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1.
DNA double-strand breaks are thought to precede the formation of most radiation-induced micronuclei. Phosphorylation of the histone H2AX is an early indicator of DNA double-strand breaks. Here we studied the phosphorylation status of the histone H2AX in micronuclei after exposure of cultured cells to ionizing radiation or treatment with colchicine. In human astrocytoma SF268 cells, after exposure to gamma radiation, the proportion of gamma-H2AX-positive to gamma-H2AX-negative micronuclei increases. The majority of the gamma-H2AX-positive micronuclei are centromere-negative. The number of gamma-H2AX-positive micronuclei continues to increase even 24 h postirradiation when most gamma-H2AX foci in the main nucleus have disappeared. In contrast, in normal human fibroblasts (BJ), the proportion of gamma-H2AX-positive to gamma-H2AX-negative micronuclei remains constant, and the majority of the centromere-negative cells are gamma-H2AX-negative. Treatment of both cell lines with colchicine results in mostly centromere-positive, gamma-H2AX-negative micronuclei. Immunostaining revealed co-localization of MDC1 and ATM with gamma-H2AX foci in both main nuclei and micronuclei; however, other repair proteins, such as Rad50, 53BP1 and Rad17, that co-localized with gamma-H2AX foci in the main nuclei were not found in the micronuclei. Combination of the micronucleus assay with gamma-H2AX immunostaining provides new insights into the mechanisms of the formation and fate of micronuclei.  相似文献   

2.
DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming γH2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear γH2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of γH2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of γH2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, γH2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of γH2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of γH2AX foci in mouse tissues.Download video file.(284M, mp4)  相似文献   

3.
The induction of DNA double-strand breaks (DSBs) by genotoxic treatment leads to hightoxicity and genetic instability. Various approaches have been undertaken to quantify thenumber of breaks and to follow the kinetic of DSB repair. Recently, the phosphorylation ofthe variant histone H2AX (named γH2AX), quantified by specific immunodetectionapproaches, has provided a valuable and highly sensitive method to monitor DSBs formation.Although it is admitted that the number of γH2AX foci reflected that of DSBs, contradictoryreports leave open the question of a link between the disappearance of γH2AX signal andDSBs repair. We determined γH2AX expression (i) in cells either proficient or not in DSBsrepair capacity, (ii) after exposure to ionizing radiation (IR) or calicheamicin γ1, aradiomimetic compound, (iii) and by three different immunodetection methods, focinumbering, flow cytometry or Western blotting. We showed here that γH2AX loss correlateswith DSB repair activity only at low cytotoxic doses, when less than 100-150 DSBs breaksper genome are produced, independently of the method used. In addition, in DNA repairproficient cells, the early decrease in the number and intensity of γH2AX foci observed after a2 Gy exposure was not associated with a significant change in the global γH2AX level asdetermined by Western blotting or flow cytometry. These results suggest that thedephosphorylation step of γH2AX may be limiting and that the loss of foci is mediated notonly by γH2AX dephosphorylation but also through its redistribution towards the chromatin.  相似文献   

4.
Cell cycle checkpoints induced by DNA damage play an integral role in preservation of genomic stability by allowing cells to limit the propagation of deleterious mutations. The retinoblastoma tumor suppressor (RB) is crucial for the maintenance of the DNA damage checkpoint function because it elicits cell cycle arrest in response to a variety of genotoxic stresses. Although sporadic loss of RB is characteristic of most cancers and results in the bypass of the DNA damage checkpoint, the consequence of RB loss upon chemotherapeutic responsiveness has been largely uninvestigated. Here, we employed a conditional knockout approach to ablate RB in adult fibroblasts. This system enabled us to examine the DNA damage response of adult cells following acute RB deletion. Using this system, we demonstrated that loss of RB disrupted the DNA damage checkpoint elicited by either cisplatin or camptothecin exposure. Strikingly, this bypass was not associated with enhanced repair, but rather the accumulation of phosphorylated H2AX (γH2AX) foci, which indicate DNA double-strand breaks. The formation of γH2AX foci was due to ongoing replication following chemotherapeutic treatment in the RB-deficient cells. Additionally, peak γH2AX accumulation occurred in S-phase cells undergoing DNA replication in the presence of damage, and these γH2AX foci co-localized with replication foci. These results demonstrate that acute RB loss abrogates DNA damage-induced cell cycle arrest to induce γH2AX foci formation. Thus, secondary genetic lesions induced by RB loss have implications for the chemotherapeutic response and the development of genetic instability.  相似文献   

