detectable DNA damage in each cell cycle phase of HeLa and CEM cells was 10 cGy. The lowest H2O2 concentration that induced detectable DNA damage in each cell cycle phase was 0.5 µM in HeLa cells, and 12.5 TµM in CEM cells. For both HeLa cells and CEM cells, DNA damage in each cell cycle compartment increased approximately linearly with increasing doses of
radiation and H2O2. Although untreated HeLa and CEM cells in S phase consistently exhibited greater DNA unwinding than did G1 or G2 cells (presumably due to DNA strand breaks associated with replication forks), there was no difference between the susceptibility of G0/G1, S and G2/M phase cells to DNA damage induced by
radiation or H2O2, or in the rate of repair of this damage. In each cell cycle phase, the susceptibility to
radiation-induced DNA damage was greater in CEM cells than in HeLa cells. In contrast to the lack of cell cycle phase-specific DNA damage induced by exposure to
radiation or H2O2, the cancer chemotherapeutic drug doxorubicin (adriamycin) predominantly induced DNA damage in G2 phase cells.
Abbreviations: ANOCoVA, analysis of co-variance; ANOVA, analysis of variance; DSB, double-strand breaks; dsDNA, double-stranded DNA; SB, strand breaks; SCGE, single-cell gel electrophoresis; ssDNA, single-stranded DNA; SSB, single-strand breaks.
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Introduction
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Measuring damage to DNA resulting from exposure to mutagens, carcinogens or cancer chemotherapy drugs is central to many studies in cancer biology, environmental health and toxicology. The susceptibility of DNA to damage and its repair can differ between cell types, between quiescent and proliferating cells of a single cell type, and between cells of different proliferative or metabolic status within a single sample (14). Furthermore, the extent of DNA damage can vary between treated cells of a seemingly homogeneous population, an indication that a relatively resistant or sensitive subpopulation may be present (4,5). This variation can confound results from methods that measure only the average amount of DNA damage in a cell population, hindering the accurate assessment of treatment effects. Accordingly, single-cell assays of DNA damage have been developed that can reveal the extent of heterogeneity within treated cell populations in addition to measuring the mean response. We have optimized a flow cytometric version of one of these assays, enhancing the sensitivity and, importantly, have provided a means of simultaneously identifying any cell cycle or ploidy-dependent susceptibility to DNA damage.
The assay described in this manuscript is based on alkaline unwinding of double-stranded DNA (dsDNA), into single-stranded DNA (ssDNA), from double-strand breaks (DSB), single-strand breaks (SSB), and from strand breaks (SB) associated with alkali-labile abasic (apurinic/apyrimidinic) sites, excision repair sites, and strand discontinuities at transcription forks (6,7). The amount of ssDNA formed in each cell then is proportional to the number of strand breaks present. A series of assays capitalizing upon this characteristic of DNA have been developed to evaluate damage and repair in individual cells. In the single-cell microgel assay (8), DNA in agarose-embedded lysed cells is denatured with alkali, ssDNA and dsDNA segments are differentially stained, and the ssDNA:dsDNA ratio in isolated cells is then calculated from microscope fluorimetry. An increase in this ratio in treated cells relative to untreated cells indicates that DNA damage has been induced, and a relatively higher ratio indicates a greater extent of DNA damage. The related microelectrophoresis assay (9) gave rise to the single-cell gel electrophoresis (SCGE) assay (comet assay) (10,11) where image analysis of DNA migration from the nucleus toward the anode is used to determine the extent of DNA damage induced in individual cells (4,6). The microgel assay was also later adapted for analysis by flow cytometry; in this report we describe the optimization of the flow cytometric DNA alkaline unwinding assay, improving the sensitivity in detecting DNA damage ~10-fold above that of the original (12) and modified (13,14) methods.
The flow cytometric DNA alkaline unwinding assay has the advantage of permitting rapid and automated analysis of a large number (>104) of individual cells per sample, in contrast to the relatively few cells (
hundreds) analyzed per SCGE sample (15), insuring that the parent population is accurately represented and that statistical analyses are valid. By measuring the extent of DNA damage in single cells, both the SCGE and the flow cytometric assays permit the distribution of intercellular heterogeneity of DNA damage and repair within each sample to be determined; therefore, any subpopulation of resistant or sensitive cells observed can be ascribed to a biologic difference rather than to experimental variation (16,17). Cell cycle position may be determined by simultaneous measurement of DNA content; although cell cycle-dependent susceptibility to a treatment is not commonly evaluated in SCGE studies, we show in this report that this is easily performed using the flow cytometric assay.
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Materials and methods
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Cell culture
HeLa human cervical carcinoma cells (American Type Culture Collection (ATCC), Manassas, VA) were cultured as monolayers in complete DMEM (BioWhittaker, Walkersville, MD) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Hyclone, Logan, UT) and CEM human T lymphoblastoid cells (ATCC) were cultured in complete RPMI 1640 (BioWhittaker) supplemented with 16% FBS, each with 100 U/ml penicillin, 100 µg/ml streptomycin, and 1 mM L-glutamine (media) in humidified air with 5% CO2 at 37°C. Cells were given fresh media 1224 h before treatment. 38 x 105 cells from cultures in exponential growth were used per sample. All samples were treated in duplicate.
Treatment with
radiation and hydrogen peroxide (H2O2)
Prior to treatment, HeLa cells were rinsed with 37°C versene and 0.05% trypsin/EDTA, held ~3 min until cells detached and then incubated in fresh media for 30 min to allow repair of any trypsin-induced DNA damage, although this was likely minimal (18). HeLa and CEM cell suspensions were pelleted at 200 g, resuspended in DMEM with 1 mg/ml endonuclease-free bovine serum albumin (BSA; Worthington Biochemical, Lakewood, NJ), held on ice, and treated within 15 min.
