the bromodeoxyuridine comet assay: detection of maturation of … · the bromodeoxyuridine comet...

[CANCER RESEARCH 59, 5912–5916, December 1, 1999]

Advances in Brief

The Bromodeoxyuridine Comet Assay: Detection of Maturation of RecentlyReplicated 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 simplemethod of measuring DNA single strand breaks and alkali-labile sites inindividual cells. We have combined this with bromodeoxyuridine(BrdUrd) labeling of DNA and immunolocalization of the BrdUrd toassess DNA replicative integrity on a single-cell basis. We show that theexistence of strand discontinuities in recently replicated domains of DNA,caused during semiconservative replication or exacerbated by the arrestof replicative polymerases at UV irradiation- or chemical-induced lesions,can be detected in individual cells. Data obtained from BrdUrd-Cometsare consistent with biochemical data derived with a range of techniquesshowing that DNA replication involves the creation of strand breaks orgaps adjacent to recently replicated material, and that DNA damageprolongs the duration of such discontinuities where DNA polymerases arestalled opposite lesions (R. T. Johnsonet 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 andautomatable assessment of replicative integrity in very small numbers ofmammalian cells, such as may be obtained by biopsy.

Introduction

In DNA replication, strand discontinuities inevitably occur at theleading and lagging strand growth points. In normal replication, DNApolymerases rapidly move on. Label incorporated into DNA during abrief pulse is initially close to strand discontinuities, but duringpulse-chase experiments, such label is soon incorporated into highmolecular weight, continuous DNA, far distant from any discontinui-ties. The process by which this occurs is known as DNA maturation.

After DNA damage, replication is affected. DNA polymerasesinitialy stall at noncoding damage sites (1–3). Through the process ofPRR,3 polymerases bypass lesions or avoid them through retrospec-tive gap filling (4). One striking case of deficiency in DNA maturationis the Indian muntjac line SVM84, which (compared with normalDM87 Indian muntjac cells) are hypersensitive to UV irradiation andare deficient, although not entirely defective, in UV PRR capacity (5).In DM87 cells, the PRR capacity is largely caffeine sensitive, whereasin SVM 84 cells, the residual PRR is caffeine insensitive (6).

PRR in these cases, and in other mammalian cells, has normallybeen studied by biochemical methods: [3H]thymidine pulse-labeling,followed by alkaline sucrose gradient centrifugation (7), or by chro-matography on BND-cellulose or hydroxyapatite (8). These tech-

niques, however, are time consuming and require both radioactivelabeling of DNA and the use of large cell populations (;105 per datapoint) for which only an average figure can be obtained.

An alternative method for measuring DNA strand breaks and alkali-labile sites in single cells, the Comet assay, has been in use for someyears (9, 10). In this assay, single cells are embedded in agarose onfrosted slides, lysed in denaturing buffer, subjected to brief electro-phoresis, stained with DNA intercalating dye, and observed withfluorescence microscopy. Undamaged DNA is unable to enter theagarose 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 quantitatethe distribution of DNA between damaged Comet tails and undam-aged Comet “heads.” Modifications of the Comet assay have beendeveloped to detect more complex DNA lesions, provided they areendonuclease sensitive (11). More recently, we have combined theComet assay with fluorescencein situhybridization to allow detectionof damage within defined gene sequences (12).

We have combined this method with pulse-labeling of replicatingDNA with the thymidine analogue BrdUrd and immunological local-ization of BrdUrd within the heads and tails of Comets to assessreplicative integrity on a single-cell basis. The BrdUrd-Comet assay isdescribed 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 wereextracted from whole blood according to the method described previously (13).For all experiments using human T lymphocytes, the Raji (TK1) Burkitt’slymphoma cell line (14) was included as a control and maintained in expo-nential growth in RPMI 1640 supplemented with 15% fetal bovine serum andantibiotics.

Fibroblast Growth and Irradiation. The Indian muntjac fibroblast celllines, DM87 (American Type Culture Collection, , MD) and SVM84 (a giftfrom Professor K. Sperling, Berlin, Germany) were plated at a density of1 3 105 cells/60-mm Petri dish in complete medium as described (5) andgrown for 48 h to ensure exponential growth prior to irradiation. Cells wereexposed to UV irradiation (254 nm) using 2 J/m2 and incubated in completemedium 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 added30 min before UV irradiation, where indicated.