5.
In this study, we investigated the formation of radiation-induced foci in normal human fibroblasts exposed to X rays or 130 keV/mum nitrogen ions using antibodies to phosphorylated protein kinase ataxia telangiectasia mutated (ATMp) and histone H2AX (gamma-H2AX). High-content automatic image analysis was used to quantify the immunofluorescence of radiation-induced foci. The size of radiation-induced foci increased for both proteins over a 2-h period after nitrogen-ion irradiation, while the size of radiation-induced foci did not change after exposure to low-LET radiation. The number of radiation-induced ATMp foci showed a more rapid rise and greater frequency after X-ray exposure and was resolved more rapidly such that the frequency of radiation-induced foci decreased by 90% compared to 60% after exposure to high-LET radiation 2 h after 30 cGy. In contrast, the kinetics of radiation-induced gamma-H2AX focus formation was similar for high- and low-LET radiation in that it reached a plateau early and remained constant for up to 2 h. High-resolution 3D images of radiation-induced gamma-H2AX foci and dosimetry computation suggest that multiple double-strand breaks from nitrogen ions are encompassed within large nuclear domains of 4.4 Mbp. Our work shows that the size and frequency of radiation-induced foci vary as a function of radiation quality, dose, time and protein target. Thus, even though double-strand breaks and radiation-induced foci are correlated, the dynamic nature of both contradicts their accepted equivalence for low doses of different radiation qualities.  相似文献   

6.
Histone H2AX is phosphorylated and forms foci in response to exposure to ionizing radiation. It has been thought that phosphorylated histone H2AX foci reflect unrepaired DNA double-strand breaks; however, we report here the localization of phosphorylated histone H2AX foci at the site of rejoined DNA double-strand breaks. We observed that phosphorylated histone H2AX foci remained even 96 h after exposure to X rays in interphase cells. To clarify the localization of residual phosphorylated histone H2AX foci, we examined localization of focus formation on mitotic chromosomes irradiated with X rays. We found that phosphorylated histone H2AX foci were located not only on chromosomal fragments but also on intact metaphase chromosomes without fragments. In anaphase cells, chromosomal bridges, which resulted from illegitimate rejoining of DNA broken ends, had phosphorylated histone H2AX foci. These foci were detected as individual small spots 30 min after X irradiation, but foci detected 20 or 96 h after X irradiation were clustered along the chromosomal bridges. These results indicate that phosphorylated histone H2AX foci persist if DNA breaks are rejoined. It is suggested that "residual" foci indicate an aberrant chromatin structure by illegitimate rejoining but not a DNA double-strand break itself.  相似文献   

7.
Rapid phosphorylation of histone H2AX after exposure of cells to ionizing radiation occurs at DSB sites and extends to a region including as much as 30 Mbp of chromatin to form visible microscopic structures called gamma-H2AX foci. Although the kinetics of total cellular histone H2AX phosphorylation after irradiation has been characterized, we still know little about the phosphorylation kinetics of individual gamma-H2AX foci. In addition, there are hundreds of smaller gamma-H2AX foci that are not associated with DNA double-strand breaks. We refer to these sites as DSB-unrelated gamma-H2AX foci. By using indirect immunofluorescence microscopy, deconvolution and three-dimensional image analysis, we established an objective method to quantitatively analyze each gamma-H2AX focus as well as to discriminate DSB-related gamma-H2AX foci from DSB-unrelated gamma-H2AX foci. Using this method, we found that histone H2AX phosphorylation at different DSB sites was asynchronous after exposure to ionizing radiation. This may reflect the heterogeneous characteristic of free DNA ends that are generated under these conditions. In addition, we found that increased histone H2AX phosphorylation also occurred outside of DSB sites after exposure to ionizing radiation. The function of this DSB-unassociated phosphorylation is not known.  相似文献   