Samples were exposed on ice to
radiation from a 137Cs source (Gammacell 40, Atomic Energy of Canada Limited, Mississauga, Ontario) at a dose rate of 1.628 cGy/s and then immediately fixed. To evaluate repair of DNA damage, treated samples were diluted with media at 37°C, and then either immediately fixed (0 min repair) or incubated at 37°C for increasing lengths of time before fixation. Untreated (control) samples were collected at the start and end of the incubation period.
Treatment with H2O2 was performed by adding an aliquot of a 0.03% or 0.003% H2O2 (Sigma, St Louis, MO) in phosphate-buffed saline (PBS) to samples on ice, held for 10 min and then fixed; control samples were treated with the highest corresponding volume of PBS alone. Repair of DNA damage was evaluated in samples treated for 10 min (pelleted at 4°C during the final 5 min of treatment), resuspended in 37°C media and either immediately fixed (0 min repair) or incubated at 37°C for increasing lengths of time before fixation. Untreated samples were taken at the start and end of the incubation period.
Treatment with doxorubicin
Working solutions of doxorubicin (Sigma) were made by diluting a 10 mM stock in dimethyl sulfoxide (DMSO; Sigma) with PBS. HeLa cells in flasks with complete media were treated with 1 µM of the drug for 0.56 h at 37°C. Negative control samples were treated with the corresponding volume of DMSO/PBS; the final DMSO concentration was <0.25%. Irradiated samples were used as positive controls. Thirty min prior to the conclusion of drug treatment, the media was removed and retained, and the cells were trypsinized as above and then resuspended in the original media for 30 min to allow repair of any trypsin-induced DNA damage. The cells were then pelleted, resuspended in DMEM/BSA and fixed. Untreated samples were taken at the start and end of the incubation period.
Viability analysis
Prior to use and at the conclusion of each treatment, a 50 µl aliquot from each sample was mixed with 2.5 µl of 1 mg/ml propidium iodide (PI, Sigma) and held on ice in the dark for a minimum of 15 min. Fluorescence microscopy (530 nm excitation, >590 nm emission) was used to determine the percentage of PI-positive (nonviable) cells. Prior to treatment, the viability of HeLa cells and CEM cells was >95% and >90%, respectively. At the conclusion of the repair period after treatment with
radiation and H2O2, the viability of HeLa cells was >90% and >85%, respectively, and the viability of CEM cells was >93% and >86%, respectively. The viability of HeLa cells treated with doxorubicin was >94% at the end of treatment.
Sample preparation for DNA damage analysis
At the end of each treatment, samples were cooled on ice and 3 ml of ice-cold 40% ethanol (30% final) was then added drop-wise while gently vortexing. After a minimum 15 min fixation on ice, samples were pelleted at 200 g for 10 min at 4°C, gently resuspended in 0.5 ml 30% ethanol, and 5 ml 0.15 N NaCl was added drop-wise while vortexing. Samples were re-pelleted, drained and resuspended in 35 µl buffer (20 mM Tris, 1 mM EDTA, 100 mM NaCl, pH 7.4) and then 70 µl 50°C 2.25% (w/v) ultra-low gelling temperature (ULGT) agarose (Seaprep, FMC Bioproducts, Rockland, ME) in PBS was added. Each cell suspension was cast into a
dram glass vial (VWR, West Chester, PA) pre-coated with a dried layer of 30 µl 0.1% (w/v) LGT agarose (Metaphor, FMC Bioproducts) in PBS to improve adhesion. The vials were held on ice, or at 4°C overnight, to allow the cell suspension/agar to gel. Samples were then subjected to DNA denaturation by exposure to alkali as given below.
DNA denaturation
An alkaline solution (300 mM NaOH, 300 mM KCl, 50 µM EDTA) was adjusted to pH 13.00 with 4 N HCl. 1 ml of this solution was layered over the gelled cell suspension/agar plug in each vial, and the samples were held at room temperature in the dark for 20 min. All the following steps were performed under subdued light. The alkaline solution was removed, and residual alkali in the agar was neutralized by rinsing twice with 1 ml 150 mM Tris in 0.15 N NaCl, pH 7.4 (Tris/NaCl). The samples were then covered with 1 ml Tris/NaCl and held in the dark at 4°C for a minimum of 20 min and a maximum of 48 h.
DNA staining with acridine orange
Just prior to use, the Tris/NaCl solution covering each sample was removed and the samples were briefly (<60 s) melted in a 65°C heat block. 50 µl of the cell suspension was held in reserve and the remaining 50 µl was mixed with 100 µl of an ice-cold solution of 0.1% Triton X-100, 0.08 N HCl, and 0.15 N NaCl, pH 1.3, and held on ice for 30 s (19). 300 µl of an ice-cold solution of 126 mM Na2HPO4, 37 mM citric acid, 0.15 N NaCl, and 1 mM EDTA, pH 6.0, (19) with 22.1 µM Acridine Orange (AO, Molecular Probes, Eugene, OR) was then added (14.6 µM final AO concentration) followed by addition of 2.5 µl chicken erythrocyte nuclei (CEN) reference standard (Biosure Controls/Reese Enterprises, Grass Valley, CA). Samples were held on ice for a minimum of 5 min for DNA staining.
Analysis of DNA damage by flow cytometry
Samples were analyzed at 4°C on a Epics Elite XL flow cytometer (Beckman Coulter, Fullerton, CA) using a single 488 nm argon-ion laser at a sample flow rate of ~200 events/s. AO green fluorescence (dsDNA) was collected at 500530 nm and AO red fluorescence (ssDNA) was collected at >645 nm (19). Data from a minimum of 104 cells per sample was collected in list mode.
