Mutagenesis vol. 25 no. 3 pp. 299–303, 2010
Advance Access Publication 16 February 2010
doi:10.1093/mutage/geq006
Comet sensitivity in assessing DNA damage and repair in different cell cycle stages
Darragh G. McArt1,*, George McKerr2, Kurt Saetzler2,
C. Vyvyan Howard2, C. Stephen Downes2 and Gillian
R. Wasson2
1
Bioinformatics Unit, Centre for Cancer Research and Cell Biology, School of
Medicine Dentistry and Biomedical Sciences, Queen’s University Belfast, 97
Lisburn Road, Belfast BT9 7BL, UK and 2Biomedical Sciences Research
Institute, University of Ulster, Coleraine Campus, Cromore Road, Coleraine
BT521SA, UK
*To whom correspondence should be addressed. Bioinformatics Unit, Centre
for Cancer Research and Cell Biology, School of Medicine Dentistry and
Biomedical Sciences, Queen’s University Belfast, 97 Lisburn Road, Belfast
BT9 7BL, UK. Tel: þ44 (0)28 90975816; Fax: þ44 (0)28 90972776; Email:
d.mcart@qub.ac.uk
Received on November 26, 2009; revised on January 14, 2010;
accepted on January 27, 2010
The comet assay is a sensitive tool for estimation of DNA
damage and repair at the cellular level, requiring only a very
small number of cells. In comparing the levels of damage or
repair in different cell samples, it is possible that small
experimental effects could be confounded by different cell
cycle states in the samples examined, if sensitivity to DNA
damage, and repair capacity, varies with the cell cycle. We
assessed this by arresting HeLa cells in various cell cycle
stages and then exposing them to ionizing radiation. Unirradiated cells demonstrated significant differences in strand
break levels measured by the comet assay (predominantly
single-strand breaks) at different cell cycle stages, increasing
from G1 into S and falling again in G2. Over and above this
variation in endogenous strand break levels, a significant
difference in susceptibility to breaks induced by 3.5 Gy
ionizing radiation was also evident in different cell cycle
phases. Levels of induced DNA damage fluctuate throughout
the cycle, with cells in G1 showing slightly lower levels of
damage than an asynchronous population. Damage increases
as cells progress through S phase before falling again towards
the end of S phase and reaching lowest levels in M phase. The
results from repair experiments (where cells were allowed to
repair for 10 min after exposure to ionizing radiation) also
showed differences throughout the cell cycle with G1-phase
cells apparently being the most efficient at repair and
M-phase cells the least efficient. We suggest, therefore, that
in experiments where small differences in DNA damage and
repair are to be investigated with the comet assay, it may be
desirable to arrest cells in a specific stage of the cell cycle or
to allow for differential cycle distribution.
Introduction
DNA is under constant threat of damage from both exogenous
sources in the environment, such as solar radiation, and
endogenous agents, such as oxygen free radicals (1), and it is
essential that cells resolve this damage through DNA repair
mechanisms. However, the amount of DNA damage and the
efficiency with which the cell deals with this damage may vary
throughout the cell cycle (2). This may be due to a number of
factors including changes in chromatin conformation that may
make the DNA more susceptible to assault and reduce levels of
repair (3). In the case of ionizing radiation, the DNA damage
induced includes single-strand breaks (SSBs) and double-strand
breaks (DSBs) that, if not accurately repaired, can lead to cell
death or chromosomal instability. SSBs account for 90% of the
breaks caused by ionizing radiation (4,5) and are dealt with
using the single-strand break repair pathway that uses some of
the base excision repair pathway enzymes (6). DSBs can be
repaired by two different pathways: homologous recombination
(HR) and non-homologous end joining (NHEJ). In S and G2
phases of the cell cycle, DSBs are generally repaired by HR,
but in G1 phase, NHEJ is more commonly used in account of
the lack of the homologous sister chromatid (2).
