[CANCER RESEARCH 59, 5912–5916, December 1, 1999]
Advances in Brief
The Bromodeoxyuridine Comet Assay: Detection of Maturation of Recently
Replicated DNA in Individual Cells1
Angela P. McGlynn, Gillian Wasson, Jacqueline O’Connor, Valerie J. McKelvey-Martin, and C. Stephen Downes2
Cancer and Ageing Research Group, School of Biomedical Sciences, University of Ulster, Coleraine BT52 1JD, Northern Ireland
Abstract
The single-cell gel electrophoresis (Comet) assay is a relatively simple
method of measuring DNA single strand breaks and alkali-labile sites in
individual cells. We have combined this with bromodeoxyuridine
(BrdUrd) labeling of DNA and immunolocalization of the BrdUrd to
assess DNA replicative integrity on a single-cell basis. We show that the
existence of strand discontinuities in recently replicated domains of DNA,
caused during semiconservative replication or exacerbated by the arrest
of replicative polymerases at UV irradiation- or chemical-induced lesions,
can be detected in individual cells. Data obtained from BrdUrd-Comets
are consistent with biochemical data derived with a range of techniques
showing that DNA replication involves the creation of strand breaks or
gaps adjacent to recently replicated material, and that DNA damage
prolongs the duration of such discontinuities where DNA polymerases are
stalled opposite lesions (R. T. Johnson et al., The Legacy of Cell Fusion,
pp. 50 – 67, Oxford: Science Publications, 1994; R. B. Painter, J. Mol. Biol.,
143: 289 –301, 1980.). Compared with standard biochemical techniques,
the BrdUrd-Comet assay is simple and suitable for the accurate and
automatable assessment of replicative integrity in very small numbers of
mammalian cells, such as may be obtained by biopsy.
Introduction
In DNA replication, strand discontinuities inevitably occur at the
leading and lagging strand growth points. In normal replication, DNA
polymerases rapidly move on. Label incorporated into DNA during a
brief pulse is initially close to strand discontinuities, but during
pulse-chase experiments, such label is soon incorporated into high
molecular weight, continuous DNA, far distant from any discontinuities. The process by which this occurs is known as DNA maturation.
After DNA damage, replication is affected. DNA polymerases
initialy stall at noncoding damage sites (1–3). Through the process of
PRR,3 polymerases bypass lesions or avoid them through retrospective gap filling (4). One striking case of deficiency in DNA maturation
is the Indian muntjac line SVM84, which (compared with normal
DM87 Indian muntjac cells) are hypersensitive to UV irradiation and
are deficient, although not entirely defective, in UV PRR capacity (5).
In DM87 cells, the PRR capacity is largely caffeine sensitive, whereas
in SVM 84 cells, the residual PRR is caffeine insensitive (6).
PRR in these cases, and in other mammalian cells, has normally
been studied by biochemical methods: [3H]thymidine pulse-labeling,
followed by alkaline sucrose gradient centrifugation (7), or by chromatography on BND-cellulose or hydroxyapatite (8). These tech-
niques, however, are time consuming and require both radioactive
labeling of DNA and the use of large cell populations (;105 per data
point) for which only an average figure can be obtained.
An alternative method for measuring DNA strand breaks and alkalilabile sites in single cells, the Comet assay, has been in use for some
years (9, 10). In this assay, single cells are embedded in agarose on
frosted slides, lysed in denaturing buffer, subjected to brief electrophoresis, stained with DNA intercalating dye, and observed with
fluorescence microscopy. Undamaged DNA is unable to enter the
agarose gel and is retained in the cavity formed by the lysed cell;
damaged DNA streams down the electrophoretic field and forms the
“tail” of a Comet. Software packages have been devised to quantitate
the distribution of DNA between damaged Comet tails and undamaged Comet “heads.” Modifications of the Comet assay have been
developed to detect more complex DNA lesions, provided they are
endonuclease sensitive (11). More recently, we have combined the
Comet assay with fluorescence in situ hybridization to allow detection
of damage within defined gene sequences (12).
