An optimized method for measurement of gamma-H2AX in blood mononuclear and cultured cells

Phosphorylation of histone protein H2AX on serine 139 (gamma-H2AX) occurs at sites flanking DNA double-stranded breaks (DSBs) and can provide a measure of the number of DSBs within a cell. We describe a flow cytometry-based method optimized to measure gamma-H2AX in nonfixed mononuclear blood cells as well as in cultured cells, which is more sensitive and involves less steps compared with protocols involving fixed cells. This method can be used to monitor induction of gamma-H2AX in mononuclear cells from cancer patients undergoing radiotherapy and for detection of gamma-H2AX throughout the cell cycle in cultured cells. The method is based on the fact that H2AX like other histone proteins are retained in the nucleus when cells are lysed at physiological salt concentrations. Cells are therefore added without fixation to a solution containing detergent to lyse the cells along with a fluorescein isothiocyanate-labeled monoclonal gamma-H2AX antibody, DNA staining dye and blocking agents. The stained nuclei can be analyzed by flow cytometry to monitor the level of gamma-H2AX to determine the level of DSBs and DNA content and to determine the cell cycle stage. The omission of fixation simplifies staining and enhances the sensitivity. This protocol can be completed within 4-6 h.     

Quantitative detection of (125)IdU-induced DNA double-strand breaks with gamma-H2AX antibody

When mammalian cells are exposed to ionizing radiation and other agents that introduce DSBs into DNA, histone H2AX molecules in megabase chromatin regions adjacent to the breaks become phosphorylated within minutes on a specific serine residue. An antibody to this phosphoserine motif of human H2AX (gamma-H2AX) demonstrates that gamma-H2AX molecules appear in discrete nuclear foci. To establish the quantitative relationship between the number of these foci and the number of DSBs, we took advantage of the ability of (125)I, when incorporated into DNA, to generate one DNA DSB per radioactive disintegration. SF-268 and HT-1080 cell cultures were grown in the presence of (125)IdU and processed immunocytochemically to determine the number of gamma-H2AX foci. The numbers of (125)IdU disintegrations per cell were measured by exposing the same immunocytochemically processed samples to a radiation-sensitive screen with known standards. Under appropriate conditions, the data yielded a direct correlation between the number of (125)I decays and the number of foci per cell, consistent with the assumptions that each (125)I decay yields a DNA DSB and each DNA DSB yields a visible gamma-H2AX focus. Based on these findings, we conclude that gamma-H2AX antibody may form the basis of a sensitive quantitative method for the detection of DNA DSBs in eukaryotic cells.     

Characteristics of gamma-H2AX foci at DNA double-strand breaks sites.

Phosphorylated H2AX (gamma-H2AX) is essential to the efficient recognition and (or) repair of DNA double strand breaks (DSBs), and many molecules, often thousands, of H2AX become rapidly phosphorylated at the site of each nascent DSB. An antibody to gamma-H2AX reveals that this highly amplified process generates nuclear foci. The phosphorylation site is a serine four residues from the C-terminus which has been evolutionarily conserved in organisms from giardia intestinalis to humans. Mice and yeast lacking the conserved serine residue demonstrate a variety of defects in DNA DSB processing. H2AX Delta/Delta mice are smaller, sensitive to ionizing radiation, defective in class switch recombination and spermatogenesis while cells from the mice demonstrate substantially increased numbers of genomic defects. gamma-H2AX foci formation is a sensitive biological dosimeter and presents new and exciting opportunities to understand important biological processes, human diseases, and individual variations in radiation sensitivity. These potentialities demonstrate the importance of understanding the parameters and functions of gamma-H2AX formation.     

DNA double-strand breaks and gamma-H2AX signaling in the testis

Within minutes of the induction of DNA double-strand breaks in somatic cells, histone H2AX becomes phosphorylated at serine 139 and forms gamma-H2AX foci at the sites of damage. These foci then play a role in recruiting DNA repair and damage-response factors and changing chromatin structure to accurately repair the damaged DNA. These gamma-H2AX foci appear in response to irradiation and genotoxic stress and during V(D)J recombination and meiotic recombination. Independent of irradiation, gamma-H2AX occurs in all intermediate and B spermatogonia and in preleptotene to zygotene spermatocytes. Type A spermatogonia and round spermatids do not exhibit gamma-H2AX foci but show homogeneous nuclear gamma-H2AX staining, whereas in pachytene spermatocytes gamma-H2AX is only present in the sex vesicle. In response to ionizing radiation, gamma-H2AX foci are generated in spermatogonia, spermatocytes, and round spermatids. In irradiated spermatogonia, gamma-H2AX interacts with p53, which induces spermatogonial apoptosis. These events are independent of the DNA-dependent protein kinase (DNA-PK). Irradiation-independent nuclear gamma-H2AX staining in leptotene spermatocytes demonstrates a function for gamma-H2AX during meiosis. gamma-H2AX staining in intermediate and B spermatogonia, preleptotene spermatocytes, and sex vesicles and round spermatids, however, indicates that the function of H2AX phosphorylation during spermatogenesis is not restricted to the formation of gamma-H2AX foci at DNA double-strand breaks.     