The single-cell gel electrophoresis technique (comet assay)
is an established method of detecting DNA damage and repair
(7–9). Its versatility and requirement for only small numbers of
cells allow it to be used in a wide variety of areas from
environmental biomonitoring to assessing dietary effects,
typically using lymphocytes and cells from disaggregated
tissues (10–12). The method is relatively low in cost and
simple in procedure compared with other assays for DNA
damage. Cells in this procedure are fixed in agarose to a slide,
lysed to remove their cellular content and electrophoresed to
extract damaged DNA. The slides are then analysed by
fluorescence microscopy, typically with analytical software.
One of the main concerns of the comet assay has been about
sensitivity as well as reproducibility (13,14). The fact that an
asynchronous cell population may contain a number of
subpopulations with varying sensitivity to DNA may cause
a degree of variability in the results of comet assay experiments
(15,16). Previous results by Olive and Banáth (17) have
concluded that differences in DNA structure decrease the
sensitivity of DSB detection for cells in S phase.
In this study, we have used the alkaline comet assay to
analyse the number of strand breaks caused by ionizing
radiation and the efficiency of their repair at different stages of
the cell cycle. Cells are trapped in G1 phase, S phase and
released and tested every hour until G2 phase and finally at the
M phase. Cells are undamaged, exposed to 3.5 Gy irradiation
or irradiated and then allowed 10 min repair time. All
samplings were tested using flow cytometry to assure cell
cycle status. For comet analysis, we have employed the
systematic random sampling (SRS) method of selection that
gives a more precise measure of the estimates of the DNA
damage on a given slide and a more accurate interpretation of
information from a given population (18). The technique
operates by a random start point followed by a systematic
traversal of the slide making each other stop point a random
occurrence also. It allows the experimenter to gain unbiased
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299
D. G. McArt et al.
estimates of damage and a better statistical inference on the
data obtained.
Materials and methods
Cultivation and extraction
HeLa cells were obtained from the European Collection of Cell Cultures
(Wiltshire, UK) and were cultured in minimum essential medium (MEM)
supplemented with 10% foetal bovine serum and 1% (v/v) penicillin–
streptomycin (5.0 IU penicillin and 5.0 lg streptomycin/ml).
G1-phase arrest. On Day 1, cells were plated onto 90-mm Petri dishes with 5
ml of MEM. After 24 h, the medium was removed and the plate washed three
times with 4 ml phosphate-buffered saline (PBS) before adding 5 ml of
isoleucine-depleted medium supplemented with 10% dialysed foetal calf serum
and 1% penicillin–streptomycin. After 28 h incubation, the Petri dishes were
washed three times with PBS and cells were removed from their monolayer
using 1 trypsin–ethylenediaminetetraacetic acid (EDTA) (Invitrogen).
S phase. As before, cells were plated in 90-mm Petri dishes with MEM on Day
1. On Day 2, MEM was removed and the plate was washed with PBS before
adding 5 ml of MEM containing 2 mM thymidine. After 19.5 h incubation, the
MEM/thymidine was removed and the cells were washed three times with 4 ml
PBS. Then 4.5 ml of fresh normal MEM was added and the dishes were placed
back in the 37°C incubator for 9.5 h. The MEM was then removed and the cells
were washed with 4 ml PBS before a second thymidine block was carried out
using 5 ml of MEM containing 2 mM thymidine for 18 h. The MEM was then
removed and the cells were washed three times with PBS. The majority of cells
should be in S phase at this point. MEM was added to all dishes except the time
point (t) 5 0 dish which was removed from its monolayer using 1 trypsin–
EDTA. All other dishes were trypsinized on the hour until t 5 7, represented by
S(t) in figures and interpretation.
Mitosis. Cells were grown in 175 mm2 tissue culture flasks until they were
subconfluent; the medium was removed and the cell monolayer washed with
PBS. MEM containing 40 ng/ml nocodazole was then added to the flask and the
cells were incubated for 4 h at 37°C. At hourly intervals, the flask was tapped to
encourage detachment of rounded-up mitotic cells and their subsequent
removal from their monolayer. After 4 h, medium and rounded-up cells were
removed by pipette, spun down and re-suspended in cold PBS.
