Correspondence to: William M. Bonner, NIH-NCI, Bldg. 37, Rm. 5D17, Bethesda, MD 20892. Tel:(301) 496-5942 Fax:(301) 402-0752 E-mail:wmbonner{at}helix.nih.gov.
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Abstract |
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The loss of chromosomal integrity from DNA double-strand breaks introduced into mammalian cells by ionizing radiation results in the specific phosphorylation of histone H2AX on serine residue 139, yielding a specific modified form named -H2AX. An antibody prepared to the unique region of human
-H2AX shows that H2AX homologues are phosphorylated not only in irradiated mammalian cells but also in irradiated cells from other species, including Xenopus laevis, Drosophila melanogaster, and Saccharomyces cerevisiae. The antibody reveals that
-H2AX appears as discrete nuclear foci within 1 min after exposure of cells to ionizing radiation. The numbers of these foci are comparable to the numbers of induced DNA double-strand breaks. When DNA double-strand breaks are introduced into specific partial nuclear volumes of cells by means of a pulsed microbeam laser,
-H2AX foci form at these sites. In mitotic cells from cultures exposed to nonlethal amounts of ionizing radiation,
-H2AX foci form band-like structures on chromosome arms and on the end of broken arms. These results offer direct visual confirmation that
-H2AX forms en masse at chromosomal sites of DNA double-strand breaks. The results further suggest the possible existence of units of higher order chromatin structure involved in monitoring DNA integrity.
Key Words: ionizing radiation, histone H2AX, DNA double-strand breaks, phosphorylation, indirect immunofluorescence
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Introduction |
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THE universal presence of ionizing radiation poses a constant threat to the chromosomal integrity of living organisms. One of the most serious cellular lesions induced by radiation is the random DNA double-strand break. This lesion jeopardizes a chromosome's physical integrity, which is essential for its correct segregation during mitosis and meiosis, as well as its informational redundancy, which is critical for maintaining accurate encoding of cellular components. Thus, it is not surprising that multiple mechanisms exist for dealing with this serious lesion, including nonhomologous end-joining, homologous recombination, and apoptosis (
In mammals, nonhomologous end-joining appears to be the major mechanism of rejoining DNA ends. This process involves a DNA-activated protein kinase complex with three subunits, a catalytic subunit, DNA-PKcs (
Whereas much is known about the rejoining of DNA double-strand breaks, much less is known about how these breaks are initially recognized. Our group reported previously that mammalian cells and mice respond to agents that introduce DNA double-strand breaks with the immediate and substantial phosphorylation of histone H2AX (-H2AX (
-phosphorylated after exposure of cells to ionizing radiation, with half-maximal amounts reached at 13 min. At the maximum, 1030 min after irradiation, the stoichiometry suggests that hundreds to several thousand
-H2AX molecules are present per DNA double-strand break in mammals. In this study, we show that the H2AX molecules are
-phosphorylated en masse at the sites of DNA double-strand breaks.
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Materials and Methods |
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Cell Culture
Cell lines were obtained from the American Type Culture Collection unless otherwise noted. IMR90 normal human fibroblast cells were grown in MEM Alpha (No. 12561; GIBCO BRL) containing 10% FBS. MCF7 human breast cancer and SF268 human astrocytoma cells (National Cancer Institute, Division of Cancer Treatment and Diagnosis, Developmental Therapeutics Program, National Institutes of Health) were grown in RPMI 1640 (No. 11875; GIBCO BRL) containing 10% FBS. Indian muntjac, Muntiacus muntjak, normal skin fibroblasts were grown in F-10 Ham's nutrient mixture (No. 11550; GIBCO BRL) containing 20% FBS. Xenopus laevis A6 normal kidney cells were grown in medium NCTC-109 (No. 21340; GIBCO BRL) containing 15% deionized water and 10% FBS. Cultures were maintained at room temperature, ~24°C, in an atmosphere of 5% CO2. Drosophila melanogaster epithelial cells, a gift of C. Wu (National Cancer Institute, Division of Basic Sciences, Laboratory of Molecular Cell Biology, National Institutes of Health, Bethesda, MD), were grown in Schneider's Drosophila medium (No. 11720; GIBCO BRL) containing 10% heat-inactivated FBS at room temperature.
Antibody Production
Anti- was prepared by Genosys Biotechnologies Inc. The peptide CKATQAS(PO4)QEY was synthesized, conjugated to keyhole limpet hemocyanin, and injected into rabbits. The immune serum from the third bleed was passed through a column containing immobilized CKATQASQEY to absorb antibodies to unphosphorylated H2AX.