Dephosphorylation of histone gamma-H2AX during repair of DNA double-strand breaks in mammalian cells and its inhibition by calyculin A

The induction of DNA double-strand breaks (DSBs) by ionizing radiation in mammalian chromosomes leads to the phosphorylation of Ser-139 in the replacement histone H2AX, but the molecular mechanism(s) of the elimination of phosphorylated H2AX (called gamma-H2AX) from chromatin in the course of DSB repair remains unknown. We showed earlier that gamma-H2AX cannot be replaced by exchange with free H2AX, suggesting the direct dephosphorylation of H2AX in chromatin by a protein phosphatase. Here we studied the dynamics of dephosphorylation of gamma-H2AX in vivo and found that more than 50% was dephosphorylated in 3 h, but a significant amount of gamma-H2AX could be detected even 6 h after the induction of DSBs. At this time, a significant fraction of the gamma-H2AX nuclear foci co-localized with the foci of RAD50 protein that did not co-localize with replication sites. However, gamma-H2AX could be detected in some cells treated with methyl methanesulfonate which accumulated RAD18 protein at stalled replication sites. We also found that calyculin A inhibited early elimination of gamma-H2AX and DSB rejoining in vivo and that protein phosphatase 1 was able to remove phosphate groups from gamma-H2AX-containing chromatin in vitro. Our results confirm the tight association between DSBs and gamma-H2AX and the coupling of its in situ dephosphorylation to DSB repair.     

Comparison of the induction and disappearance of DNA double strand breaks and gamma-H2AX foci after irradiation of chromosomes in G1-phase or in condensed metaphase cells

The induction and disappearance of DNA double strand breaks (DSBs) after irradiation of G1 and mitotic cells were compared with the gamma-H2AX foci assay and a gel electrophoresis assay. This is to determine whether cell cycle related changes in chromatin structure might influence the gamma-H2AX assay which depends on extensive phosphorylation and dephosphorylation of the H2AX histone variant surrounding DSBs. The disappearance of gamma-H2AX foci after irradiation was much slower for mitotic than for G1 cells. On the other hand, no difference was seen for the gel electrophoresis assay. Our data may suggest the limited accessibility of dephosphorylation enzyme in irradiated metaphase cells or trapped gamma-H2AX in condensed chromatin.     

Replication protein A and gamma-H2AX foci assembly is triggered by cellular response to DNA double-strand breaks

Human replication protein A (RPA p34), a crucial component of diverse DNA excision repair pathways, is implicated in DNA double-strand break (DSB) repair. To evaluate its role in DSB repair, the intranuclear dynamics of RPA was investigated after DNA damage and replication blockage in human cells. Using two different agents [ionizing radiation (IR) and hydroxyurea (HU)] to generate DSBs, we found that RPA relocated into distinct nuclear foci and colocalized with a well-known DSB binding factor, gamma-H2AX, at the sites of DNA damage in a time-dependent manner. Colocalization of RPA and gamma-H2AX foci peaked at 2 h after IR treatment and subsequently declined with increasing postrecovery times. The time course of RPA and gamma-H2AX foci association correlated well with the DSB repair activity detected by a neutral comet assay. A phosphatidylinositol-3 (PI-3) kinase inhibitor, wortmannin, completely abolished both RPA and gamma-H2AX foci formation triggered by IR. Additionally, radiosensitive ataxia telangiectasia (AT) cells harboring mutations in ATM gene product were found to be deficient in RPA and gamma-H2AX colocalization after IR. Transfection of AT cells with ATM cDNA fully restored the association of RPA foci with gamma-H2AX illustrating the requirement of ATM gene product for this process. The exact coincidence of RPA and gamma-H2AX in response to HU specifically in S-phase cells supports their role in DNA replication checkpoint control. Depletion of RPA by small interfering RNA (SiRNA) substantially elevated the frequencies of IR-induced micronuclei (MN) and apoptosis in human cells suggestive of a role for RPA in DSB repair. We propose that RPA in association with gamma-H2AX contributes to both DNA damage checkpoint control and repair in response to strand breaks and stalled replication forks in human cells.     