Data analysis
Data were analyzed using ListView (Phoenix Flow Systems, San Diego, CA) and Multiplus (Phoenix Flow Systems) software programs. Analysis gates were set on forward light scatter (proportional to cellular diameter) versus orthogonal light scatter (granularity) to exclude debris, and on the peak versus integrated area of AO green fluorescence to exclude cell aggregates. A cytogram of the ssDNA (AO red fluorescence) versus dsDNA (AO green fluorescence) in each cell was then plotted (Figure 1A
), and re-plotted as a cytogram of total DNA content (the sum of AO red fluorescence plus AO green fluorescence) versus the ratio of ssDNA to dsDNA (Figure 1B
). The sum and ratio were each multiplied by a scaling constant before plotting.

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Fig. 1. Flow cytometric DNA alkaline unwinding assay. (A) Cells in the G0/G1, S and G2/M phases of the cell cycle and the CEN flow cytometric reference fluorescence standard are indicated on the cytogram of dsDNA (green AO fluorescence) versus ssDNA (red AO fluorescence from DNA unwound from strand breaks and alkali-labile sites) in ~5 x 104 untreated HeLa cells. (B) The same data replotted as a cytogram of total DNA content (ssDNA plus dsDNA; abscissa) versus DNA denaturation (ssDNA:dsDNA ratio; ordinate). The regions containing the analyzed CEN, G0/G1, S and G2/M phase cells are indicated (solid lines), as are the regions containing G1-S and S-G2 phase cells excluded from analysis (dotted lines). The mean ssDNA:dsDNA ratio (AO red:green fluorescence ratio) of CEN, G2/M, G0/G1 and S phase cells, are indicated by arrowheads along the ordinate from bottom to top, respectively.
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Using the total DNA content parameter (excluding CEN, which have lower fluorescence), the percentages of cells in the G0/G1, S and G2/M phases of the cell cycle and the mean DNA content of each phase were determined. Using this information, regions encompassing each cell cycle compartment were set on the cytogram of DNA content versus DNA strand breakage (Figure 1B
). To minimize the effect of overlap with S phase cells, the G0/G1 region was restricted to cells with a DNA content
the mean DNA content of G0/G1 phase cells, and the G2/M region was restricted to cells with a DNA content
the mean DNA content of G2/M phase cells. Similarly, the S phase analysis region was restricted to the middle 50% of S phase cells in the interval between the G1and G2 means. An additional analysis region was restricted to the CEN (Figure 1B
).
For each sample, the mean and standard deviation of the DNA strand breakage (ssDNA:dsDNA ratio) in G0/G1, in S and in G2/M phase cells were recorded (Figure 1B
). Each mean was then divided by the mean ssDNA:dsDNA ratio of the CEN to normalize the data to CEN. The average CEN-normalized DNA strand breakage and coefficient of variation (CV) of G0/G1, S and G2/M phase cells were calculated from the duplicate samples used per condition. Comparisons between the extent of DNA damage in G0/G1 versus S versus G2/M phase cells induced by a treatment were complicated by cell cycle differences in baseline DNA strand breakage (see below). Baseline-corrected DNA strand breakage values for G0/G1, S and G2/M phase cells in treated samples were therefore calculated by subtracting the average DNA strand breakage value for each cell cycle compartment in the duplicate untreated samples from the DNA strand breakage value of the corresponding cell cycle compartment in each treated sample.
Statistical analysis
Comparisons within and between data sets were performed using analysis of variance (ANOVA) and least significant difference t-tests. Comparisons between specific groups within data sets were made by paired t-test. Multiple comparisons with controls were made by Dunnett's t-test. Simple linear regression analyses were performed on all data using the least squares method and ANOVA. Comparisons between regression curves were made by analysis of co-variance (ANOCoVA). Trend tests were made by ANOVA and linear contrast t-test. F < 0.05 and P < 0.05 were considered statistically significant. The CV in reproducibility studies analysis (20) was used to determine the reproducibility of repeated experiments.
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Results
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Flow cytometric analysis of HeLa cells after alkaline unwinding (denaturation) of DNA is illustrated in Figure 1
. AO green fluorescence is proportional to dsDNA content and AO red fluorescence is proportional to ssDNA content (Figure 1A
), the latter indicating the extent of DNA unwinding from SSB, DSB and abasic sites (collectively referred to as DNA damage). The data were replotted (Figure 1B
) as the sum of the red and green AO fluorescence (total DNA content, abscissa) versus the calculated ratio of red (ssDNA) to green (dsDNA) AO fluorescence (ordinate) producing a display of DNA denaturation as a function of DNA content (cell cycle position). The magnitude of DNA alkaline unwinding in each cell cycle compartment (G0/G1, S and G2/M phases) may be readily determined from these plots. In untreated samples, the extent of DNA unwinding was found to be greatest in S phase cells, presumably due to the presence of SSBs at each replication fork (Figure 1B
). The CEN added to each sample, after alkaline treatment, served as a fluorescence intensity standard. The cellular AO red/green fluorescence ratio (ssDNA/dsDNA) was standardized to the CEN AO red/green fluorescence ratio, allowing cellular DNA damage to be quantified in a consistent manner within and between experiments.
Treatment with
radiation
When HeLa cells were exposed to increasing doses of
radiation, DNA damage in each cell cycle compartment increased relatively homogeneously (Figure 2
). No subpopulation of resistant cells was seen, which would have appeared as a group of cells that remained lower on the ordinate relative to the major population of cells. The DNA unwinding within S phase cells was elevated, relative to G0/G1 and G2/M phase cells, in both untreated and treated samples.

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Fig. 2. Cytograms of DNA content versus DNA denaturation for ~5 x 104 HeLa cells in an untreated sample (A) and in samples exposed on ice to increasing doses of radiation as indicated (BF). The regions containing G0/G1, S and G2/M phase cells are indicated in (A). The dotted arrows indicate the position of the CEN flow cytometric standard and are located in the same position in each panel.