Flow cytometry. Cells that were removed from their monolayer using 1
trypsin or nocodazole were immediately spun down, re-suspended in 5 ml
cold PBS and had 4 ml removed and placed into 70% ethanol at 20°C for
flow cytometric analysis. One millilitre was processed for the comet assay.
Each of the nine stages of the cell cycle points assessed was checked by flow
cytometry to establish validity. For this, the cells in 70% ethanol were spun
down and ethanol was poured off before washing twice with ice-cold PBS.
Cells were then re-suspended in 1 ml propidium iodide staining buffer (10 ll/
ml propidium iodide, 0.1 mg/ml RNAse A, 0.1% IgePal, 50 lg/ml trisodium
citrate in PBS, H2O), incubated for 30 min at room temperature in the dark and
analysed by histogram plots of count versus propidium iodide-A and dot plots
of forward scatter versus side scatter on a BD FACSCalibur.
Irradiation and alkaline comet assay
Comet assay slides were prepared using the protocol described by Wasson et al.
(19). Briefly, 200 ll of normal-melting-point agarose was pipetted onto frosted
slides and spread by the lowering of coverslips (22 50 mm) to ensure an even
agarose spread and left briefly to solidify. Coverslips were then removed. Cells
were re-suspended in 100 ll of low-melting-point agarose that was in turn pipetted
onto the normal-melting-point agarose layer, spread with coverslips and left
briefly to solidify. Slides that were to be damaged were placed on ice and irradiated
with 3.5 Gy c-radiation at a dose rate of 2 cGy/s using a Mainance Cs137 source.
After irradiation, slides had their coverslips gently removed and were
submerged into a Coplin jar containing freshly prepared cold neutral lysis
solution (2.5 M lithium chloride, 0.03 M EDTA, 10 mM Tris, 0.1% lithium
lauryl sulphate, pH 8) and 600 ll of Proteinase K, along with control slides.
The Coplin jar was then placed into a 4°C room for 1 h to prevent repair while
initial lysis was occurring, then into a 37°C water bath overnight. Slides that
were allowed to repair were placed in a Coplin jar containing medium in
a water bath at 37°C for 10 min and then into the neutral lysis as above. All
slides were then transferred into a Coplin jar containing alkaline lysis solution
(2.5 M sodium chloride, 100 mM EDTA, 10 mM Tris, pH 10, with 1% Triton
X-100) and returned to 4°C for 1 h.
300
From this stage, all work was carried out in a 4°C room, to allow for
standardization of the experiments. The slides were immersed in freshly
prepared cold electrophoresis solution (300 mM sodium hydroxide, 1 mM
EDTA, pH .13). This solution was filled to a level that covered the slides by
0.5–1 cm. Slides were then left in the solution to unwind the DNA for 20 min.
Electrophoresis was conducted at 25 V and 0.3 A for 20 min. Slides were taken
out and washed three times gently with neutralization solution (0.4 M Tris, pH
7.5) for 5 min per wash, to stabilize the pH. Slides were drained and stained
with 30 ll of 2 lg/ml ethidium bromide, spread under a coverslip, placed in
a humidified chamber and stored in the dark in a 4°C room, prior to analysis.
All solutions, unless otherwise stated, are from Sigma-Aldrich, Poole, UK.
Image analysis
Comet analysis was performed using a Nikon Eclipse E400 epifluorescence
microscope fitted with a Nikon 20 fluorescence objective lens (Plan Apo 20
0.75) and visualized with a Hamamatsu Orca digital CCD camera with
a filter set for ethidium bromide (excitation 510 nm, emission 595 nm). Comet
acquisition and analysis were carried out using Kometþþ with Orca1, version
1, Kinetic Imaging Ltd. The measurement assessed in this study was that of %
tail DNA. Slides were analysed using the SRS method (18).
