DNA breakage detection-fish (DBD-FISH): effect of unwinding time

Mutation Research 453 (2000) 83–88

DNA breakage detection-fish (DBD-FISH): effect of unwinding time Fernando Vázquez-Gund´ın a , Jaime Gosálvez b , Joaquina de la Torre b , José Luis Fernández a,∗ a

Laboratorio de Genética Molecular y Radiobiolog´ıa, Centro Oncológico de Galicia, Avda de Montserrat s/n 15009, La Coruña, Spain b Unidad de Genética, Facultad de Biolog´ıa, Universidad Autónoma de Madrid, 15679 Madrid, Spain Received 28 February 2000; received in revised form 29 May 2000; accepted 6 June 2000

Abstract DBD-FISH is a new procedure that allows detection and quantification of DNA breakage in situ within specific DNA target sites. Cells embedded in an agarose matrix on a slide are treated in an alkaline unwinding solution to transform DNA breaks into single-stranded DNA (ssDNA). After removal of proteins, DNA probes are hybridized and detected. DNA breaks increase the ssDNA and relax supercoiling of DNA loops, so more probe hybridizes, thereby increasing the surface area and fluorescence intensity of the FISH signal. The probe selects the chromatin area to be analysed. In order to restrict the extension of unwound ssDNA to a region closer to the origin of the DNA break, human leukocytes were processed for DBD-FISH with a whole genome probe, after a 10 Gy dose of X-rays, for various unwinding times: 5, 2 min and 30 s. Two cell populations were detected after 30 s, but not with the 5 or 2 min unwinding times. One cell group had small to medium haloes corresponding to the relaxation of DNA supercoiling after DAPI staining, and strong DBD-FISH labelling of induced DNA breaks, whereas the other cell group showed big haloes of DNA loop unfolding and an absence of DBD-FISH labelling. The latter group was similar to cells processed by DBD-FISH without the unwinding step. Thus, they should correspond to cells unaffected by the alkaline unwinding solution, possibly because very brief unwinding times do not allow the diffusion of the alkali into the cells deep within the gel, thus biasing the results. Taking this into account, 2 min seems to be the minimum unwinding time required for an accurate detection of a signal by DBD-FISH. © 2000 Elsevier Science B.V. All rights reserved. Keywords: DBD-Fish; DNA breaks; Alkaline unwinding

1. Introduction Recently, a new procedure (DBD-FISH: DNA Breakage Detection–FISH [1]), which allows cell by cell detection and quantification of DNA breakage within specific DNA sequence areas, has been devel∗ Corresponding author. Tel.: +34-981-287499; fax: +34-981287122. E-mail address: [email protected] (J.L. Fern´andez).

oped. This procedure integrates microgel embedding [2], DNA unwinding [3] and FISH techniques [4]. Cells trapped within an agarose matrix on a slide are incubated in an alkaline unwinding solution, deproteinized, and hybridized with DNA probes, which are detected by immunofluorescence. As DNA breaks increase within a specific chromatin target or whole nucleus, the alkaline unwinding solution causes more ssDNA to be produced and a greater release of DNA supercoiling. As a consequence, more probe

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hybridizes, leading to an increase in the whole or mean fluorescence intensity and surface area of the FISH signal. Current unwinding procedures use relatively long incubation periods in alkali, e.g. 0.03 M NaOH, 1 M NaCl 30 min at 0◦ C [5] or 10–30 min at 22◦ C [6,7]. Under these conditions, the amount of unwound ssDNA generated by the alkali from a single break may be considerable. As a consequence, DNA breaks in the vicinity or even relatively distant from the probed sequence may produce ssDNA that could extend to this probed locus, so quantification of DNA breakage level by DBD-FISH is not absolutely restricted to the specific chromatin area, especially when this target is small. We have assayed three successively shorter unwinding times (5, 2 min, and 30 s) for DBD-FISH of the whole genome in human blood leukocytes after a 10 Gy X-ray exposure. This should progressively decrease the length of melted ssDNA from the origin of the break, allowing the feasibility of this reduction for quantification purposes to be evaluated.

2. Materials and methods 2.1. Slide preparation Human peripheral blood was obtained from a healthy male and anticoagulated with preservative-free heparin. Leukocytes in buffy coat were employed in all experiments. Sometimes isolated granulocytes were also processed. In this case, they were separated on Polymorphprep (Nycomed) obtaining a 97% of polymorphonuclear neutrophils, and resuspended in blood plasma. The cell suspension was mixed with low melting point agarose, thereby giving a final concentration of 0.7%, at 37◦ C. The mixture (200 ␮l) were pipetted onto polystyrene slides (Nunc), covered with glass coverslips (24 mm×60 mm) and left at 4◦ C for 5 min. After gel solidification, half the area of the slides was irradiated with a Philips RT-100 X-ray machine (100 kVp) administering a dose of 10 Gy. The other half was a control-unirradiated area. Coverslips were carefully removed and slides were immediately immersed in abundant, freshly prepared alkaline unwinding solution (0.03 M NaOH, 1 M NaCl) for 5, 2 min or 30 s, in the dark, at 22◦ C. Afterwards, they were transferred to excess of neutralizing and lysing