Ionizing Radiation
Cells growing in 10-cm dishes, on Labtek II slides (Nalge Nunc International), or on coverslips were exposed to the indicated amount of ionizing radiation from a 137Cs source in a Mark I irradiator (J.L. Shepherd and Associates). Doses >20 Gy were given at a rate of 15.7 Gy/min. Doses of 2 and 0.6 Gy were given in 1 min.
Laser-introduced DNA Double-Strand Breaks
Except for the source of UVA irradiation, the method of
Laser Scanning Confocal Microscopy
Cells were grown on Labtek II slides or coverslips. After irradiation and recovery at 37°C, the cell preparations were fixed in 2% paraformaldehyde in PBS for 5 min, washed in PBS, permeabilized in 100% methanol at -20°C for 5 min, washed, blocked with 8% BSA for 1 h, incubated with the -H2AX first antibody at 800-fold dilution for 2 h, washed, incubated with a Cy2-conjugated goat antirabbit second antibody (Jackson Immunolabs) at 200-fold dilution for 1 h, washed, mounted with or without propidium iodide, and viewed with a PCM2000 laser scanning confocal microscope (Nikon Inc.) using a 100x objective. Optical sections (0.5 µm) through the thickness of the sample were imaged and combined in a maximum projection with Simple32 software (Compix Inc.) so that all of the visible foci and bands in a nucleus or mitotic figure were recorded. The projection was saved as a BMP file and brought into Paint Shop Pro 5 (Jasc Software, Inc.) and Powerpoint (Microsoft Corp.) for presentation.
Immunoblotting
Polyvinylidene difluoride (PVDF) membranes containing transferred proteins were blocked with 1% dried nonfat milk for 1 h, incubated with the -H2AX first antibody at 12,000-fold dilution for 2 h, washed, incubated with peroxidase goat antirabbit second IgG (Calbiochem-Novabiochem Corp.) at 3,000-fold dilution for 1.5 h and washed. Anti-
binding was visualized by chemiluminescence (ECL RPN 2209; Amersham Pharmacia Biotech).
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Results |
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Antibody to -H2AX COOH Terminus
To examine the spatial distribution of -H2AX in the chromatin of irradiated cells, a polyclonal antibody (anti-
) was raised in rabbits against a synthetic phosphorylated peptide containing the mammalian
-H2AX COOH-terminal sequence. On immunoblots of total protein extracts from irradiated MCF7 cells, anti-
detected one band at the position expected for
-H2AX (Figure 1 A). No binding was detected in irradiated samples when the immunizing peptide was present as competitor (Figure 1 A, P-pep) or with preimmune serum (Figure 1 A, pre). Although anti-
binding was not apparent in unirradiated MCF7 samples at film exposures optimal for
-H2AX detection (Figure 1 A, 0 Gy), small amounts of binding were detectable on highly exposed immunoblots. Results from our laboratory show that
-H2AX is present in apoptotic cells with fragmented DNA (Rogakou, E.P., W. Nieves-Neira, C. Boon, Y. Pommier, and W.M. Bonner, manuscript submitted for publication), indicating a possible source of
-H2AX in unirradiated cultures. Another possibility is that anti-
cross-reacts slightly with unmodified H2AX. However, two-dimension gels which separate these two protein species (Figure 1 B) showed that anti-
bound only
-H2AX (Figure 1 C, solid line) with no detectable cross-reaction to unmodified H2AX. This result indicates that the binding of anti-
in extracts of unirradiated cells is due to the presence of
-H2AX in some of the cells in those cultures.
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H2AX as well as H2A1, the most plentiful of the H2A species in mammals, can be phosphorylated on serine residue 1 and acetylated on lysine residue 5 (Figure 1 B, dotted lines). H2AX molecules modified in the NH2-terminal region bound anti- only when they were
-modified (Figure 1 C, dotted line). Also noticeable was the lack of anti-
binding to H2A1, whose sequence, except for the COOH-terminal motif, is almost identical to the H2AX.
Since the H2AX COOH terminus is highly conserved, immunoblots were prepared from irradiated cell cultures of various species to examine whether anti- could detect
-H2AX homologues. Anti-
detected one band that migrated as expected for the appropriate
-H2AX homologue not only in other examined mammalian species such as the mouse, rat, hamster, (data not shown), and M. muntjak (Figure 1 D), but also in X. laevis (Figure 1 E), D. melanogaster (Figure 1 F), and S. cerevisiae (Figure 1 G). These experiments show that not only H2AX itself, but also its phosphorylation in response to ionizing radiation, have been highly conserved during evolution.