Heterochromatin is refractory to gamma-H2AX modification in yeast and mammals

Double-strand break (DSB) damage in yeast and mammalian cells induces the rapid ATM (ataxia telangiectasia mutated)/ATR (ataxia telangiectasia and Rad3 related)-dependent phosphorylation of histone H2AX (gamma-H2AX). In budding yeast, a single endonuclease-induced DSB triggers gamma-H2AX modification of 50 kb on either side of the DSB. The extent of gamma-H2AX spreading does not depend on the chromosomal sequences. DNA resection after DSB formation causes the slow, progressive loss of gamma-H2AX from single-stranded DNA and, after several hours, the Mec1 (ATR)-dependent spreading of gamma-H2AX to more distant regions. Heterochromatic sequences are only weakly modified upon insertion of a 3-kb silent HMR locus into a gamma-H2AX-covered region. The presence of heterochromatin does not stop the phosphorylation of chromatin more distant from the DSB. In mouse embryo fibroblasts, gamma-H2AX distribution shows that gamma-H2AX foci increase in size as chromatin becomes more accessible. In yeast, we see a high level of constitutive gamma-H2AX in telomere regions in the absence of any exogenous DNA damage, suggesting that yeast chromosome ends are transiently detected as DSBs.     

Ionizing radiation-dependent gamma-H2AX focus formation requires ataxia telangiectasia mutated and ataxia telangiectasia mutated and Rad3-related

The histone variant H2AX is rapidly phosphorylated at the sites of DNA double-strand breaks (DSBs). This phosphorylated H2AX (gamma-H2AX) is involved in the retention of repair and signaling factor complexes at sites of DNA damage. The dependency of this phosphorylation on the various PI3K-related protein kinases (in mammals, ataxia telangiectasia mutated and Rad3-related [ATR], ataxia telangiectasia mutated [ATM], and DNA-PKCs) has been a subject of debate; it has been suggested that ATM is required for the induction of foci at DSBs, whereas ATR is involved in the recognition of stalled replication forks. In this study, using Arabidopsis as a model system, we investigated the ATR and ATM dependency of the formation of gamma-H2AX foci in M-phase cells exposed to ionizing radiation (IR). We find that although the majority of these foci are ATM-dependent, approximately 10% of IR-induced gamma-H2AX foci require, instead, functional ATR. This indicates that even in the absence of DNA replication, a distinct subset of IR-induced damage is recognized by ATR. In addition, we find that in plants, gamma-H2AX foci are induced at only one-third the rate observed in yeasts and mammals. This result may partly account for the relatively high radioresistance of plants versus yeast and mammals.     

Slow elimination of phosphorylated histone gamma-H2AX from DNA of terminally differentiated mouse heart cells in situ

Phosphorylation of replacement histone H2AX occurs in megabase chromatin domains around double-strand DNA breaks (DSBs) and this modification (called gamma-H2AX) may serve as a useful marker of genome damage and repair in terminally differentiated cells. Here using immunohistochemistry we studied kinetics of gamma-H2AX formation and elimination in the X-irradiated mouse heart and renal epithelial tissues in situ. Unirradiated tissues have 3-5% gamma-H2AX-positive cells and in tissues fixed 1h after X-irradiation gamma-H2AX-positive nuclei are induced in a dose-dependent manner approaching 20-30% after 3 Gy of IR. Analysis of mouse tissues at different times after 3 Gy of IR showed that maximal induction of gamma-H2AX in heart is observed 20 min after IR and then is decreased slowly with about half remaining 23 h later. In renal epithelium maximum of the gamma-H2AX-positive cells is observed 40 min after IR and then decreases to control values in 23 h. This indicates that there are significant variations between non-proliferating mammalian tissues in the initial H2AX phosphorylation rate as well as in the rate of gamma-H2AX elimination after X-irradiation, which should be taken into account in the analysis of radiation responses.     

Phosphorylation of histone H2AX in human lymphocytes as a possible marker of effective cellular response to ionizing radiation

DNA double-strand breaks (DSBs) which occurs in cells after ionizing radiation (IR) or chemical agents are the most dangerous lesions in eukariotic cells, which leads to cell death or chromosome abberations and cancer. One of the earliest response of cells to DSBs formation is phosphorylation by 139 serine of core variant histone H2AX in megabase chromatin domains around DSB (gamma-H2AX), which amplify signal and makes it possible to identify even one DSB in genome. Effective formation of gamma-H2AX is very important for maintenance of genome stability. Here, using immunofluorescent and Western blotting techniques, we studied dynamics of gamma-H2AX formation in human lymphocytes of various individuals irradiated ex vivo. We have found that dynamics of gamma-H2AX formation in lymphocytes differ between individuals but have similar kinetics and statistically is independent on people age.     