8.
Cellular senescence, an irreversible proliferation arrest evoked by stresses such as oncogene activation, telomere dysfunction, or diverse genotoxic insults, has been implicated in tumor suppression and aging. Primary human fibroblasts undergoing oncogene-induced or replicative senescence are known to form senescence-associated heterochromatin foci (SAHF), nuclear DNA domains stained densely by DAPI and enriched for histone modifications including lysine9-trimethylated histone H3. While cellular senescence occurs also in premalignant human lesions, it is unclear how universal is SAHF formation among various cell types, under diverse stresses, and whether SAHF occur in vivo. Here, we report that human primary fibroblasts (BJ and MRC-5) and primary keratinocytes undergoing replicative senescence, or premature senescence induced by oncogenic H-Ras, diverse chemotherapeutics and bacterial cytolethal distending toxin, show differential capacity to form SAHF. Whereas all tested cell types formed SAHF in response to activated H-Ras, only MRC-5, but not BJ fibroblasts or keratinocytes, formed SAHF under senescence induced by etoposide, doxorubicin, hydroxyurea, bacterial intoxication or telomere attrition. In addition, DAPI-defined SAHF were detected on paraffin sections of Ras-transformed cultured fibroblasts, but not human lesions at various stages of tumorigenesis. Overall, our results indicate that unlike the widely present DNA damage response marker γH2AX, SAHF is not a common feature of cellular senescence. Whereas SAHF formation is shared by diverse cultured cell types under oncogenic stress, SAHF are cell-type-restricted under genotoxin-induced and replicative senescence. Furthermore, while the DNA/DAPI-defined SAHF formation in cultured cells parallels enhanced expression of p16ink4a, such ‘prototypic’ SAHF are not observed in tissues, including premalignant lesions, irrespective of enhanced p16ink4a and other features of cellular senescence.  相似文献   

9.
Damage to DNA that engenders double-strand breaks (DSBs) triggers phosphorylation of histone H2AX on Ser-139. Expression of phosphorylated H2AX (_H2AX) can be revealed immunocytochemically; the intensity of ?H2AX immunofluorescence (IF) measured by cytometry was reported to correlate with the frequency of DSBs induced by X-ray radiation or by DNA damaging antitumor drugs. The aim of the present study was to measure expression of ?H2AX following exposure of HeLa and HL-60 cells to a wide range of doses of UV-B light (6.1 J/m2-3.45 kJ/m2) and using multiparameter flow and laser scanning cytometry (LSC) to correlate DNA damage with cell cycle phase and induction of apoptosis. In both cell lines, the highest degree of H2AX phosphorylation induced by UV was seen in S-phase cells, particularly during early portion of S. In cells that did not replicate DNA (G1, G2 and M) the degree of H2AX phosphorylation was markedly lower than that in S-phase cells, and was strongly UV dose-dependent. Furthermore, the level of UV-induced γH2AX in G1, G2 and M was much higher in HeLa- than in HL-60-cells. Apoptotic cells become apparent >2h after exposure to UV and exhibited nearly an order of magnitude higher intensity of γH2AX IF than that initially induced by UV; predominantly S-phase cells underwent apoptosis. While the suppression of DNA replication aphidicolin prevented the induction of H2AX phosphorylation by UV in most S phase cells, it had no effect on a small cohort of cells that appeared to be entering S-phase, that expressed very high levels of γH2AX. Furthermore, aphidicolin itself induced γH2AX in early-S phase cells. The induction of γH2AX by UV was inhibited, but the incidence of apoptosis increased, by 5 mM caffeine, a known inhibitor of PI-3-related kinases. The data are consistent with the notion that H2AX phosphorylation observed throughout S phase reflects formation of DSBs due to the collision of replication forks with the UV-induced primary DNA lesions. Induction of γH2AX in GG1, GG2 and M is likely a response to the primary DSBs generated during UV exposure and/or DNA repair. It is unclear why the latter process was more pronounced in HeLa than in HL-60 cells.  相似文献   

10.

Background

Significant data supports the health benefits of selenium although supplementation trials have yielded mixed results. GPx-1, whose levels are responsive to selenium availability, is implicated in cancer etiology by human genetic data. Selenium's ability to alter the phosphorylation of the H2AX, a histone protein that functions in the reduction of DNA damage by recruiting repair proteins to the damage site, following exposure to ionizing radiation and bleomycin was investigated.

Methods

Human cell lines that were either exposed to selenium or were transfected with a GPx-1 expression construct were exposed to ionizing radiation or bleomycin. Phosphorylation of histone H2AX was quantified by flow cytometry and survival by the MTT assay. Phosphorylation of the Chk1 and Chk2 checkpoint proteins was quantified by western blotting.