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To determine the sensitivity and range of the flow cytometric DNA alkaline unwinding assay, the extent of DNA damage was quantified in HeLa cells (Figure 3A
) and CEM cells (Figure 3B
) exposed to 01012 cGy of
radiation. Ten cGy was found to be the minimum dose that produced a detectable increase in DNA damage in G0/G1, S and G2/M phase HeLa and CEM cells (Figure 3A
and Figure 3B
, respectively; each one-tail t-test P <0.05) when compared with the corresponding cells in untreated samples. In each cell cycle compartment, DNA damage in HeLa cells (Figure 3A
) and CEM cells (Figure 3B
) increased approximately linearly with increasing
radiation dose (each R2 > 0.91, P < 0.0001 and R2 > 0.96, P < 0.0001, respectively). The reproducibility CVs were <11% for HeLa cells and <22% for CEM cells, indicating that inter-experiment reproducibility was acceptable (20). Above 1012 cGy, the extent of DNA damage in HeLa and CEM cells saturated the assay under the protocol conditions used (data not shown).

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Fig. 3. DNA unwinding as a function of radiation dose. (A) HeLa cells. The mean ± SEM of the ssDNA:dsDNA ratio in G0/G1, S and G2/M phase cells relative to the CEN standard for five experiments (one experiment at 1012 cGy). (B) CEM cells. The mean ± SEM of the ssDNA:dsDNA ratio in G0/G1, S and G2/M phase cells relative the CEN standard for three experiments (two experiments at 1012 cGy). Regression lines are drawn through the data from G0/G1 (solid), S (dashed) and G2/M (dotted) phase cells (A and B). The data from 050 cGy are expanded for clarity (insets, A and B).
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Since the extent of DNA unwinding in untreated samples was greater in S phase cells than in G0/G1 and G2/M phase cells, these baseline values were subtracted from the DNA unwinding values in treated samples to allow comparisons of treatment effects between cell cycle compartments. Comparisons of the baseline-adjusted DNA unwinding values at each radiation dose suggested that there was no difference between the sensitivity of G0/G1, S and G2/M phase cells to DNA damage produced by
radiation in HeLa or CEM samples (Figure 4
; each ANOVA P > 0.14 and ANOVA P > 0.54, respectively). Similarly, comparisons between the slopes of the regression lines indicated that G0/G1, S and G2/M phase cells were equally susceptible to DNA damage produced by
radiation in HeLa samples (Figure 3A
; each ANOCoVA P > 0.14) and in CEM samples (Figure 3B
; each ANOCoVA P > 0.32). These comparisons did, however, suggest that HeLa cells were less susceptible to DNA damage produced by
radiation than were CEM cells (Figure 4
; each ANOCoVA P < 0.001).
Treatment with H2O2
The extent of DNA damage in HeLa cells is shown to increase with H2O2 concentration in Figure 5
. While the intercellular heterogeneity in the extent of DNA damage was greater in these samples than was found in the irradiated samples (Figure 5
versus Figure 2
), a distinct subpopulation of resistant cells was not observed.

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Fig. 5. Cytograms of DNA content versus DNA denaturation for ~5 x 104 HeLa cells in untreated (A) and samples treated on ice for 10 min with the indicated concentration of H2O2 (BF). The regions containing G0/G1, S and G2/M phase cells are indicated in (A). The dotted arrows indicate the position of the CEN standard and are located in the same position in each panel.
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The minimum H2O2 concentration found to produce detectable DNA damage in G0/G1, S and G2/M phase HeLa cells compared with untreated samples was 0.5 µM (Figure 6A
; each one-tailed t-test P < 0.05), while the detection limit in CEM samples was 1 µM for G0/G1 and G2/M phase cells and 2.5 mM for S phase cells (Figure 6B
; each one-tailed t-test P < 0.04). The DNA damage in HeLa cells (Figure 6A
) and CEM cells (Figure 6B
) in each cell cycle compartment increased approximately linearly with increasing H2O2 concentration (each R2 > 0.89, P < 0.0001 and R2 > 0.97, P < 0.0001, respectively). The relatively low reproducibility CVs for both HeLa cells (<21%) and CEM cells (<23%) indicated that inter-experiment reproducibility was acceptable. As was noted following treatment with relatively high doses of radiation, DNA damage produced by treatment with H2O2 concentrations above 25 µM saturated the assay (data not shown).

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Fig. 6. DNA denaturation as a function of H2O2 concentration. (A) HeLa cells. The mean ± SEM of the ssDNA:dsDNA ratio in G0/G1, S and G2/M phase cells relative to CEN for three experiments. (B) CEM cells. The mean ± SEM of the ssDNA:dsDNA ratio in G0/G1, S and G2/M phase cells relative to CEN for three experiments. Regression lines are drawn through the data from G0/G1 (solid), S (dashed) and G2/M (dotted) phase cells (A and B). The data from 05 µM are expanded for clarity (insets, A and B).
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Comparisons between the slopes of the regression lines (Figure 6A
and Figure 6B
) and comparisons of the baseline-adjusted data at each H2O2 concentration (Figure 7
) suggested that there was no difference between the susceptibility of G0/G1, S and G2/M phase cells to H2O2-induced DNA damage in HeLa samples (each ANOCoVA P > 0.10 and ANOVA P > 0.24, respectively) or in CEM samples (each ANOCoVA P > 0.39 and ANOVA P > 0.28, respectively). Comparisons of the slopes of the regression lines also indicated that although there was no difference between the susceptibility of S and G2/M phase HeLa cells to H2O2-induced DNA damage compared with S and G2/M phase CEM cells (Figure 7
; each ANOCoVA P > 0.06), G0/G1 phase HeLa cells were more susceptible to this treatment than were G0/G1 phase CEM cells (Figure 7
; ANOCoVA P = 0.03).