BrdUrd Labeling and Processing of Cells.Medium was removed fromexponentially growing cells (with or without irradiation) and replaced withfresh medium containing 100mM BrdUrd (Sigma) at 37°C for 15 min. Mediumcontaining BrdUrd was washed off with PBS (pH 7.4); cells were eitherprocessed for the Comet assay or, in chase experiments, given mediumcontaining each of 200mM thymidine, 29-deoxycytidine, 29-deoxyguanosine,and 29-deoxyadenosine (Sigma). Fibroblasts were harvested by brief trypsini-sation, and lymphocytes were harvested by centrifugation, both were andresuspended in ice-cold PBS at 53 104 to 13 105 cells/ml, respectively. Cellswere washed in cold PBS, and the cell pellet was resuspended in 100ml of0.75% low melting point agarose at 37°C. The cell suspension was spread ontoa standard Comet assay slide (15), and the slides were immersed in a neutrallysis buffer, adapted from that described by Blin and Stafford (16): 2.5M LiCl,

Received 6/1/99; accepted 9/3/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was entirely supported by Contract D215 from the Ministry for AgricultureFood and Fisheries, U.K.

2 To whom requests for reprints should be addressed, at Cancer and Ageing ResearchGroup, University of Ulster, School of Biomedical Sciences, Coleraine BT52 1JD,Northern Ireland. Phone: 44-1265-324121; Fax: 44-1265-324965; E-mail: [email protected].

3 The abbreviations used are: PRR, post replicative repair; BrdUrd, bromodeoxyuri-dine.

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0.03 M EDTA, 10 mM Tris, and 0.1% LiDS (pH 8.0), prewarmed to 37°Cbefore the addition of 0.03 mg/ml proteinase K. Slides were incubated at 37°Covernight for protein digestion and cell lysis. This lysis solution was usedbecause of the superior solubility of LiDS compared with SDS. Slides werethen transferred to alkaline lysis solution (2.5 mM NaCl, 100 mM Na2EDTA,and 10 mM Tris, pH 10) for 1 h at4°C.

Comet Electrophoresis and Staining.Slides were removed from lysissolution, drained, and placed in a horizontal electrophoresis tank, side by sidewith gaps filled with blank slides. The tank was filled with fresh electrophore-sis buffer (0.3M 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. Electro-phoresis 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 anddetergents that would interfere with immunostaining. The incorporated BrdUrdwas immunolocalized by adding 25ml/gel of mouse monoclonal anti-BrdUrd(10 mg/ml; Boehringer Mannheim) to the gels, with coverslips (183 18-mm)applied on top of the gels, allowing the antibody to spread evenly. Slides wereincubated in the dark at room temperature for 1 h. After removal of thecoverslips, the primary antibody was gently washed off with three changes ofPBS and one wash with PBS/0.1% BSA before addition of 25ml/gel ofsecondary antibody (5mg/ml sheep anti-mouse IgG, fluorescein conjugated;Boehringer Mannheim), which was incubated and washed off as before. Thegels were then counterstained with 25ml of propidium iodide (0.75mg/ml;Sigma) and covered with a coverslip (223 50-mm) for image analysis.

Comet Analysis. Observations were made at a final magnification of3400(Nikon 340 Fluor lens) using an epifluorescence microscope (Olympus BH2)equipped with an Hitachi KP571 CCD camera interfaced through a Matrox IP8board, using Hewlett Packard Super VGA and Komet 3.0 software (KineticImaging Ltd., Liverpool, United Kingdom). Propidium iodide was visualizedusing a Chroma filter at excitation 540 nm and emission 635 nm. Fluoresceinwas visualized using a Chroma filter at excitation 480 nm and emission 535nm. Densitometric and geometric parameters of each Comet were calculatedand recorded for statistical analysis using Microsoft Excel, version 5.0, soft-ware.

The percentage of DNA in the Comet tail for each cell, which is indicativeof the number of replicons in the replicating DNA, was measured. Results wereexpressed as the distribution of the percentage of Comet tail DNA in 25 cellson each Comet slide. The incorporation of the BrdUrd label into the Comethead after the chase period is indicative of DNA maturation and is representedby a reduction in percentage of tail DNA for each of 25 Comets.

Results

BrdUrd-Comet Assay Development.Several improvements ofthe initially tested assay were made by altering lysis and stainingconditions. The standard Comet assay for the detection of DNA strandbreaks is carried out under alkaline conditions (15); however, use ofneutral lysis facilitated nuclear matrix protein digestion using protein-ase K at 37°C. This improvement to the technique, which is notnormally used in the alkaline Comet assay, greatly improved cell lysisand BrdUrd-Comet formation (Fig. 1). In addition, the use of LiCl andLiDS in place of NaCl and SDS, commonly used for cell lysis, alsoprovided greater cell lysis because of the greater solubility of LiDScompared with SDS; in neutral lysis, SDS was found to precipitate at37°C. Comet detection was improved with the application of a sec-ond-stage antibody to detect BrdUrd-labeled DNA. The resultingComets, using the improved protocol, displayed greater movement ofDNA strands through the agarose gel with greater differentiationbetween Comet head and tail.