Statistical analysis
Analysis was carried out initially with Microsoft Excel, with SPSS being used
for specific tests. One-way analysis of variance (ANOVA) was used to establish
differences between groups with Tukey’s post hoc test used to negate the
problem with multiple comparisons. Tukey’s method constructs confidence
intervals of all pairs of means by their differences to allow simultaneous
coverage probability. The duality of confidence intervals and tests can then be
examined for significant differences between pairs (20). Independent t-tests
were carried out on any paired tests.
Results
Levels of endogenous strand breaks were measured at different
stages of the cell cycle in HeLa cells with % tail DNA ranging
from 4.3% in G2/M to 10.7% in S3 (Figure 1A). Analysis by
ANOVA showed that there was a significant difference in the
basal levels of DNA damage in different cell cycle stages (P ,
0.001). Utilizing Tukey’s post hoc test, basal levels of strand
breaks can be split into three subsets with similar levels of
strand breaks: with S3 and S5 forming one group; asynchronous, G1, S0 and S7 a second group; with G1 and G2, showing
similar levels with M phase, the third group. When cells were
treated with 3.5 Gy of irradiation, % tail DNA results again
varied significantly throughout the cell cycle (P , 0.001),
ranging from 8.3% in M to 23.8% in S3 (Figure 2A and B).
Tukey’s test gave rise to four subsets with M, S3 and S5 each
being significantly different from the others and asynchronous,
G1, S0 and S7 having similar levels of damage (P . 0.9). After
10 min repair, % tail DNA ranged from 7.1% in M to 17.5% in
S3 and again varied to a significant extent throughout the cell
cycle (P , 0.001) when analysed by ANOVA. Tukey’s test
gave three subsets: S3 and S5 being similar; asynchronous, G1,
M and S0 forming another subset and S7 being similar to S0.
Figure 1B shows the amount of DNA damage remaining
when basal levels are deducted; it shows that mid-S phase had
the highest amount of damage inflicted by the irradiation when
examined with .12% tail DNA as opposed to ,10% for G1
and 8% in G2, which was enhanced using dual lysis and
incorporating Proteinase K and LiDs.
To establish the levels of repair, the average basal levels of
strand breaks were subtracted from both their respective
irradiated and repaired values. This showed a mixed ability
of the cells to repair DNA damage throughout the cell cycle.
Asynchronous, G1 and S0 cells were able to repair .70% of the
strand breaks in 10 min but this repair rate fell throughout S
phase to M, with levels of 50% (S3), 46% (S5), 45% (S7) and
29% (M) (Figure 1C and D).
Comet sensitivity in assessing DNA damage and repair
Fig. 1. (A) Line chart depicting the pooled averages of three trials for the cell cycle showing three levels of measurement: control (ct), damaged (3.5 Gy) and
repaired (10 min). (B) Chart with joint error bars using standard error (SE) of means, showing the differences in the cell cycle in damage accrued once the ct level of
damage for that cycle is deducted. (C) Chart with joint error bars (SE of means), which illustrates the repair throughout the cell cycle of the comet assay if the repair
averages are subtracted from its corresponding irradiated cycle stage, which shows a visual decline in repair levels. (D) Graph representing the % repair going
through the cell cycle stages.
It can be seen from Figure 1A that cells at the entrance to S
phase and exit to G2 have similar levels of basal strand breaks and
also similar levels of strand breaks after irradiation. Independent
t-tests (two-tailed) showed that these were not different (P 5 0.49
for control levels and P 5 0.524 for irradiated). However, repair
levels for both were significantly different (P 5 0.002), with G1
and early S-phase cells having more efficient repair.
M phase showed the lowest levels of DNA strand breaks
either at basal level or after 3.5 Gy of irradiation and also gave
the lowest levels of repair. However, analysis by independent ttest suggested that, although there was only 1% difference in
means (8.3% in irradiated and 7.1% in repair), repair in the
cells was significant (P 5 0.012).