Repair of DNA damage produced by treatment with
radiation
The flow cytometric DNA alkaline unwinding assay was used to determine if repair of DNA damage differed between cells in different phases of the cell cycle. HeLa cells were exposed to 245 cGy of
radiation and incubated for 0180 min to allow DNA repair (Figure 8A
). The repair curves of G0/G1, S and G2/M phase cells each appeared to be biphasic, with DNA damage decreasing from the maximum approximately linearly with repair time through 60 min (Figure 8A
; each R2 > 0.67, P < 0.0001) and exhibiting a repair half-time of 21 ± 8, 21 ± 9 and 20 ± 9 min, respectively. Comparisons between the slopes of the regression lines through the linear portion of the repair curves indicated that G0/G1, S and G2/M phase cells did not differ in the rate of DNA repair (Figure 8A
; each ANOCoVA P > 0.68). In agreement with this finding, the extent of DNA damage remaining in G0/G1, S and G2/M phase cells at each repair time point did not significantly differ (Figure 8A
; each ANOVA P > 0.13). The DNA damage decreased by 120 min to the level found in untreated samples (Figure 8A
; each one-tail Dunnett's t-test P > 0.05).
DNA repair was also evaluated in CEM cells exposed to 506 cGy of
radiation (Figure 8B
). As was found for HeLa cells, G0/G1, S and G2/M phase CEM cells exhibited biphasic repair curves, here with DNA damage decreasing approximately linearly with repair time through 60 min (Figure 8B
; each R2 > 0.75, P < 0.0001) and exhibiting a repair half-time of 19 ± 5, 21 ± 6 and 20 ± 7 min, respectively. Comparisons between the slopes of the regression lines through the linear portion of the repair curves and comparisons between the extent of DNA damage remaining at each repair time point indicated that there was no difference between the rate of DNA repair in G0/G1, S and G2/M phase cells (Figure 8B
; each ANOCoVA P > 0.53 and ANOVA P > 0.68, respectively). The DNA damage decreased to the level found in the corresponding untreated cells by 60 min (Figure 8B
; each one-tail Dunnett's t-test P > 0.05).
Repair of DNA damage produced by treatment with H2O2
To further evaluate repair of DNA damage as a function of cell cycle position, the flow cytometric assay was used to examine HeLa cells treated with 10 µM H2O2 and then incubated to allow DNA repair (Figure 9A
). Maximal DNA damage was observed in G0/G1, S and G2/M phase cells allowed 5 min of repair, although this difference was not statistically significant compared with samples without repair (Figure 9A
; each two-tail t-test P > 0.34). The DNA damage in each cell cycle compartment decreased approximately linearly with repair time through 30 min (Figure 9A
; each R2 > 0.74, P < 0.001) and exhibited a repair half-time of 14 ± 4, 13 ± 4 and 13 ± 4 min in G0/G1, S and G2/M phase cells, respectively. Comparisons between the slopes of the regression lines and between the extent of DNA damage remaining at each time point suggested that the DNA damage repair rate did not vary with cell cycle phase (Figure 9A
; each ANOCoVA P > 0.90 and ANOVA P > 0.10, respectively). The DNA damage in G0/G1, S and G2/M phase cells decreased to the control levels by 60 min (Figure 9A
; each one-tail Dunnett's t-test P > 0.05).

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Fig. 9. Repair of DNA damage in (A) HeLa cells and (B) CEM cells, induced by exposure to 10 µM and 25 µM H2O2, respectively, as a function of post-exposure incubation time. The mean ± SEM of the ssDNA:dsDNA ratio in treated G0/G1, S and G2/M phase cells relative to untreated samples are shown for two experiments (A) and three experiments (B). Data were calculated as described in Figure 4 .
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DNA repair in CEM cells treated with 25 µM H2O2 was also evaluated (Figure 9B
). As was observed in HeLa cells, maximal DNA damage was observed in G0/G1, S and G2/M phase cells allowed 5 min of repair, although again this difference was not statistically significant compared with samples without repair (Figure 9B
; each two-tail t-test P > 0.39). DNA damage in G0/G1, S and G2/M phase cells decreased from the maximum approximately linearly with repair time through 45 min (Figure 9B
; each R2 > 0.68, P < 0.0001) and exhibited a repair half-time of 16 ± 5, 15 ± 4 and 16 ± 5 min, respectively. As was found in HeLa cell samples, comparisons between the slopes of the regression lines and between the extent of DNA damage remaining at each time point indicated that the rate of DNA repair did not vary with cell cycle phase (Figure 9B
; each ANOCoVA P > 0.70 and ANOVA P > 0.83, respectively). The DNA damage in G0/G1, S and G2/M phase cells decreased by 45 min post-treatment to the values found in untreated cells (Figure 9B
; each one-tail Dunnett's t-test P > 0.05).
Treatment with doxorubicin
The flow cytometric assay of DNA alkaline unwinding was used to examine cells treated with doxorubicin. Although DNA damage was seen to increase in each cell cycle compartment with increasing treatment time, G2/M phase cells appeared to be the most susceptible to the drug (Figure 10
).

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Fig. 10. Cytograms of DNA content versus DNA unwinding for ~1 x 104 HeLa cells in an untreated sample (A) and in samples treated with 1 µM doxorubicin for the duration indicated (BF). The regions containing G0/G1, S and G2/M phase cells are indicated in (A). The dotted arrows indicate the position of the CEN standard and are located in the same position in each panel.
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Quantification of the DNA damage in HeLa cells treated with 1 µM doxorubicin for increasing times (0.56 h) is shown in Figure 11
. As suggested in Figure 10
, the extent of DNA damage in G0/G1, S and G2/M phase cells in samples treated for 16 h was greater than in untreated samples (Figure 11
; each one-tail Dunnett's t-test P < 0.05). Furthermore, at these timepoints the induced DNA damage was greater in G2/M phase cells than in either G0/G1 phase cells or S phase cells (Figure 11
; each ANOVA P < 0.05 and two-tail t-test P < 0.04 and P < 0.03, respectively). The extent of DNA damage induced in G0/G1 and S phase cells did not significantly differ (Figure 11
; each two-tail t-test P > 0.48). Cell cycle analysis indicated that in samples treated with doxorubicin for 6 h the percentage of G0/G1 cells was diminished, and the percentage of G2/M cells was increased, relative to the cell cycle distribution in untreated samples (each Dunnett's two-tail t-test P < 0.05).