solution 1 (0.4 M Tris–HCl, 0.8 M DTT, 1% SDS, 0.05 M EDTA, pH 7.5) for 20 min at room temperature, and then to neutralizing and lysing solution 2 (0.4 M Tris–HCl, 2 M NaCl, 1% SDS, pH 7.5) for 10 min at room temperature. Lysing solutions extract nuclear proteins and the DNA remains within a residual nucleus-like structure, the nucleoid. After washing in excess of TBE buffer (0.04 M Tris–borate, 0.002 M EDTA, pH 7.5) for 15 min at room temperature, the gels were dehydrated by sequential 70, 90 and 100% ethanol baths, 2 min each, and air-dried. 2.2. Fish A human whole genome probe biotin-labelled by nick-translation was denatured and incubated on dried gels. After overnight incubation at room temperature, the slides were washed twice at room temperature in each of 50% formamide/2×SSC, pH 7, for 5 min, and then in 2×SSC, pH7, for 3 min. Bound probe was detected by a 30 min incubation with streptavidin-Cy3 (1:200, Sigma) and nucleoids counterstained with 45 ␮l of DAPI (1 ␮g/ml) in Vectashield (Vector). 2.3. Fluorescence microscopy and digital image analysis Signals were viewed under a DMRB epifluorescence microscope (Leica) equipped with a DMRD photoexposer, using PL Fluotar 100× or 40× objectives and appropriate fluorescence filters. Images were acquired using a high sensitivity CCD camera (Ultrapix 1600, Astrocam) which distinguishes over 16 000 grey levels and allows subtraction of the current dark image and correction for nonuniform sample illumination. Groups of 100–200 digital images (16 bit) were taken for each experimental point under similar conditions, stored in the file format of the camera (.apf) and then converted to .img files. Image analysis was performed using a macro designed with Visilog 5.1 software (Noesis). This allows for thresholding, background subtraction, and measures the surface area (in pixels), mean fluorescence intensity (mean grey level) and whole fluorescence intensity (surface area×mean fluorescence intensity) of the signals. One and two-way ANOVA (p<0.05) were employed for statistical analysis.

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Fig. 1. DAPI counterstaining (left) and DBD-FISH with a whole genome probe (right) in human leukocytes exposed to 10 Gy of X-rays. a, b: Cells unwound for 5 min and then lysed. Cells are homogeneous, with strong and dispersed DBD-FISH signals. c, d: Cells unwound for 2 min and then lysed. Cells are homogeneous, with less strong and dispersed DBD-FISH signals. e, f: Cells unwound for 30 s and then lysed. Different cell types are evident. A: cells with small haloes after DAPI counterstaining and with strong labelling by DBD-FISH. A0 : cell with medium sized halo after DAPI staining and with restricted labelling by DBD-FISH. B: cell with big halo after DAPI staining and without labelling by DBD-FISH. g, h: Cells not unwound but lysed. Cells (C) are homogeneous and similar to cell type B in e–f. i, j: Cells unwound for 30 s but not treated with lysing solutions. Two cell types are detected. D: cell with small halo after DAPI staining and with DBD-FISH signal. E: cell without unfolding of DNA loops and without DBD-FISH signal.

3. Results and discussion The aim of this work was to restrict the unwound ssDNA starting from the DNA break of origin in order to focus as much as possible on those breaks specifically generated within or very close to the DNA

sequence to be probed by DBD-FISH. A whole genome probe was assayed to evaluate the effect of decreasing the unwinding time on the FISH signal obtained in human blood leukocytes exposed to 10 Gy of X-rays. Fig. 1 shows images of nuclei unwound for 5, 2 min and 30 s. The decrease in the surface

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Table 1 Values of the parameters from the different cell populations observed in human leukocytes exposed to 10 Gy of X-rays and processed by DBD-FISH in the whole genome, with incubation or not in the unwinding solution for 30 s and/or lysing solutionsa Treatment

30 s unwinding ↓ Lysis (No unwinding) ↓ Lysis 30 s unwinding ↓ (No lysis) a

Cells

DAPI-staining

DBD-FISH

Type

Mean (%)

Surface

Surface

Whole fluorescence intensity

A A0 B C

46 5 49 100

12554.8±2116.9 14908.1±3692.9 34675.9±3699.1 35397.7±3179.4

31816.2±13004.8 3446.2±484.2 0 0

37507797.5±23578954.9 8008179.9±4552186.2 0 0

9592.5±1712.3 4574.8±1744.6 3993.1±600.9

25836.7±10118.1 1502.0±157.5 0

34825302.1±15912097.9 9611228.6±3794672.1 0

D D0 E

66 5.6 28.4

The percentage of the cell types is the mean of three–four experiments.