We have combined this method with pulse-labeling of replicating
DNA with the thymidine analogue BrdUrd and immunological localization of BrdUrd within the heads and tails of Comets to assess
replicative integrity on a single-cell basis. The BrdUrd-Comet assay is
described using UV-irradiated or caffeine-treated muntjac fibroblasts,
DM87 and SVM84, and human T lymphocytes.
Materials and Methods
Isolation and Growth of Human T Lymphocytes. Lymphocytes were
extracted from whole blood according to the method described previously (13).
For all experiments using human T lymphocytes, the Raji (TK1) Burkitt’s
lymphoma cell line (14) was included as a control and maintained in exponential growth in RPMI 1640 supplemented with 15% fetal bovine serum and
antibiotics.
Fibroblast Growth and Irradiation. The Indian muntjac fibroblast cell
lines, DM87 (American Type Culture Collection, , MD) and SVM84 (a gift
from Professor K. Sperling, Berlin, Germany) were plated at a density of
1 3 105 cells/60-mm Petri dish in complete medium as described (5) and
grown for 48 h to ensure exponential growth prior to irradiation. Cells were
exposed to UV irradiation (254 nm) using 2 J/m2 and incubated in complete
medium at 37°C for 1 h to recover prior to BrdUrd pulsing. Caffeine (Sigma,
Dorset, United Kingdom) was used at a concentration of 2 mM and was added
30 min before UV irradiation, where indicated.
BrdUrd Labeling and Processing of Cells. Medium was removed from
exponentially growing cells (with or without irradiation) and replaced with
fresh medium containing 100 mM BrdUrd (Sigma) at 37°C for 15 min. Medium
containing BrdUrd was washed off with PBS (pH 7.4); cells were either
Received 6/1/99; accepted 9/3/99.
processed for the Comet assay or, in chase experiments, given medium
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
containing each of 200 mM thymidine, 29-deoxycytidine, 29-deoxyguanosine,
18 U.S.C. Section 1734 solely to indicate this fact.
and 29-deoxyadenosine (Sigma). Fibroblasts were harvested by brief trypsini1
This work was entirely supported by Contract D215 from the Ministry for Agriculture
sation, and lymphocytes were harvested by centrifugation, both were and
Food and Fisheries, U.K.
2
To whom requests for reprints should be addressed, at Cancer and Ageing Research
resuspended in ice-cold PBS at 5 3 104 to 1 3 105 cells/ml, respectively. Cells
Group, University of Ulster, School of Biomedical Sciences, Coleraine BT52 1JD,
were washed in cold PBS, and the cell pellet was resuspended in 100 ml of
Northern Ireland. Phone: 44-1265-324121; Fax: 44-1265-324965; E-mail: CS.Downes@
0.75% low melting point agarose at 37°C. The cell suspension was spread onto
ulst.ac.uk.
3
a standard Comet assay slide (15), and the slides were immersed in a neutral
The abbreviations used are: PRR, post replicative repair; BrdUrd, bromodeoxyurilysis buffer, adapted from that described by Blin and Stafford (16): 2.5 M LiCl,
dine.
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SINGLE-CELL DETECTION OF DNA MATURATION
0.03 M EDTA, 10 mM Tris, and 0.1% LiDS (pH 8.0), prewarmed to 37°C
before the addition of 0.03 mg/ml proteinase K. Slides were incubated at 37°C
overnight for protein digestion and cell lysis. This lysis solution was used
because of the superior solubility of LiDS compared with SDS. Slides were
then transferred to alkaline lysis solution (2.5 mM NaCl, 100 mM Na2EDTA,
and 10 mM Tris, pH 10) for 1 h at 4°C.