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Fig. 11. DNA damage induced in HeLa cells by 1 µM doxorubicin as a function of treatment time. The mean ± SEM of the ssDNA:dsDNA ratio in treated G0/G1, S and G2/M phase cells relative to untreated cells are shown for six experiments. Data were calculated as described in Figure 4 .
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Intercellular variation
Intercellular variability in the extent of DNA damage present in each cell cycle compartment was evaluated by comparing the CVs of the DNA unwinding. In irradiated samples, the CVs of G0/G1, S and G2/M phase HeLa and CEM cells decreased as the
radiation dose increased (each R2 > 0.52, P < 0.0001 and R2 > 0.29, P < 0.002, respectively). In samples allowed to repair radiation-induced damage, the CVs of the DNA unwinding in G0/G1, S and G2/M phase HeLa and CEM cells immediately after treatment were less than the CVs of controls (each Dunnett's two-tail t-test P < 0.05), and then progressively increased with increasing repair time (each R2 > 0.45, P < 0.005 and R2 > 0.67, P < 0.0001, respectively). We believe that these results are consistent with the hypothesis that within each cell cycle compartment the extent of DNA damage induced by ionizing radiation is more uniform than is the endogenous level of strand breakage; with increasing radiation dose, the endogenous strand breakage becomes a progressively smaller proportion of the total, and hence, intercellular variability decreases. In contrast, there was a trend for the CVs of G0/G1, S and G2/M phase HeLa and CEM cells to increase as the H2O2 concentration increased (each R2 > 0.30, P < 0.05 and R2 > 0.41, P < 0.02, respectively). There was no clear trend for the CVs of DNA damage in G0/G1, S and G2/M phase HeLa and CEM cells allowed to repair H2O2-induced DNA damage, although the values returned to control levels by the conclusion of the repair period (each two-tail Dunnett's t-test P > 0.05). The CVs of doxorubicin-induced DNA damage in G0/G1, S and G2/M phase HeLa cells did not differ from the CVs of untreated samples (each two-tail Dunnett's t-test P > 0.05).
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Discussion
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Although the susceptibility of proliferating cells to carcinogens and cancer chemotherapy drugs has been extensively investigated (6,18,21), DNA damage induced by genotoxins is rarely studied as a function of cell cycle phase. To address this issue, we optimized the flow cytometric DNA alkaline unwinding assay, originally developed by Rydberg (12) and subsequently modified by Affentranger and Burkart (13,14), to increase the sensitivity in detecting low levels of DNA damage and to permit the extent of DNA damage within each cell cycle compartment to be determined. The critical modifications included ethanol fixation, increased alkalinity of the DNA unwinding solution, optimized DNA staining conditions, and use of a DNA fluorescence standard. The extent of DNA damage in HeLa and CEM cells in each cell cycle phase increased approximately linearly with increasing doses of
radiation and H2O2. In each cell cycle compartment, 10 cGy was the lowest
radiation dose that induced detectable DNA damage in HeLa and CEM cells. The lowest concentration of H2O2 that induced detectable DNA damage was 0.5 µM in G0/G1, S and G2/M phase HeLa cells, 1 µM H2O2 in G0/G1 and G2/M phase CEM cells, and 2.5 mM in S phase CEM cells. Although untreated HeLa and CEM cells in S phase consistently exhibited elevated DNA unwinding, presumably due to SSBs associated with replication forks, there was no difference between the susceptibility of G0/G1, S and G2/M phase cells to DNA damage by these agents, or in their rate of DNA repair. In each cell cycle compartment, CEM cells were more susceptible to
radiation-induced DNA damage than were HeLa cells. G0/G1 phase HeLa cells were more susceptible to H2O2-induced DNA damage than were the corresponding CEM cells. In HeLa and CEM cells in each cell cycle compartment, the intercellular heterogeneity of DNA damage was greater in H2O2-treated samples than in irradiated samples. Treatment of HeLa cells with the cancer chemotherapy drug doxorubicin predominantly induced DNA damage in G2 phase cells.
The alkaline solution conditions described in this manuscript were optimal for DNA denaturation over the range of treatments used here; different conditions might increase the sensitivity or expand the assay range (19,2225). Although Rydberg (12) used a high salt concentration to increase the efficiency of DNA alkaline denaturation by promoting dissociation of DNA-bound proteins that could interfere with unwinding, we found that this salt concentration increased background DNA unwinding. The low levels of induced DNA damage that we were able to discern did, however, indicate that protein-mediated interference with DNA denaturation was minimal in the salt conditions of our protocol, in agreement with previous studies (22,26,27). The CEN added to each sample served as an internal standard of fluorescence to control for inherent variations in the assay, allowing data obtained from separate experiments to be compared. Cell cycle phase was determined by standard DNA content analysis; any free DNA fragments were likely retained in the nucleus by entangling with matrix-attached segments (26,28). We and others (19) found that careful control of the staining conditions produced histograms with adequate CVs, allowing the cell cycle phases to be readily identified except in samples with the most extreme levels of DNA damage. The entire assay was typically performed in one day. As with many flow cytometric procedures, this assay has the advantage of analyzing large numbers of cells chosen at random, thereby avoiding any subjective selection based on characteristics such as morphology or staining quality.