-H2AX Foci in Cells Subjected to Ionizing Radiation
With the demonstration that anti- is specific for
-H2AX, the distribution of
-H2AX in irradiated cells was examined. Cells of the normal human fibroblast line IMR90 and the human breast cancer line MCF7 both responded to ionizing radiation with the formation of discrete foci containing
-H2AX throughout the nuclei (Figure 2). Some cells of both lines contained foci in the absence of irradiation; most of the unirradiated MCF7 cells contained one to two foci (Figure 2 I), whereas fewer of the IMR90 cells did (Figure 2 A). The amount of
-H2AX present in these foci in unirradiated cells is evidently below the level of detection of the immunoblots shown in Figure 1 A, but may account for the signal seen on highly exposed immunoblots. No foci were apparent in unirradiated or irradiated cells when 1 µM immunizing peptide was included in the first antibody solution (data not presented). The relationship between the presence of
-H2AX in unirradiated and irradiated cells is being examined.
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With IMR90 cells, foci were apparent 3 min after irradiation with 0.6 Gy (Figure 2 B), persisted at 1560 min (Figure 2, CE), then decreased in number at 180 min (Figure 2 F). With MCF7 cells, the time course of foci appearance and disappearance was similar (Figure 2, IN). A more detailed analysis of IMR90 cells is presented in Figure 3, in which individual nuclei in fields of cells were scored for the number of foci. Compared with the unirradiated control cells, which contained an average of 1.5 ± 2.4 (14 nuclei) foci per nucleus, all the IMR90 cells 3 min after exposure to 0.6 Gy contained numerous small foci, with an average of 16.3 ± 3.6 (11 nuclei) foci per nucleus. The foci became fewer in number but better defined after 15 min, 10.1 ± 3.9 (17 nuclei) foci per nucleus; 30 min, 11.6 ± 5.3 (18 nuclei) foci per nucleus; and 60 min, 11.4 ± 6.1 (15 nuclei) foci per nucleus. After 180 min recovery, the number of foci again decreased to 4.8 ± 3.3 (17 nuclei) foci per nucleus, and at 270 min there were 4.5 ± 5.3 (26 nuclei) foci per nucleus. In addition, after 270 min recovery, 2 of the 26 scored nuclei appeared to be free of foci, possibly suggesting that in these two cells all of the introduced DNA double-strand breaks had been rejoined. This time course obtained by counting foci is very similar to that obtained by -H2AX was measured after CHO cells had been irradiated with 200 Gy. This similarity suggests that the processes of
-H2AX appearance and disappearance are the same at these two very different amounts of ionizing radiation.
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Larger amounts of radiation resulted in larger numbers of foci in both IMR90 (Figure 2G and Figure H) and MCF7 (Figure 2O) cultures. Compared with IMR90 cultures 15 min after 0.6 Gy, with 10.1 to ± 3.9 (17 nuclei) foci per nucleus, IMR90 cultures 15 min after 2 Gy contained 24 ± 5.7 (10 nuclei) foci per nucleus. For MCF7 cultures, the comparable numbers are 12.2 ± 5.7 (26 nuclei) foci per nucleus 15 min after 0.6 Gy, and 27.1 ± 10.8 (24 nuclei) foci per nucleus 15 min after 2 Gy. For both IMR90 and MCF7 cultures, the values for 2 Gy are somewhat less than expected for linear proportionality with respect to the amount of radiation. This discrepancy may be at least partly explained if two foci at different levels in the nuclei overlap in the maximum projection and are scored as a single focus. This overlapping is more likely as the number of foci increases. In addition, it is relevant to mention that the SD values are presented as a means of displaying the range of the values in different nuclei, not as a measure of reproducibility. DNA breakage by ionizing radiation is a stochastic process, and thus the values of foci per nucleus are expected to follow a Poisson distribution. The distribution of values shown in Figure 3 for each time point as well as the calculated SDs are consistent with those of Poisson distributions.