Comet Electrophoresis and Staining. Slides were removed from lysis
solution, drained, and placed in a horizontal electrophoresis tank, side by side
with gaps filled with blank slides. The tank was filled with fresh electrophoresis buffer (0.3 M NaOH, 1 mM EDTA, pH 13) to a level just covering the slides,
and the DNA was left to unwind for 40 min before electrophoresis. Electrophoresis was conducted for 20 min at 25 V and 300 mA. After electrophoresis,
the slides were drained and flooded with three changes of neutralization buffer
(0.4 M Tris), followed by one wash in PBS to allow removal of alkali and
detergents that would interfere with immunostaining. The incorporated BrdUrd
was immunolocalized by adding 25 ml/gel of mouse monoclonal anti-BrdUrd
(10 mg/ml; Boehringer Mannheim) to the gels, with coverslips (18 3 18-mm)
applied on top of the gels, allowing the antibody to spread evenly. Slides were
incubated in the dark at room temperature for 1 h. After removal of the
coverslips, the primary antibody was gently washed off with three changes of
PBS and one wash with PBS/0.1% BSA before addition of 25 ml/gel of
secondary antibody (5 mg/ml sheep anti-mouse IgG, fluorescein conjugated;
Boehringer Mannheim), which was incubated and washed off as before. The
gels were then counterstained with 25 ml of propidium iodide (0.75 mg/ml;
Sigma) and covered with a coverslip (22 3 50-mm) for image analysis.
Comet Analysis. Observations were made at a final magnification of 3400
(Nikon 340 Fluor lens) using an epifluorescence microscope (Olympus BH2)
equipped with an Hitachi KP571 CCD camera interfaced through a Matrox IP8
board, using Hewlett Packard Super VGA and Komet 3.0 software (Kinetic
Imaging Ltd., Liverpool, United Kingdom). Propidium iodide was visualized
using a Chroma filter at excitation 540 nm and emission 635 nm. Fluorescein
was visualized using a Chroma filter at excitation 480 nm and emission 535
nm. Densitometric and geometric parameters of each Comet were calculated
and recorded for statistical analysis using Microsoft Excel, version 5.0, software.
The percentage of DNA in the Comet tail for each cell, which is indicative
of the number of replicons in the replicating DNA, was measured. Results were
expressed as the distribution of the percentage of Comet tail DNA in 25 cells
on each Comet slide. The incorporation of the BrdUrd label into the Comet
head after the chase period is indicative of DNA maturation and is represented
by a reduction in percentage of tail DNA for each of 25 Comets.
Results
BrdUrd-Comet Assay Development. Several improvements of
the initially tested assay were made by altering lysis and staining
conditions. The standard Comet assay for the detection of DNA strand
breaks is carried out under alkaline conditions (15); however, use of
neutral lysis facilitated nuclear matrix protein digestion using proteinase K at 37°C. This improvement to the technique, which is not
normally used in the alkaline Comet assay, greatly improved cell lysis
and BrdUrd-Comet formation (Fig. 1). In addition, the use of LiCl and
LiDS in place of NaCl and SDS, commonly used for cell lysis, also
provided greater cell lysis because of the greater solubility of LiDS
compared with SDS; in neutral lysis, SDS was found to precipitate at
37°C. Comet detection was improved with the application of a second-stage antibody to detect BrdUrd-labeled DNA. The resulting
Comets, using the improved protocol, displayed greater movement of
DNA strands through the agarose gel with greater differentiation
between Comet head and tail.
Calibration of Assay Against Fibroblasts of Known PRR Propensity. Treated and control SVM84 and DM87 fibroblasts were
pulsed with BrdUrd for a short time and either processed directly for
the BrdUrd comet assay, or the incorporated BrdUrd was chased with
normal nucleotides for a period of 1 h.