Calibration of Assay Against Fibroblasts of Known PRR Pro-pensity. Treated and control SVM84 and DM87 fibroblasts werepulsed with BrdUrd for a short time and either processed directly forthe BrdUrd comet assay, or the incorporated BrdUrd was chased withnormal nucleotides for a period of 1 h.

After UV irradiation (2 J/m2) of SVM84 cells, the amount ofBrdUrd-labeled DNA moving into the Comet head was reduced

compared with the nonirradiated cells. No further effect was seen inthe presence of caffeine (Fig. 2). These results confirm the maturationdeficiency of UV-irradiated SVM84 fibroblasts (5).

In the nonirradiated DM87 cells, the level of Comet tail BrdUrd-labeled DNA was reduced in the DM87 cells after 1-h chase. Thiscompetency in DNA maturation is similar, regardless of the presenceof caffeine in the nonirradiated DM87 cell line (Fig. 3, nonirradiatedcells).

In the absence of caffeine, UV irradiation alone (2 J/m2) induced anincrease in the percentage of DNA in the BrdUrd-Comet tail of DM87cells (Fig. 3, irradiated cells). However, after a 1-h chase period withnormal nucleotides, there is a retardation of maturation in the presenceof caffeine compared with cells exposed to UV irradiation alone in theabsence of caffeine (Fig. 3, irradiated cells). These results confirm thereduced efficiency of DNA maturation in UV-irradiated, but notnonirradiated, DM87 cells exposed to caffeine (6).

PRR after Low-Dose Hydrogen Peroxide Detected by theBrdUrd Comet Assay in Human T Lymphocytes. UV irradiationproduces large helix-distorting lesions (17); we have also exposedisolated T lymphocytes to hydrogen peroxide, an agent that inducessmall base lesions. After 72 h of culture, H2O2 induced a measurableincrease in BrdUrd-Comet tail moment in these cells. This is indica-tive of a delay in maturation occurring in these cells after exposure toa low dose of the DNA-damaging agent, H2O2 and occurs prior to theonset of measurable or lethal DNA damage, as detected by Cometanalysis of bulk DNA (Fig. 4).

Discussion

In this study, we have developed a novel assay for the detection ofDNA synthesis and maturation in single cells. We have demonstratedthat the novel BrdUrd-Comet assay can be further used to detectdelays in replication on a damaged template after UV irradiation orafter exposure to caffeine. Caffeine has been shown to potentiatecytotoxic, mutagenic, and chromosome-damaging effects of DNA-damaging agents by alternative mechanisms in different cell types;

Fig. 1. Optimization of the BrdUrd-Comet assay. BrdUrd-Comets of untreated fibro-blasts using original (a andb) and improved (c andd) conditions for BrdUrd pulse, celllysis, and BrdUrd detection are shown. Comets shown ind show improved cell lysis andelectrophoretic mobility of recently replicated DNA fragments. Conditions used were:aandb, 30 min of BrdUrd incorporation, followed by alkaline lysis in NaCl/SDS buffer at4°C, followed by one-step immunolocalization of BrdUrd;c and d, 15 min of BrdUrdpulse, 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 ofBrdUrd. Greenfluorescein staining (BrdUrd labeling) andred propidium iodide staining(DNA labeling) were used.

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SINGLE-CELL DETECTION OF DNA MATURATION



inhibition of postreplication recovery has been reported in caffeine-sensitive DM fibroblasts (6). In the present study, caffeine-treatedDM87 cells demonstrated a marked increase in the amount of BrdUrd-labeled DNA in the Comet tail (Fig. 3), which is indicative of anincrease in the number of replicons, a known mechanism for theaction of caffeine in DNA maturation delay (6). Caffeine retardedmaturation on a damaged, but not on an undamaged, template inDM87 cells exposed to UV irradiation (Fig. 3). This is consistent with

the alkaline sucrose sedimentation data described in muntjac fibro-blasts (6), where caffeine was shown to block any bypass of UV-induced damage during replication. SVM cells, on the other hand,have been shown to be replication incompetent after exposure to lowfluences of UV irradiation and were unaffected by the presence ofcaffeine (7, 8). Our data confirm these findings (Fig. 2).

Data shown in this study, using the BrdUrd-Comet assay, is there-fore parallel to the biochemical data derived earlier and offer a simple

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 withBrdUrd in culture for 15 min and either processed immediately for the BrdUrd-Comet assay or chased with 200mM 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; andc, BrdUrd pulse with chasein the presence of caffeine;B, representative Comet images, where identical fields are shown withgreenfluorescein staining (BrdUrd labeling) andredpropidium iodide staining (DNAlabeling) using appropriate fluorescent filters;a, BrdUrd pulse alone;b, BrdUrd pulse after UV irradiation;c, BrdUrd pulse with chase; andd, BrdUrd pulse with chase after UVirradiation.