area and whole fluorescence intensity of the FISH signal with reduced unwinding times was evident. Nevertheless, whereas almost all nuclei processed for 5 (Fig. 1a,b) and 2 min (Fig. 1c,d) showed FISH signals, only a fraction of them appeared labelled after 30 s. Thus, after 30 s, two clearly differentiated cell populations were evident (Fig. 1e,f; Table 1). One cell population (cell type A) appeared with strong and dispersed labelling of induced DNA breaks, corresponding to nucleoids with small haloes of relaxed supercoiling of DNA loops after DAPI staining. This population included a small subgroup of cells (cell type A0 ) (one per 10 A type cells) whose labelling was strong but restricted to the original nuclear area, showing medium-sized haloes of DNA unfolding after DAPI staining. The other cell population (cell type B) exhibited big haloes of DNA loop relaxation but without labelling of induced DNA breaks (Fig. 1e,f; Table 1). The proportion of both cell types varied considerably between slides and between experiments (40–72% unlabelled cells). This effect was not related to the leukocyte cell type since the same experiment performed on isolated granulocytes (98% of polymorphonuclear neutrophils) gave rise to the same two cell populations. When 10 Gy-irradiated leukocytes were exposed to the lysing solutions without the previous unwinding step, their DNA breaks appeared unlabelled as expected, and DAPI staining revealed nucleoids with big haloes of decondensation (cell type C) (Fig. 1g,h; Table 1). Their appearance was similar to the unlabelled cell population described above (cell

type B), and there was no significant statistical difference between their surface area with DAPI staining (Fig. 2). This suggests that the unlabelled population (cell type B) corresponded to cells unaffected by the alkaline unwinding solution. Finally, 10 Gy-irradiated leukocytes exposed for 30 s to the unwinding solution and then neutralized, but without lysing steps, also showed two cell populations. Nevertheless, in this case, the result was opposite to that described for 30 s unwinding followed by extensive protein removal (Fig. 1i,j; Table 1). The cell population with strong and dispersed FISH signals (cell type D) now showed small haloes of decondensation under DAPI staining, whereas the unlabelled population (cell type E) appeared without unfolding of nuclear DNA loops. Again, great variation in the proportion of both cell populations was evident (27–71.5% of labelled cells, depending on slide or experiment), and both cell types were also obtained in isolated polymorphonuclear neutrophils. The results obtained from cells irradiated with 10 Gy of X-rays, exposed for 30 s to the alkaline unwinding solution, and then lysed, can be explained as follows. The alkaline solution penetrates the gel, affecting the cells in the periphery, but exposure is too brief to reach more deeply located cells. Although the DNA breaks are distributed in a Poisson fashion among the cells, they are only transformed into ssDNA areas in superficial cells, being also partially lysed by the alkaline solution. Therefore, cells affected by the alkali appear labelled by DBD-FISH

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Fig. 2. Graphic representation of 30 s unwound cells and non-unwound cells. In each cell, the whole fluorescence intensity (arbitrary units) after DBD-FISH with a whole genome probe is plotted against its respective surface area (pixels) with DAPI counterstaining. It is evident that 30 s unwound cells show two clear subpopulations, one with a FISH signal and DAPI surface area between 10 000 and 20 000, and another one without FISH signal (0 intensity) and a greater DAPI surface area, between 25 000 and 45 000. The non-unwound cells coincide with the latter subpopulation.

and show small haloes of unfolding with DAPI staining (cell type D). Conversely, those cells unaffected by the alkali remain unlabelled and without haloes of decondensation (cell type E). When the neutral lysing solutions are employed after the brief unwinding step, extensive protein removal allows the relaxation of the supercoiling of DNA loops in all irradiated cells. Cells affected by the alkali now appear with the labelled DNA more extensively dispersed (cell type A). Though the surface area of DAPI staining increases,

it is not as great as that of the labelled chromatin because it is mainly composed of irregularly renatured ssDNA that curls up and becomes entangled, being less stained by DAPI especially in the nucleoid (cell type A) [8,9]. Otherwise, irradiated nuclei unaffected by the alkali also relax their chromatin after extensive neutral protein removal, but DAPI stains the extended loops more effectively, since they do not contain ssDNA (cell type B) [8,9]. This must be a dynamic process. In fact, some cells appear with interme-

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diate haloes of decondensation and more restricted DBD-FISH labelling of DNA breakage (cell type A0 ). Overall, the practical consequence is that a very brief unwinding treatment to restrict as much as possible the ssDNA produced starting from a break is not feasible for quantitative purposes under the conditions employed here. In fact, we found that all 10 Gy-irradiated leukocytes exposed for 2 min to the alkali solution appeared with DBD-FISH labelling in the alphoid DXZ1 locus, while after 30 s of unwinding treatment, the signal appeared only in those cells that showed small haloes with DAPI staining, which were otherwise quite variable in proportion among slides. The proportion of 10 Gy-irradiated cells unlabelled by the whole genome probe and with big haloes was negligible (1/312 cells) after 2 min of the unwinding step.

Acknowledgements We are grateful to Dr. Phil Mason for improving the English style of the manuscript. This work was supported by the Consejo de Seguridad Nuclear (CSN) and by an EC Nuclear Fission Safety-Radiation Protection Program grant.

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