After UV irradiation (2 J/m2) of SVM84 cells, the amount of
BrdUrd-labeled DNA moving into the Comet head was reduced
Fig. 1. Optimization of the BrdUrd-Comet assay. BrdUrd-Comets of untreated fibroblasts using original (a and b) and improved (c and d) conditions for BrdUrd pulse, cell
lysis, and BrdUrd detection are shown. Comets shown in d show improved cell lysis and
electrophoretic mobility of recently replicated DNA fragments. Conditions used were: a
and b, 30 min of BrdUrd incorporation, followed by alkaline lysis in NaCl/SDS buffer at
4°C, followed by one-step immunolocalization of BrdUrd; c and d, 15 min of BrdUrd
pulse, followed by overnight neutral lysis in LiCl/LiDS buffer with proteinase K at 37°C,
followed by alkaline lysis as above at 4°C for 1 h, and two-step immunolocalization of
BrdUrd. Green fluorescein staining (BrdUrd labeling) and red propidium iodide staining
(DNA labeling) were used.
compared with the nonirradiated cells. No further effect was seen in
the presence of caffeine (Fig. 2). These results confirm the maturation
deficiency of UV-irradiated SVM84 fibroblasts (5).
In the nonirradiated DM87 cells, the level of Comet tail BrdUrdlabeled DNA was reduced in the DM87 cells after 1-h chase. This
competency in DNA maturation is similar, regardless of the presence
of caffeine in the nonirradiated DM87 cell line (Fig. 3, nonirradiated
cells).
In the absence of caffeine, UV irradiation alone (2 J/m2) induced an
increase in the percentage of DNA in the BrdUrd-Comet tail of DM87
cells (Fig. 3, irradiated cells). However, after a 1-h chase period with
normal nucleotides, there is a retardation of maturation in the presence
of caffeine compared with cells exposed to UV irradiation alone in the
absence of caffeine (Fig. 3, irradiated cells). These results confirm the
reduced efficiency of DNA maturation in UV-irradiated, but not
nonirradiated, DM87 cells exposed to caffeine (6).
PRR after Low-Dose Hydrogen Peroxide Detected by the
BrdUrd Comet Assay in Human T Lymphocytes. UV irradiation
produces large helix-distorting lesions (17); we have also exposed
isolated T lymphocytes to hydrogen peroxide, an agent that induces
small base lesions. After 72 h of culture, H2O2 induced a measurable
increase in BrdUrd-Comet tail moment in these cells. This is indicative of a delay in maturation occurring in these cells after exposure to
a low dose of the DNA-damaging agent, H2O2 and occurs prior to the
onset of measurable or lethal DNA damage, as detected by Comet
analysis of bulk DNA (Fig. 4).
Discussion
In this study, we have developed a novel assay for the detection of
DNA synthesis and maturation in single cells. We have demonstrated
that the novel BrdUrd-Comet assay can be further used to detect
delays in replication on a damaged template after UV irradiation or
after exposure to caffeine. Caffeine has been shown to potentiate
cytotoxic, mutagenic, and chromosome-damaging effects of DNAdamaging agents by alternative mechanisms in different cell types;
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SINGLE-CELL DETECTION OF DNA MATURATION
Fig. 2. BrdUrd-Comet analysis of SVM84 fibroblasts after exposure to UV irradiation. Nonirradiated and UV-irradiated (2 J/m2 with 1-h recovery period) cells were pulsed with
BrdUrd in culture for 15 min and either processed immediately for the BrdUrd-Comet assay or chased with 200 mM of all four deoxynucleotides for 1 h prior to Comet processing.
Results are expressed as: A, frequency distribution plots of BrdUrd-Comet percentage of tail DNA; a, BrdUrd pulse alone; b, BrdUrd pulse with chase; and c, BrdUrd pulse with chase
in the presence of caffeine; B, representative Comet images, where identical fields are shown with green fluorescein staining (BrdUrd labeling) and red propidium iodide staining (DNA
labeling) using appropriate fluorescent filters; a, BrdUrd pulse alone; b, BrdUrd pulse after UV irradiation; c, BrdUrd pulse with chase; and d, BrdUrd pulse with chase after UV
irradiation.