Our data (e.g. Figure 1
) and that of others (4,16) indicate that the level of endogenous SB varies with cell cycle growth phase. Since we avoided technical factors known to increase baseline DNA unwinding, such as exposure of denatured DNA to visible light, shear forces, elevated temperature, and extended storage (22), the limited DNA unwinding in untreated cells likely arose from SB associated with replication, abasic sites, and from SB induced by reactive by-products of endogenous processes (4,16,29,30). This intrinsic DNA damage has also been observed in proliferating cells analyzed by SCGE (e.g. refs 1,31,32), and, in further agreement with our observations, detailed investigations found this damage to be elevated in S phase cells relative to G1 and G2 phase cells (17). The S phase-associated SB have been shown to be associated with recently replicated DNA and attributed to DNA unwinding from strand discontinuities at replication forks (4,17,22). Contrary to our findings, the previous studies using the flow cytometric DNA alkaline unwinding assay did not consistently observe the relatively elevated SB in S phase cells (1214), a difference likely related to our use of a higher pH DNA denaturation solution (17).
There are 7501100 replication forks estimated to be active during early S phase and fewer in mid-to-late S phase (33,34). It has been suggested that each of the two DNA discontinuities at a replication fork act as a SSB under alkaline conditions (16). Assuming 1 Gy induces ~7501000 SB/cell (17,32) and using the average 1.7 arbitrary units of DNA damage induced per Gy found here (Figure 4
), the 1.8 arbitrary units difference measured between untreated S phase cells versus untreated G1 and G2 phase cells (Figure 3A
) suggests there were ~9001100 additional SB per mid-S phase HeLa cell. A similar calculation suggests there were ~600800 additional SB per mid-S phase CEM cell. These values are equivalent to ~450550 and 300400 replication forks, respectively, and appear to be reasonable approximations of the likely number of SB per mid-S phase cell. The practical importance of these endogenous SB is that, unless DNA damage is identified by cell cycle compartment, they could confound conclusions about the extent or heterogeneity of an effect from treatments that induce low levels of damage or disrupt cell cycle progression, or of the extent of DNA repair (16). Further, we and others (4,35) found that some compounds have a propensity to induce DNA damage in specific cell cycle phases; this as well could confound results from studies of proliferating cells analyzed as a homogenous population and has been avoided here by measuring damage as a function of cell cycle position.
The minimum
radiation dose (10 cGy) which induced DNA damage detectable by the flow cytometric assay in G0/G1, S and G2/M phase HeLa and CEM cells was equivalent to ~100 SB/cell (17,32). In contrast, the minimum radiation dose found in SCGE studies to induce DNA damage in proliferating cells analyzed as a homogeneous population has been reported to be 50300 cGy (32,38,39). We attribute the relatively low limit of detection of the flow cytometric assay to the separate analysis of S phase cells (with their higher baseline). This contention is supported by reports that DNA damage can be detected in quiescent (G0) cells treated with 525 cGy using conventional SCGE (6,10,36) and <15 cGy using the most sensitive versions of that assay (2325,37). The current limit of detection of DNA damage by the flow cytometric assay might be improved by incorporating some further refinements used in SCGE assays (23,25).
Alternatives to using the flow cytometric assay to determine the extent of DNA damage as a function of cell cycle position include synchronizing or sorting samples by cell cycle phase (ref. 17 and references therein), however, use of these techniques has been limited because synchronization is imperfect and sorting is both time-consuming and may be associated with toxicity (40,41). A credible SCGE method of measuring DNA damage in each cell cycle compartment uses the total fluorescence of each comet to determine DNA content (and thus cell cycle position) coupled with the conventional SCGE analysis of DNA damage (4,16,17,28,42). Using this method, the extent of DNA alkaline unwinding can clearly be seen in each cell cycle compartment, including the elevated SB in untreated S phase cells (4,16,17,28); these plots closely resemble the cytograms presented here (e.g. Figure 2
). The comet/cell cycle assay has not been widely applied, however, perhaps due to the complexity of analysis and the increased number of cells that must be evaluated to provide sufficient data for determining the response in each cell cycle phase. The flow cytometric DNA alkaline unwinding assay overcomes this limitation by measuring thousands of cells, in accord with previous studies showing that accurate cell cycle analysis from DNA content distributions requires 500010 000 cells (43,44).
radiation is thought to induce DNA damage via radiolysis of water into DNA-reactive hydroxyl radicals (OH·) and, to a lesser extent, through ionization of DNA bases (45). The linear relationship between DNA damage and
radiation dose, which we observed in both HeLa and CEM cells (Figure 4
), has been seen previously in SCGE studies of irradiated proliferating cells (11,17,28,32,38,39,46). Our results also indicated that, for both HeLa and CEM cells, the susceptibility to radiation-induced DNA damage did not vary between cell cycle compartments. Although this was in agreement with previous studies using pooled semi-synchronized cells exposed to significantly higher radiation doses (ref. 27 and references therein), radiation doses similar to those used here have been observed to induce greater DNA damage in G1 phase cells than in G2 phase cells (17); it was suggested, however, that this may have been an artifact of the assay conditions and that the DNA damage did not likely vary with cell cycle position, a conclusion supported by our observations. At each
radiation dose, histograms by cell cycle phase of the extent of DNA damage versus cell number each resembled a normal distribution, similar to reports using SCGE (37,47).
The breadth (CV) of the distribution of DNA unwinding may be used to determine the likelihood of the presence of a subpopulation of resistant or susceptible cells. We did not observe distinct subpopulations within any cell cycle compartment of irradiated samples, in agreement with the findings of SCGE studies of irradiated proliferating cells analyzed either as a homogenous population (46) or by cell cycle phase (17,28). However, the ability to identify a subpopulation in a cell cycle compartment by SCGE may be compromised by the limited number of cells analyzed per sample (4,15,37) and the yet smaller number of cells in each cell cycle phase. This concern is obviated in the flow cytometric assay by the relatively large number of cells that are analyzed.