Discussion
The comet assay’s ability to measure strand breaks in
individual cells allows us to look at DNA damage at a cellular
level and to detect heterogeneity within a sample. However,
differences in susceptibility to damage and ability to repair at
different stages of the cell cycle may influence the results
obtained from the comet assay.
Our results suggested that cells in G1 are more susceptible to
ionizing radiation-induced strand breaks than cells in G2, with
G1 cells showing 63.6% damage above basal levels as opposed
to 55.9% at G2 (S7), as a percentage of total damage divided by
the basal levels. Levels of repair also varied between the stages
with 75% of breaks being repaired in G1 within 10 min
compared to 45% in G2. These results are not inconsistent with
previous studies using yeast and mammalian cells, which have
shown corresponding inverse differences in the susceptibility
of cells in G2 and G1 to UV-induced DNA damage (21,22);
however, unlike our study that looked directly at DNA damage
and repair, these studies looked at mutation frequencies and
lethality, which are the outcome of a long and complex process
of which repair is only a part. Our results, taken together with
these previous studies, suggest a trend towards poor G2 repair
(23). One possible explanation for this is that cells in G2 have
already replicated their DNA in preparation for mitosis and
therefore have double the amount of DNA without increasing
their repair capacity. Therefore, the perceived difference in
repair levels may be due to the greater amount of DNA in G2
rather than any increased repair proficiency in G1.
Previous studies on DNA damage susceptibility throughout
the cell cycle of HeLa cells irradiated with UV showed that
increased number of dimers may be induced as cells pass
through the G1 phase of the cell cycle and into early S phase
(3). Similar results were found for ionizing radiation in our
study; we observed a 63.6% damage increase in G1 phase after
3.5 Gy of ionizing irradiation. Overall the most SSBs were
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D. G. McArt et al.
Fig. 2. (A) Comets (HeLa cells) that were subjected to 3.5 Gy synchronized at
G2/M. (B) Comets that were subjected to 3.5 Gy synchronized at S phase (T3).
Showing illustratively the difference in both size and damage sustained.
observed in early to mid-S phase with basal levels of % tail
DNA reaching as high as 10.7% and ionizing radiation causing
a % tail DNA of 23.4% at time point S3. These overall damage
levels reduced as the cell cycle reached the latter stages of S
phase, G2 and particularly M.
It is noticeable that comet results for S phase for all three
conditions, basal, damaged and repair, showed elevated levels
of strand breaks compared to other areas of the cell cycle. The
trend in the results went from a low at the beginning of S
phase, increasing to mid-S phase and decreasing again going
towards G2, with the cells’ ability to repair also decreasing. The
high basal level of strand breaks in S phase is unsurprising
given the number of free ends and strand breaks generated
during DNA replication. Graubmann and Dikomey (24)
suggested that the levels of SSB induction do not vary in the
cell cycle, and indeed this may be the case, but chromatin
decondensation increases to allow replication, and the
consequent increase in nuclear volume must produce a greater
absorption of radiation by the nucleus.
Cells in M phase had the lowest amount of basal-level strand
breaks and the lowest amount of damage after exposure to
ionizing radiation (8.3%); they also had the lowest amount of
repair (7.1%) of the strand breaks. This could be a direct
consequence of the fact that DNA is highly condensed at this
stage of the cell cycle; there is a smaller volume to absorb
radiation but also, when damage occurs, carrying out repair
may be more difficult. Chromatin is more decondensed in S
phase and G1 than in M, with more loosely bound DNA
increasing absorption rates and therefore increasing damage.
Oleinick et al. describe this differential sensitivity in the active
302
and non-active chromatin as an accessibility differential.
Oleinick et al. also describe it as a hypothesis of differential
accessibility where active sequences receive more rapid repair.
They found that metaphase cells of Chinese hamster V79-379
also had slower repair levels in radiation-induced strand breaks
than in interphase cells (25).