inhibition of postreplication recovery has been reported in caffeinesensitive DM fibroblasts (6). In the present study, caffeine-treated
DM87 cells demonstrated a marked increase in the amount of BrdUrdlabeled DNA in the Comet tail (Fig. 3), which is indicative of an
increase in the number of replicons, a known mechanism for the
action of caffeine in DNA maturation delay (6). Caffeine retarded
maturation on a damaged, but not on an undamaged, template in
DM87 cells exposed to UV irradiation (Fig. 3). This is consistent with
the alkaline sucrose sedimentation data described in muntjac fibroblasts (6), where caffeine was shown to block any bypass of UVinduced damage during replication. SVM cells, on the other hand,
have been shown to be replication incompetent after exposure to low
fluences of UV irradiation and were unaffected by the presence of
caffeine (7, 8). Our data confirm these findings (Fig. 2).
Data shown in this study, using the BrdUrd-Comet assay, is therefore parallel to the biochemical data derived earlier and offer a simple
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SINGLE-CELL DETECTION OF DNA MATURATION
Fig. 3. Frequency distribution curves of
BrdUrd-Comets of DM fibroblasts exposed to caffeine. Nonirradiated and UV-irradiated (2 J/m2
with 1-h recovery period) cells were pulsed with
BrdUrd in culture for 15 min and either processed
immediately for the BrdUrd-Comet assay or chased
with 200 mM of all four deoxynucleotides for 1 h
prior to Comet processing. Results are expressed as
frequency distribution plots of BrdUrd-Comet percentage of tail DNA. Where indicated, caffeine (2
mM) was present for 30 min prior to irradiation and
during BrdUrd pulse and chase periods. a, BrdUrd
pulse only; b, BrdUrd pulse with chase; c, BrdUrd
pulse with chase in the presence of caffeine.
alternative to other laborious and time-consuming biochemical methods that involve either tedious centrifugation or meticulous preparation of human DNA. In addition, small numbers of cells are sufficient
for analysis, and the technique is therefore potentially suitable for the
analysis of single cells derived from small patient biopsies.
We have also exposed isolated T lymphocytes to hydrogen peroxide, an agent that induces small base lesions (18) and plays a role in
a variety of physiological and pathological processes, in particular
aging and carcinogenesis (19). Cells exposed to a low dose of the
DNA-damaging agent had more DNA breaks adjacent to fluorescently
labeled repair sites visible in the Comet tail than control cells, indicating the sublethality of this low dose and the ability of the cells to
repair any strand breaks formed. Indeed, this effect is visible prior to
the onset of measurable DNA damage, evident by the lack of DNA
Fig. 4. BrdUrd-Comet images of isolated human T
lymphocytes. Cells were cultured for 72 h and processed
for BrdUrd-Comet analysis. Control cells were stained
with PI (a) and BrdUrd (b) and double exposed for
propidium iodide and BrdUrd staining (c) cells treated
with 10 mM H2O2 at 4°C prior to processing and stained
with PI (d) and for BrdUrd (e) are shown.
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SINGLE-CELL DETECTION OF DNA MATURATION
damage (Comet formation) in propidium iodide-stained whole DNA
in these cells.
The present data on PRR in the two muntjac cell lines represent a
semiquantitative analysis of the rates of DNA maturation. Further
development of the assay are ongoing to determine the lengths and
activity of labeled DNA molecules. Furthermore, the BrdUrd-Comet
assay is applicable to the detection of DNA repair after early DNA
damage induced by hydrogen peroxide in isolated T lymphocytes.
References
1. Johnson, R. T., Downes, C. S., Godfrey, D. B., Hatton, D. H., Ryan, A. J., and
Squires, S. Some insights into the replication of damaged DNA in mammalian cells.
In: S. Gordon (ed.), The Legacy of Cell Fusion, pp. 50 – 67. Oxford: Science
Publications, 1994.
2. Painter, R. B. Effect of caffeine on DNA synthesis in irradiated and unirradiated
mammalian cells. J. Mol. Biol., 143: 289 –301, 1980.