Each Gy introduces one double-strand break per 0.2 x 109 bp DNA when irradiated cells are analyzed before any repair can take place (
Thus, depending on their position in the cell cycle, IMR90 cells are predicted to contain 1836 initial and 714 persistent DNA double-strand breaks after exposure to 0.6 Gy. At 3 min after irradiation, most of the IMR90 cells displayed between 10 and 20 foci (Figure 3), with an average of 16.3 ± 3.6 foci per nucleus, values near the low number of expected initial breaks (1836) and the high number of expected persistent breaks (714). At 15 and 30 min after irradiation, most of the IMR90 cells displayed between 5 and 15 foci (Figure 3) with averages of 10.1 ± 3.9 and 11.6 ± 5.3 foci per nucleus, respectively. These values agree well with the number of expected persistent breaks (714). As there is likely to be a wide range in the severity of the DNA damage at locally multiply damaged sites (-H2AX foci seen at 3 min after irradiation cease growing or disappear because those DNA double-strand breaks were rejoined very quickly. These observations show that there is a close correlation between the numbers of
-H2AX foci and the numbers of expected DNA double-strand breaks, leading to the conclusion that each
-H2AX focus may represent a DNA double-strand break in vivo.
Laser-induced DNA Double-Strand Breaks
If each -H2AX focus identifies a DNA double-strand break, then the two should coincide. To determine this, advantage was taken of the finding that
-H2AX was formed when DNA double-strand breaks were introduced into cells by the BrdU dyeUVA light procedure of
-H2AX foci (Figure 4 A).
-H2AX formation was dependent on the presence of BrdU; when BrdU was absent but dye still present,
-H2AX foci were consistently found only in the cells traversed with the laser at 30% relative power (Figure 4 B). This experiment demonstrates that
-H2AX foci form at the sites of DNA double-strand breaks.
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Notably, there appears to be no difference in the efficacy of 1, 10, and 30% relative power in the formation of -H2AX foci (Figure 4 A), possibly because even at 1% the exposure was more than sufficient to lead to the complete conversion of H2AX in the path of the laser to
-H2AX.
-H2AX occurs in the beam path at 1% relative power and that more is not formed at the higher power settings. It is relevant to mention that cells are generally quite transparent to these UVA wavelengths, thus most of the laser radiation probably passes completely through the cell, inflicting little if any damage except for that absorbed by the dye.
It is also notable that there are more -H2AX foci in the regions adjacent to the path of the microbeam laser in the cells grown with BrdU than in those without (compare Figure 4 A and Figure 4 B), probably because the BrdU and dye present in the former make the whole nucleus very sensitive to any UVA light that might be scattered from the laser beam. Thus, to limit DNA damage to more defined regions, it may be more appropriate to omit BrdU and use higher laser power settings.
Foci in Irradiated Mitotic Cells
Mitotic MCF7 cells were present in some of the cultures analyzed in Figure 2. The mitotic cell noted in Figure 2 J (m) is of particular interest because this culture was fixed only 3 min after irradiation, a result indicating that -H2AX foci form on mitotic chromosomes as well as on interphase chromatin. When fields of cells from MCF7 cultures containing both mitotic and interphase cells were analyzed for
-H2AX foci 15 and 225 min after exposure to 0.6 Gy, the number of foci was decreased at the latter time in both interphase and mitotic cells (Figure 5, AC). These results show that the kinetics of
-H2AX foci appearance and disappearance are similar whether cells are interphase or mitotic.
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Because of the large number of small chromosomes in human mitotic cells, it is not possible to consistently visualize -H2AX foci on individual chromosomal arms. However, individual chromosome arms can be easily visualized in fibroblasts of the Indian muntjac, M. muntjak, a small deer with the typical mammalian DNA complement divided into 6 chromosomes instead of the 46 in humans (
-H2AX foci similar in number and intensity to those seen in the MCF7 cells (Figure 5 D). In addition, it is possible to see the relationship of
-H2AX foci to the chromosome arms.