Olive et al. used the neutral comet assay to analyse DNA
damage throughout the cell cycle in CHO and V79 hamster
cells. In contrast to the alkaline comet assay that can detect
both SSBs and DSBs, the neutral comet assay of Olive et al.
(26) measures DSBs only. They found that there was less
apparent damage to cells irradiated in S phase than at any other
cycle stage and suggested that this may be due to changes in
DNA packaging in early S phase inhibiting the migration of
DNA. Our results in comparison, with the exception of M,
showed that the G1 and G2 phases appeared less susceptible to
damage by radiation than S phase, as can be seen in Figure 1B.
However, our study used the alkaline version of the comet
assay that allows for greater sensitivity at lower doses of
radiation with our results describing the damage and repair due
to 3.5 Gy and Olive et al. stating that the lowest dose
discernible was 5 Gy.
An advantage of our technique is the use of both neutral lysis
solution with Proteinase K and alkaline lysis before unwinding
to allow for better and consistent cell lysis. The removal of the
cell soluble constituents, thus leaving only the cellular matrix
and DNA, increases the amount of DNA movement into the
tail. As part of an unpublished separate study, we have shown
using scanning electron microscopy that the incorporation of
the neutral lysis (LiDs) plus Proteinase K step removes
substantially more cellular content than 1-h alkaline lysis
alone. This dual lysis technique was performed by McGlynn
et al. (27) for the bromodeoxyuridine comet assay. It allowed
for better differentiation between head and tail, on account of
liberation of DNA from the nuclear matrix.
An important aspect of our study was that, like the paper by
Downes and Collins (28), it was performed on a human cell
line, in contrast to previous studies that focused on CHO and
V79 rodent cell lines (24,26). There are subtle differences in
repair activities between human and rodent cell lines (29–32)
and this is an important aspect to keep in mind when designing
experiments for use in cancer therapies. Another aspect of this
study was the use of SRS in the selection process of comets.
This method removes experimenter bias while increasing the
precision of the estimates of DNA damage (18).
As DSBs induced by ionizing radiation are thought to be the
lesions responsible for cell death due to irradiation, DSB repair
is an important aspect of cellular research. H2AX phosphorylation has been used in comet studies by Olive and Banáth
(33) as a sensitive indicator of radiosensitivity by DSBs. Kato
et al. (34) have demonstrated a reduction in the disappearance
of H2AX-c foci between G1 and mitosis and shown
hypersensitivity of the mitosis phase in comparison to G1.
They suggested that this may be as a direct result of
compaction of DNA inhibiting the access of dephosphorylation
enzymes. Susceptibility to radiation in the cell cycle was also
reported by Terzoudi et al. (35) demonstrating that cdk1/
cyclinB levels during G2 to mitosis impair DNA repair
processes and are a large factor in chromatin break numbers.
Our data suggest that that there is a decreased level of repair
between G2 and mitosis and that this is discernible from G1.
Although our study looked at SSB repair, it may further
demonstrate the condensing of DNA as a factor in repair.
Comet sensitivity in assessing DNA damage and repair
The cell’s susceptibility to exogenous damaging agents is
quite a complex issue but it is believed that the organization of
chromatin within the nucleus may have an important role in the
cell’s ability to survive, with one hypothesis suggesting that
chromatin destabilizes locally to allow repair after damage and
restoration of chromatin upon repair (36–38). The compaction
or relaxation of the chromatin during a specific cell cycle stage
may increase or decrease the volume available for absorption
of irradiation. Our results showed that there were significant
differences between different stages of the cell cycle not only at
the stage of damage or repair but even at a basal level showing
the sensitivity of the comet assay. Therefore, we would
question the use of asynchronous cells in studies where low
amounts of strand breaks are being observed, particularly if
analysed without unbiased sampling techniques.
Acknowledgements
The authors would like to thank Dr Declan McKenna and Dr Barry O’Hagan
for their help with equipment and their expertise during the experiment and
Dr Mary Jo Kurth for cell cycle information.
Conflict of interest statement: None declared.
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