H2O2 transformation into reactive oxygen species, predominantly OH·, is likely catalyzed by transition metals complexed with DNA (29). The linear increase in DNA damage in each cell cycle compartment of HeLa and CEM induced by increasing concentrations of H2O2 we observed (Figure 7
) is similar to that reported in SCGE studies of proliferating cells analyzed as a homogeneous population (2,18,39,48). We also found that although the mean extent of H2O2-induced DNA damage did not significantly differ between G0/G1, S and G2/M phase cells, intercellular heterogeneity within each cell cycle compartment after H2O2 treatment was greater than was observed in untreated or irradiated samples. This suggests that there is substantial intercellular variability in antioxidant defense mechanisms (29,30). Although the minimum H2O2 concentrations that induced DNA damage detectable by the flow cytometric assay in CEM cells were similar to those reported to be detectable by SCGE in proliferating cells analyzed as a homogeneous population (18,39,48), the flow cytometric assay appeared to be relatively more sensitive in detecting DNA damage induced by low concentrations of H2O2 in HeLa cells. Again, the higher detection limits of SCGE in proliferating cells may be due to the inclusion of endogenous S phase-associated SB. Under conditions similar to those used here, Villani and colleagues (42) did analyze SCGE results by cell cycle phase. Their report that G1 phase cells were less susceptible to H2O2-induced DNA damage than were S or G2 phase cells did not agree with our observations of H2O2-treated cells nor did their measures of the cell cycle distribution of endogenous SB (42); this disagreement may have arisen from the practice of analyzing relatively few cells in each cell cycle compartment per SCGE sample. Although we found that there was substantial intercellular heterogeneity in H2O2-induced DNA damage, we did not observe differentially susceptible subpopulations within any cell cycle compartment, in agreement with the findings of SCGE studies evaluating H2O2-treated proliferating cells either as a homogenous population (1,5,31) or by cell cycle phase (42).
Our results suggested that CEM cells were more susceptible than HeLa cells to radiation-induced DNA damage, and that G0/G1 phase HeLa cells were more sensitive to H2O2-induced DNA damage than G0/G1 phase CEM cells. Differences in sensitivity to DNA damage have also been observed between cell types in some SCGE studies of irradiated (38,49) and H2O2-treated cells (1,2,5,48). Such differences may be related to variations in antioxidant defense mechanisms, enzymatic and nonenzymatic DNA repair processes, chromatin condensation, or the distribution or levels of DNA-bound proteins (29,30,50,51).
The DNA damage repair curves for HeLa and CEM cells treated with
radiation (Figure 8
) or H2O2 (Figure 9
) were similar to those shown in SCGE studies of proliferating cells treated with radiation (11,17,28,46) or H2O2 (1,46). Repair of this DNA damage likely occurred through direct religation of breaks and base excision repair (52). The slight increase in DNA damage seen in cells allowed 5 min of repair at 37°C after treatment with H2O2 on ice may have been caused by lipid or protein peroxides released from membranes (30,53) as warming restored membrane fluidity (54), or by the warming permitting repeated reduction (cycling) of DNA-complexed transition metals and hence further OH· production (55). We did not observe a subpopulation of G0/G1 cells exhibiting a relatively increased rate of repair following irradiation as was reported in a SCGE study (17); this difference might be ascribed to our use of an ~8-fold lower radiation dose and/or differences between cell types. The repair half-times found here of ~20 ± 7 min and 14 ± 4 min following
radiation and H2O2 treatments, respectively, of HeLa and CEM cells appeared to be comparable with, or in some cases, slightly longer than those observed in SCGE studies of proliferating cells treated with
radiation (16,17,28,) or H2O2 (2,5,46).
We also used the flow cytometric DNA alkaline unwinding assay to monitor DNA damage induced in HeLa cells by treatment with a clinically relevant concentration of doxorubicin (adriamycin) (56), a drug used to treat a wide variety of malignancies (57). Our data show that the DNA damage induced by doxorubicin was predominantly found in G2/M phase cells; this is consistent with SCGE studies (3,58) reporting that proliferating cells were most sensitive to the drug, as well as with reports that doxorubicin-induced cytotoxicity was elevated in S and G2/M phase cells (59) and that the drug was capable of inducing chromosome aberrations directly in G2/M phase cells (60). Doxorubicin is believed to act primarily through immobilizing topoisomerase (topo) II-DNA complexes (61,62), including the topo II
isoform expressed primarily in S and G2 phase cells (63), thereby preventing release of DNA torsion during transcription and replication (64). The DNA damage we observed predominantly in G2/M phase cells then was likely related to the accumulation of SB arising from collision of replication machinery with immobilized topo II
-DNA complexes as well as SB arising from disruption of chromosome separation (65,66). While doxorubicin may be enzymatically reduced to a free radical (67), a concentration of the drug several fold higher than used here is believed to be necessary to induce DNA damage via this mechanism (56).
We have shown that the flow cytometric DNA alkaline unwinding assay is a simple and rapid method for measuring DNA damage in large numbers of individual cells with a sensitivity comparable to that of other single-cell DNA damage assays. The cell-cycle phase (or ploidy) of responding cells can be identified, as can subpopulations of relatively resistant or sensitive cells and the intercellular heterogeneity of DNA damage and repair. We believe that the assay may be useful in studies of oncology, genetic toxicology, and DNA damage and repair.
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Notes
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3 Current address: Department of Anatomy and Neurobiology, Washington University in St Louis, St Louis, MO 63144, USA 
4 Current address: Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA 
5 Current address: Department of Surgery, University of Washington, Seattle, WA 98195, USA 
6 To whom correspondence should be addressed at: Box 357705, Department of Pathology, University of Washington, Seattle, WA 98195, USA Email: petersr{at}u.washington.edu 
 |
Acknowledgments
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We are grateful to Judy Anderson, Hai Hong, Chong Kim, Julianne Hill, Josh Renz, and Anthony Zeimet for excellent technical assistance, to Dr Narendra Singh for critical reading of the manuscript, and to Dr Barry Storer for critical review of the statistical analyses. This work was supported by NIH grants PO1 AG00057, P30 AG13240 and PO1 CA74184.
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Received November 19, 2001;
revised November 19, 2001;
accepted November 20, 2001.