3. Painter, R. B. Inhibition and recovery of DNA synthesis in human cells after exposure
to ultraviolet light. Mutat. Res., 145: 63– 69, 1985.
4. Fujiwara, Y., and Tatsumi, M. Replicative bypass repair of ultraviolet damage to
DNA of mammalian cells: caffeine sensitive and caffeine resistant mechanisms.
Mutat. Res., 37: 91–110, 1976.
5. Pillidge, L., Downes, C. S., and Johnson, R. T. Defective post-replication recovery
and U.V. sensitivity in a simian virus 40-transformed Indian muntjac cell line. Int. J.
Radiat. Biol., 50: 119 –136, 1986.
6. Musk, S. R. R., Pillidge, L., Johnson, R. T., and Downes, C. S. Action of caffeine on
DNA replication after ultraviolet irradiation in Indian muntjac cells: no connection
with action on cell cycle delay. Biochim. Biophys. Acta., 1052: 53– 62, 1990.
7. Brown, R. F. Chlorpropamide inhibition of excision repair and postreplication repair
of ultraviolet damage in Chinese hamster ovary cells. Mutat. Res., 77: 251–258, 1980.
8. Clark, J. M., and Hanawalt, P. C. Replicative intermediates in UV-irradiated simian
virus 40. Mutat. Res., 132: 1–14, 1984.
9. Ostling, O., and Johnson, K. J. Microelectrophoretic study of radiation-induced DNA
damage in individual cells. Biochem. Biophys. Res. Commun., 123: 291–298, 1984.
10. Fairbairn, W., Olive, P. L., and O’Neil, K. L. The Comet assay: a comprehensive
review. Mutat. Res., 339: 37–59, 1995.
11. Collins, A. R., Duthie, S. J., and Dobson, V. L. Direct enzymatic detection of
endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis
(Lond.), 14: 1733–1735, 1993.
12. McKelvey-Martin, V. J., Ho, E. T. S., McKeown, S. R., Johnson, S. R., McCarthy,
P. J., Rajab, N. F., and Downes, C. S. Emerging applications of the single cell gel
electrophoresis (Comet) assay. I. Management of invasive transitional cell human
bladder carcinoma. II. Fluorescence in situ hybridisation Comets for the identification
of damaged and repaired DNA sequences in individual cells. Mutagenesis, 13: 1– 8,
1998.
13. Duell, T. H., Lengfelder, E., Fink, R., Giesen, R., Giesen, R., and Bauchinger, M.
Effect of activated oxygen species in human lymphocytes. Mutat. Res., 336: 29 –38,
1995.
14. Pulvertat, R. J. Cytology of Burkitt’s tumour (African lymphoma). Lancet, In:
238 –240, 1964.
15. Singh, N. P., McCoy, M. T., Tice, R. R., and Schneider, E. L. A simple technique for
quantitation of low levels of DNA damage in individual cells. Exp. Cell Res., 175:
184 –191, 1988.
16. Blin, N., and Stafford, D. W. A general method for isolation of high molecular weight
DNA from eukaryotes. Nucleic Acids Res., 3: 2303–2307, 1976.
17. Mitchell, D. L., and Nairn, R. S. The biology of the photoproduct. Review. Photochem. Photobiol., 49: 805– 820, 1989.
18. Frenzel, K. Carcinogen-mediated oxidant formation and oxidative DNA damage.
Pharmacol. Therapeut., 53: 127–166, 1992.
19. Cross, C., Halliwell, B., Borish, E., Pryor, W. A., Ames, B., and Harman, D. Davis
conference: oxygen radicals and human disease. Ann. Intern. Med., 107: 526 –545,
1987.
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The Bromodeoxyuridine Comet Assay: Detection of Maturation
of Recently Replicated DNA in Individual Cells
Angela P. McGlynn, Gillian Wasson, Jacqueline O'Connor, et al.
Cancer Res 1999;59:5912-5916.
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