Results

In colon-derived cells, selenium increases GPx-1 and attenuated H2AX phosphorylation following genotoxic exposures while the viability of these cells was unaffected. MCF-7 cells and transfectants that express high GPx-1 levels were exposed to ionizing radiation and bleomycin, and H2AX phosphorylation and cell viability were assessed. GPx-1 increased H2AX phosphorylation and viability following the induction of DNA damage while enhancing the levels of activated Chk1 and Chk2.

Conclusions

Exposure of mammalian cells to selenium can alter the DNA damage response and do so by mechanisms that are dependent and independent of its effect on GPx-1.

General significance

Selenium and GPx-1 may stimulate the repair of genotoxic DNA damage and this may account for some of the benefits attributed to selenium intake and elevated GPx-1 activity.  相似文献   

11.
12.
DNA double-strand breaks and gamma-H2AX signaling in the testis   总被引:6,自引:0,他引:6  
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.  相似文献   

13.
H2AX is a histone variant which is present and ubiquitously distributed throughout the genome. An immunocytochemical assay using antibodies capable of recognizing histone H2AX phosphorylated at serine 139 (gammaH2AX) is very sensitive and is a specific indicator for the existence of a DNA double strand break. Although heat stress has been reported to induce the formation of gammaH2AX foci, no gammaH2AX foci formation was observed in several mammalian cell lines after heat shock. Since this was in contrast to earlier reports, the work described here was intended to verify that heat-induced gammaH2AX foci do form in mammalian cell lines other than the cell lines used in earlier reports concerning gammaH2AX foci formation. The cell lines used in this work includes cell lines with differing p53-gene status (H1299, H1299/neo, H1299/mp53 and H1299/wtp53 cells), various cancer cell lines (HeLa, HepG2, U2-OS cells), normal human cells (HEK-293 and AG1522), and cell lines established from other species (MEF normal mouse cells and CHL normal Chinese hamster cells). Exponentially growing cells were exposed to heat shock (42 degrees C for 6 h or 45.5 degrees C for 20 min) or to X-rays (3Gy). The presence of gammaH2AX was examined with immunocytochemistry and flow cytometry. Induction of gammaH2AX foci formation was observed in all of the mammalian cell lines used here after heat-treatment as well as after X-irradiation. However, the intensity of gammaH2AX was different in the different cell lines used. These results confirm that heat can induce gammaH2AX foci formation in many mammalian cell lines.  相似文献   

14.
15.
16.
A role for histone H2AX, one of the variants of the nucleosome core histone H2A, has been demonstrated in DNA repair, tumor suppression, apoptosis, and cell cycle checkpoint function. However, the physiological function and post-translational modification of histone H2AX during vertebrate development have not been elucidated. Here, we provide evidence showing that Xenopus histone H2AX (XH2AX) has a role in the anterior neural plate for eye field formation during Xenopus embryogenesis. A loss-of-function study clearly demonstrated a critical role of XH2AX in anterior neural specification. Through a differentiation assay with Xenopus animal cap embryonic stem cells, we confirmed that XH2AX is required for the activin-induced anterior neural specification of the ectoderm. Furthermore, we found that Chk1 is an essential kinase to phosphorylate histone XH2AX at Thr16, which is involved in the biological function of this histone. Taken together, our findings reveal that XH2AX has a specific role in anterior neural formation of Xenopus, which is mediated through phosphorylation of XH2AX at Thr16 by Chk1.  相似文献   

17.
Hau PM  Siu WY  Wong N  Lai PB  Poon RY 《FEBS letters》2006,580(19):4727-4736
Polyploidization occurs during normal development as well as during tumorigenesis. In this study, we investigated if the responses to genotoxic stress in cancer cells are influenced by the ploidy. Prolonged treatment of Hep3B cells with the spindle inhibitor nocodazole resulted in mitotic slippage, followed by re-replication of the DNA to produce polyploids. Reintroduction of p53 restored the checkpoints and suppressed polyploidization. Remarkably, a stable tetraploidy cell line could be generated from Hep3B by a transient nocodazole treatment followed by a period of recovery. Using this novel tetraploid system, we found that tetraploidization increased the cell volume without significantly affecting the cell cycle. Although tetraploidization was accompanied by an increase in centrosome number, the majority of mitoses in the tetraploid cells remained bipolar. Polyploidization sensitized cells to genotoxic stress inflicted by ionizing radiation and topoisomerase inhibitors without affecting the sensitivity to spindle inhibitors. Accordingly, more gamma-H2AX foci were induced by radiation in tetraploids than in normal Hep3B cells. Likewise, primary tetraploid human fibroblasts displayed higher gamma-H2AX foci formation than diploid human fibroblasts. An implication for chemotherapy is that some cancer cells can be sensitized to genotoxic agents by a preceding step that induces polyploidization.  相似文献   