A study was performed with muntjac cell cultures exposed to 0.6 Gy and permitted to recover for various times (Figure 6). The fields were searched for mitotic cells, which were imaged. A mitotic figure with discretely visible arms taken from each recovery time period is presented in Figure 6. -H2AX foci, although not detectable after 0.3 min (Figure 6 A), were detectable as small punctate foci after 1 min and continued to grow in number and size until 30 min (Figure 6, BE). At 90 min, the
-H2AX foci were fewer in number but similar in size to those seen at 30 min. Since muntjac cells contain ~90% of the DNA per cell as human cells (
-H2AX foci per Gy would be expected in the two. If each half mitotic figure contains the G1 complement of DNA, one would expect ~6 initial and 6 persistent DNA double-strand breaks in each. The number of
-H2AX foci visible in each half of the 9- and 30-min mitotic figures (Figure 6D and Figure E) is higher than that found in the IMR90 cells (Figure 2C and Figure D, and Figure 3), and nearer the expected value for initial rather than persistent DNA double-strand breaks. This difference might reflect a greater sensitivity of detection of small
-H2AX foci in mitotic cells due to the greater compaction of the chromatin; mitotic cells often display more distinct foci than do interphase cells (compare the mitotic and interphase cells in Figure 2J and Figure M). The difference could also be due to differences in DNA double-strand break detection and rejoining between interphase and mitotic cells, or to differences between human and muntjac cell metabolism. The IMR90 cells contained more foci at 3 min after 0.6 Gy than later (Figure 2B and Figure C), whereas the opposite was the case for the muntjac mitotic cells (Figure 6C and Figure D). These findings indicate that these types of differences do exist, but whether they are due to differences in detection sensitivity, DNA compaction, species metabolism, or other factors requires further study.
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These findings show that DNA double-strand breaks are rapidly detected and marked in condensed chromosomes as well as in interphase chromatin. The mitotic cells imaged in Figure 6 appear to have been dividing normally when fixed. However, in cultures fixed at 90 min after irradiation, defective mitotic cells could be found (Figure 7, AC). Three mitotic cells containing six isolated chromosomal arm fragments are shown, each with a large -H2AX focus at one end (indicated by green arrows). In addition, no isolated chromosomal arms lacking a terminal
-H2AX focus were found in any mitotic figure. These results provide direct visual confirmation that
-H2AX forms en masse at the sites of DNA double-strand breaks.
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Ionizing radiation introduces a delay in cell cycle progression in early G2.
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Discussion |
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Collectively, these data reveal the presence of an immediate, substantial, and evolutionarily conserved response of cells to the introduction of DNA double-strand breaks. This response involves the formation of -H2AX on chromosomal regions encompassing megabase lengths of DNA adjacent to break sites. No mammalian cell line, normal or repair-defective, has been found that lacks the ability to form
-H2AX when exposed to ionizing radiation. Cell lines from patients with ataxia telangiectasia, Werner's syndrome, Bloom's syndrome, and Nijmegen breakage syndrome all form
-H2AX after exposure to ionizing radiation (
-H2AX by gel analysis, and show
-H2AX foci after exposure to ionizing radiation (Rogakou, E.P., C. Boon, and W.M. Bonner, unpublished results). A KU70 knockout cell line (
-H2AX. The lack of mutant cell lines unable to form
-H2AX indicates that it may fulfill essential functions in organisms throughout the evolutionary scale.
It has been reported that when DNA double-strand breaks were introduced into regions of fibroblast nuclei by partial volume irradiation, MRE11, one of the proteins involved in DNA double-strand break rejoining, was found in these regions 30 min after irradiation (-H2AX are formed in irradiated human fibroblasts by 10 min, amounts corresponding to regions of chromatin containing about 2 x 106 bp of DNA and 2,000
-H2AX molecules (
-H2AX foci at the sites of DNA double-strand breaks could be to serve in recruiting proteins that are involved in rejoining DNA ends such as MRE11 or RAD50 to those sites, either directly, through binding to the
-H2AX COOH terminus, or indirectly, through an altered regional chromatin structure. With the
-H2AX antibody, regions of chromatin containing DNA double-strand breaks may be isolated and components that interact with those regions may be characterized.
The -H2AX domains seen on chromosome arms are similar in appearance to chromosome bands and also appear to stop enlarging after 30 min, even though some DNA double-strand breaks are still present. Although other explanations are possible, these findings may suggest the existence of units of higher-order chromatin structures that are involved in monitoring DNA integrity.
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Acknowledgements |
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We thank Dr. Kurt Kohn (National Cancer Institute, Division of Basic Sciences, Laboratory of Molecular Pharmacology, National Institutes of Health) for his continuous support of this work, Dr. Kenneth Yamada (National Institute of Dental and Craniofacial Research, Craniofacial Developmental Biology and Regeneration Branch, National Institutes of Health) for the use of the LaserScissorsTM instrument, and Dr. Robert F. Bonner (National Institute of Child Health and Human Development, Laboratory of Integrative and Medical Biophysics, National Institutes of Health) for instruction on the LaserScissorsTM.
Submitted: 1 June 1999
Revised: 26 July 1999
Accepted: 27 July 1999
1.used in this paper: BrdU, bromo deoxyuridine
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References |
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