Photobleaching of GFP-labeled H2AX in chromatin: H2AX has low diffusional mobility in the nucleus

The Ser-139 phosphorylated form of replacement histone H2AX (gamma-H2AX) is induced within large chromatin domains by double-strand DNA breaks (DSBs) in mammalian chromosomes. This modification is known to be important for the maintenance of chromosome stability. However, the mechanism of gamma-H2AX formation at DSBs and its subsequent elimination during DSB repair remains unknown. gamma-H2AX formation and elimination could occur by direct phosphorylation and dephosphorylation of H2AX in situ in the chromatin. Alternatively, H2AX molecules could be phosphorylated freely in the nucleus, diffuse into chromatin regions containing DSBs and then diffuse out after DNA repair. In this study we show that free histone H2AX can be efficiently phosphorylated in vitro by nuclear extracts and that free gamma-H2AX can be dephosphorylated in vitro by the mammalian protein phosphatase 1-alpha. We made N-terminal fusion constructs of H2AX with green fluorescent protein (GFP) and studied their diffusional mobility in transient and stable cell transfections. In the absence or presence of DSBs, only a small fraction of GFP-H2AX is redistributed after photobleaching, indicating that in vivo this histone is essentially immobile in chromatin. This suggests that gamma-H2AX formation in chromatin is unlikely to occur by diffusion of free histone and gamma-H2AX dephosphorylation may involve the mammalian protein phosphatase 1alpha.     

gamma-H2AX formation in response to interstrand crosslinks requires XPF in human cells

To further define the molecular mechanisms involved in processing interstrand crosslinks, we monitored the formation of phosphorylated histone H2AX (gamma-H2AX), which is generated in chromatin near double strand break sites, following DNA damage in normal and repair-deficient human cells. Following treatment with a psoralen derivative and ultraviolet A radiation doses that produce significant numbers of crosslinks, gamma-H2AX levels in nucleotide excision repair-deficient XP-A fibroblasts (XP12RO-SV) increased to levels that were twice those observed in normal control GM637 fibroblasts. A partial XPA revertant cell line (XP129) that is proficient in crosslink removal, exhibited reduced gamma-H2AX levels that were intermediate between those of GM637 and XP-A cells. XP-F fibroblasts (XP2YO-SV and XP3YO) that are also repair-deficient exhibited gamma-H2AX levels below even control fibroblasts following treatment with psoralen and ultraviolet A radiation. Similarly, another crosslinking agent, mitomycin C, did not induce gamma-H2AX in XP-F cells, although it did induce equivalent levels of gamma-H2AX in XPA and control GM637 cells. Ectopic expression of XPF in XP-F fibroblasts restored gamma-H2AX induction following treatment with crosslinking agents. Angelicin, a furocoumarin which forms only monoadducts and not crosslinks following ultraviolet A radiation, as well as ultraviolet C radiation, resulted only in weak induction of gamma-H2AX in all cells, suggesting that the double strand breaks observed with psoralen and ultraviolet A treatment result preferentially following crosslink formation. These results indicate that XPF is required to form gamma-H2AX and likely double strand breaks in response to interstrand crosslinks in human cells. Furthermore, XPA may be important to allow psoralen interstrand crosslinks to be processed without forming a double strand break intermediate.     

A PP4-phosphatase complex dephosphorylates gamma-H2AX generated during DNA replication

The histone H2A variant H2AX is rapidly phosphorylated in response to DNA double-stranded breaks to produce gamma-H2AX. gamma-H2AX stabilizes cell-cycle checkpoint proteins and DNA repair factors at the break site. We previously found that the protein phosphatase PP2A is required to resolve gamma-H2AX foci and complete DNA repair after exogenous DNA damage. Here we describe a three-protein PP4 phosphatase complex in mammalian cells, containing PP4C, PP4R2, and PP4R3beta, that specifically dephosphorylates ATR-mediated gamma-H2AX generated during DNA replication. PP4 efficiently dephosphorylates gamma-H2AX within mononucleosomes in vitro and does not directly alter ATR or checkpoint kinase activity, suggesting that PP4 acts directly on gamma-H2AX in cells. When the PP4 complex is silenced, repair of DNA replication-mediated breaks is inefficient, and cells are hypersensitive to DNA replication inhibitors, but not radiomimetic drugs. Therefore, gamma-H2AX elimination at DNA damage foci is required for DNA damage repair, but accomplishing this task involves distinct phosphatases with potentially overlapping roles.     