18.
Cellular senescence is a phenotype that is likely linked with aging. Recent concepts view different forms of senescence as permanently maintained DNA damage responses partially characterized by the presence of senescence-associated DNA damage foci at dysfunctional telomeres. Irradiation of primary human dermal fibroblasts with the photosensitizer 8-methoxypsoralen and ultraviolet A radiation (PUVA) induces senescence. In the present study, we demonstrate that senescence after PUVA depends on DNA interstrand cross-link (ICL) formation that activates ATR kinase. ATR is necessary for the manifestation and maintenance of the senescent phenotype, because depletion of ATR expression before PUVA prevents induction of senescence, and reduction of ATR expression in PUVA-senesced fibroblasts releases cells from growth arrest. We find an ATR-dependent phosphorylation of the histone H2AX (gamma-H2AX). After PUVA, ATR and gamma-H2AX colocalize in multiple nuclear foci. After several days, only few predominantly telomere-localized foci persist and telomeric DNA can be coimmunoprecipitated with ATR from PUVA-senesced fibroblasts. We thus identify ATR as a novel mediator of telomere-dependent senescence in response to ICL induced by photoactivated psoralens.  相似文献   

19.
Immunocytochemistry using α-phospho-H2AX antibodies shows that hydroxyurea (HU), an inhibitor of ribonucleotide reductase, and aphidicolin (APH), an inhibitor of DNA-polymerases α and δ, may promote formation of phospho-H2AX foci in late S/G2-phase cells in root meristems of Vicia faba. Although fluorescent foci spread throughout the whole area of nucleoplasm, large phospho-H2AX aggregates in HU-treated cells allocate mainly in perinucleolar regions. A strong tendency of ATR/ATM-dependent phospho-Chk1S317 kinase to focus in analogous compartments, as opposed to phospho-Chk2T68 and to both effector kinases in APH-treated cells, may suggest that selected elements of the intra-S-phase cell cycle checkpoints share overlapping locations with DNA repair factors known to concentrate in phospho-H2AX aggregates. APH-induced phosphorylation of H2AX exhibits little or no overlap with the areas positioned close to nucleoli. Following G2-M transition of the HU- and APH-pretreated cells, altered chromatin structures are still discernible as large phospho-H2AX foci in the vicinity of chromosomes. Both in HU- and APH-treated roots, immunofluorescence analysis revealed a dominant fraction of small foci and a less frequent population of large phospho-H2AX agregates, similar to those observed in animal cells exposed to ionizing radiation. The extent of H2AX phosphorylation has been found considerably reduced in root meristem cells treated with HU and caffeine. The frequencies of phospho-H2AX foci observed during mitosis and caffeine-mediated premature chromosome condensation (PCC) suggest that there may be functional links between the checkpoint mechanisms that control genome integrity and those activities which operate throughout the unperturbed mitosis in plants.  相似文献   

20.
The DNA double-strand breaks (DSBs) are considered to be the most relevant lesions for the deleterious effects of ionizing radiation exposure. The discovery that the induction of DSBs is rapidly followed by the phosphorylation of H2AX histone at Ser-139, favoring repair protein recruitment or access, opens the possibility for a wide range of research. This phosphorylated histone, named gamma-H2AX, has been shown to form foci in interphase nuclei as well as megabase chromatin domains surrounding the DNA lesion on chromosomes. Using detection of gamma-H2AX on germ cell mitotic chromosomes 2 h after gamma-irradiation, we studied radiation-induced DSBs during the G(2)/M phase of the cell cycle. We show that 1) non-irradiated neonatal germ cells express gamma-H2AX with variable patterns at metaphase, 2) gamma-irradiation induces foci whose number increases in a dose-dependent manner, 3) some foci correspond to visible chromatid breaks or exchanges, 4) sticky chromosomes characterizing cell radiation exposure during mitosis are a consequence of DSBs, and 5) gamma-H2AX remains localized at the sites of the lesions even after end-joining has taken place. This suggests that completion of DSB repair does not necessarily imply disappearance of gamma-H2AX.  相似文献   

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