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alternative to other laborious and time-consuming biochemical meth-ods that involve either tedious centrifugation or meticulous prepara-tion of human DNA. In addition, small numbers of cells are sufficientfor analysis, and the technique is therefore potentially suitable for theanalysis of single cells derived from small patient biopsies.

We have also exposed isolated T lymphocytes to hydrogen perox-ide, an agent that induces small base lesions (18) and plays a role in

a variety of physiological and pathological processes, in particularaging and carcinogenesis (19). Cells exposed to a low dose of theDNA-damaging agent had more DNA breaks adjacent to fluorescentlylabeled repair sites visible in the Comet tail than control cells, indi-cating the sublethality of this low dose and the ability of the cells torepair any strand breaks formed. Indeed, this effect is visible prior tothe onset of measurable DNA damage, evident by the lack of DNA

Fig. 3. Frequency distribution curves ofBrdUrd-Comets of DM fibroblasts exposed to caf-feine. Nonirradiated and UV-irradiated (2 J/m2

with 1-h recovery period) cells were pulsed withBrdUrd in culture for 15 min and either processedimmediately for the BrdUrd-Comet assay or chasedwith 200 mM of all four deoxynucleotides for 1 hprior to Comet processing. Results are expressed asfrequency distribution plots of BrdUrd-Comet per-centage of tail DNA. Where indicated, caffeine (2mM) was present for 30 min prior to irradiation andduring BrdUrd pulse and chase periods.a, BrdUrdpulse only;b, BrdUrd pulse with chase;c, BrdUrdpulse with chase in the presence of caffeine.

Fig. 4. BrdUrd-Comet images of isolated human Tlymphocytes. Cells were cultured for 72 h and processedfor BrdUrd-Comet analysis. Control cells were stainedwith PI (a) and BrdUrd (b) and double exposed forpropidium iodide and BrdUrd staining (c) cells treatedwith 10 mM H2O2 at 4°C prior to processing and stainedwith PI (d) and for BrdUrd (e) are shown.

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damage (Comet formation) in propidium iodide-stained whole DNAin these cells.

The present data on PRR in the two muntjac cell lines represent asemiquantitative analysis of the rates of DNA maturation. Furtherdevelopment of the assay are ongoing to determine the lengths andactivity of labeled DNA molecules. Furthermore, the BrdUrd-Cometassay is applicable to the detection of DNA repair after early DNAdamage induced by hydrogen peroxide in isolated T lymphocytes.

References

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2. Painter, R. B. Effect of caffeine on DNA synthesis in irradiated and unirradiatedmammalian cells. J. Mol. Biol.,143: 289–301, 1980.

3. Painter, R. B. Inhibition and recovery of DNA synthesis in human cells after exposureto ultraviolet light. Mutat. Res.,145: 63–69, 1985.

4. Fujiwara, Y., and Tatsumi, M. Replicative bypass repair of ultraviolet damage toDNA 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 recoveryand 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 onDNA replication after ultraviolet irradiation in Indian muntjac cells: no connectionwith action on cell cycle delay. Biochim. Biophys. Acta.,1052: 53–62, 1990.

7. Brown, R. F. Chlorpropamide inhibition of excision repair and postreplication repairof ultraviolet damage in Chinese hamster ovary cells. Mutat. Res.,77: 251–258, 1980.

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9. Ostling, O., and Johnson, K. J. Microelectrophoretic study of radiation-induced DNAdamage in individual cells. Biochem. Biophys. Res. Commun.,123: 291–298, 1984.

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11. Collins, A. R., Duthie, S. J., and Dobson, V. L. Direct enzymatic detection ofendogenous 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 gelelectrophoresis (Comet) assay. I. Management of invasive transitional cell humanbladder carcinoma. II. Fluorescencein situhybridisation Comets for the identificationof 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.

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15. Singh, N. P., McCoy, M. T., Tice, R. R., and Schneider, E. L. A simple technique forquantitation 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 weightDNA from eukaryotes. Nucleic Acids Res.,3: 2303–2307, 1976.

17. Mitchell, D. L., and Nairn, R. S. The biology of the photoproduct. Review. Photo-chem. 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. Davisconference: oxygen radicals and human disease. Ann. Intern. Med.,107: 526–545,1987.

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1999;59:5912-5916. Cancer Res Angela P. McGlynn, Gillian Wasson, Jacqueline O'Connor, et al. of Recently Replicated DNA in Individual CellsThe Bromodeoxyuridine Comet Assay: Detection of Maturation

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