NADP increases the level of histone H2AX phosphorylation in mouse heart cells after ionizing radiation

Phosphorylation of replaceable histone H2AX occurs in megabase chromatin domains around DNA double-strand breaks (DSBs), and this modification called gamma-H2AX can be used as an effective marker for DSBs repair and DNA damage response. Using Western blotting and immunohistochemistry techniques we have studied here the influence of exogenous nicotinamide adenine dinucleotide phosphate (NADP) which could potentially increase the intracellular level of NAD+ and on the level of gamma-H2AX formation in mouse heart cells after ionizing radiation (IR). We have found that injection of NAD+ in different doses immediately after IR causes an increased level of gamma-H2AX in mouse heart cells 20 min after IR at the dose of 3 Gy compared to control mice after IR exposure. It indicates that it could be a relationship between intracellular NAD+ content and DNA damage response in vivo.     

Co-exposure to benzo[a]pyrene and UVA induces phosphorylation of histone H2AX

Phosphorylation of histone H2AX (termed gamma-H2AX) was recently identified as an early event after induction of DNA double strand breaks (DSBs). We have previously shown that co-exposure to benzo[a]pyrene (BaP), a wide-spread environmental carcinogen, and ultraviolet A (UVA), a major component of solar UV radiation, induced DSBs in mammalian cells. In the present study, we examined whether co-exposure to BaP and UVA generates gamma-H2AX in CHO-K1 cells. Single treatment with BaP (10(-9)-10(-7)M) or UVA ( approximately 2.4 J/cm(2)) did not result in gamma-H2AX, however, co-exposure drastically induced foci of gamma-H2AX in a dose-dependent manner. gamma-H2AX could be detected even at very low concentration of BaP (10(-9)M) plus UVA (0.6J/cm(2)), which did not change cell survival rates. NaN(3) effectively inhibited the formation of gamma-H2AX induced by co-exposure, indicating the contribution of singlet oxygen. This is the first evidence that co-exposure to BaP and UVA induced DSBs, involving gamma-H2AX.     

Phosphorylation of histone H2AX at M phase in human cells without DNA damage response

A variant of histone H2A, H2AX, is phosphorylated on Ser139 in response to DNA double-strand breaks (DSBs), and clusters of the phosphorylated form of H2AX (gamma-H2AX) in nuclei of DSB-induced cells show foci at breakage sites. Here, we show phosphorylation of H2AX in a cell cycle-dependent manner without any detectable DNA damage response. Western blot and immunocytochemical analyses with the anti-gamma-H2AX antibody revealed that H2AX is phosphorylated at M phase in HeLa cells. In ataxia-telangiectasia cells lacking ATM kinase activity, gamma-H2AX was scarcely detectable in the mitotic chromosomes, suggesting involvement of ATM in M-phase phosphorylation of H2AX. Single-cell gel electrophoresis assay and Western blot analysis with the anti-phospho-p53 (Ser15) antibody indicated that H2AX in human M-phase cells is phosphorylated independently of DSB and DNA damage signaling. Even in the absence of DNA damage, phosphorylation of H2AX in normal cell cycle progression may contribute to maintenance of genomic integrity.     

DNA polymerase eta reduces the gamma-H2AX response to psoralen interstrand crosslinks in human cells

DNA interstrand crosslinks are processed by multiple mechanisms whose relationships to each other are unclear. Xeroderma pigmentosum-variant (XP-V) cells lacking DNA polymerase eta are sensitive to psoralen photoadducts created under conditions favoring crosslink formation, suggesting a role for translesion synthesis in crosslink repair. Because crosslinks can lead to double-strand breaks, we monitored phosphorylated H2AX (gamma-H2AX), which is typically generated near double-strand breaks but also in response to single-stranded DNA, following psoralen photoadduct formation in XP-V fibroblasts to assess whether polymerase eta is involved in processing crosslinks. In contrast to conditions favoring monoadducts, conditions favoring psoralen crosslinks induced gamma-H2AX levels in both XP-V and nucleotide excision repair-deficient XP-A cells relative to control repair-proficient cells; ectopic expression of polymerase eta in XP-V cells normalized the gamma-H2AX response. In response to psoralen crosslinking, gamma-H2AX as well as 53BP1 formed coincident foci that were more numerous and intense in XP-V and XP-A cells than in controls. Psoralen photoadducts induced gamma-H2AX throughout the cell cycle in XP-V cells. These results indicate that polymerase eta is important in responding to psoralen crosslinks, and are consistent with a model in which nucleotide excision repair and polymerase eta are involved in processing crosslinks and avoiding gamma-H2AX associated with double-strand breaks and single-stranded DNA in human cells.