Visualization of focal nuclear sites of DNA repair synthesis induced by bleomycin in human cells

In this study, we examined DNA repair synthesis in human cells treated with the radiomimetic drug bleomycin, which efficiently induces double-strand breaks (DSBs). Using tyramide-biotin to amplify fluorescent signals, discrete nuclear foci from the incorporation of 5-iododeoxyuridine (IdU) were detected in proliferating human cells treated with bleomycin. We believe this comes from the repair of DSBs. An increase in the number of foci (>5 per nucleus) was detected in a major fraction (75%) of non-S-phase cells labeled for 30 min with IdU 1 h after the end of bleomycin treatment. The fraction of cells with multiple IdU-containing foci was found to decrease 18 h after treatment. The average number of foci per nucleus detected 1 h after bleomycin treatment was found to decrease twofold between 1 and 3.5 h, indicating that the foci may be associated with the slow component of DSB repair. The presence of DSBs in bleomycin-treated cells was confirmed using antibodies against phosphorylated histone H2AX (gamma-H2AX), which is strictly associated with this type of DNA damage. After treatment with bleomycin, non-S-phase cells also displayed heterogeneous nuclear foci containing tightly bound proliferating cell nuclear antigen (PCNA), suggesting an ongoing process of unscheduled DNA synthesis. PCNA is known to be involved in base excision repair, but a fraction of the PCNA foci may also be associated with DNA synthesis occurring during the repair of DSBs.     

Involvement of histone H1.2 in apoptosis induced by DNA double-strand breaks

It is poorly understood how apoptotic signals arising from DNA damage are transmitted to mitochondria, which release apoptogenic factors into the cytoplasm that activate downstream destruction programs. Here, we identify histone H1.2 as a cytochrome c-releasing factor that appears in the cytoplasm after exposure to X-ray irradiation. While all nuclear histone H1 forms are released into the cytoplasm in a p53-dependent manner after irradiation, only H1.2, but not other H1 forms, induced cytochrome c release from isolated mitochondria in a Bak-dependent manner. Reducing H1.2 expression enhanced cellular resistance to apoptosis induced by X-ray irradiation or etoposide, but not that induced by other stimuli including TNF-alpha and UV irradiation. H1.2-deficient mice exhibited increased cellular resistance in thymocytes and the small intestine to X-ray-induced apoptosis. These results indicate that histone H1.2 plays an important role in transmitting apoptotic signals from the nucleus to the mitochondria following DNA double-strand breaks.     

Initiation of DNA fragmentation during apoptosis induces phosphorylation of H2AX histone at serine 139

Histone H2AX is a ubiquitous member of the H2A histone family that differs from the other H2A histones by the presence of an evolutionarily conserved C-terminal motif, -KKATQASQEY. The serine residue in this motif becomes rapidly phosphorylated in cells and animals when DNA double-stranded breaks are introduced into their chromatin by various physical and chemical means. In the present communication we show that this phosphorylated form of H2AX, referred to as gamma-H2AX, appears during apoptosis concurrently with the initial appearance of high molecular weight DNA fragments. gamma-H2AX forms before the appearance of internucleosomal DNA fragments and the externalization of phosphatidylserine to the outer membrane leaflet. gamma-H2AX formation is inhibited by N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone and the inhibitor of caspase-activated DNase, and it is induced when DNase I and restriction enzymes are introduced into cells, suggesting that any apoptotic endonuclease is sufficient to induce gamma-H2AX formation. These results indicate that gamma-H2AX formation is an early chromatin modification following initiation of DNA fragmentation during apoptosis.     

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.     

Phosphorylated histone H2AX foci persist on rejoined mitotic chromosomes in normal human diploid cells exposed to ionizing radiation

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.     

Cell cycle-dependent expression of phosphorylated histone H2AX: reduced expression in unirradiated but not X-irradiated G1-phase cells

Exposure of cells to ionizing radiation causes phosphorylation of histone H2AX at sites flanking DNA double-strand breaks. Detection of phosphorylated H2AX (gammaH2AX) by antibody binding has been used as a method to identify double-strand breaks. Although generally performed by observing microscopic foci within cells, flow cytometry offers the advantage of measuring changes in gammaH2AX intensity in relation to cell cycle position. The importance of cell cycle position on the levels of endogenous and radiation-induced gammaH2AX was examined in cell lines that varied in DNA content, cell cycle distribution, and kinase activity. Bivariate analysis of gammaH2AX expression relative to DNA content and synchronization by centrifugal elutriation were used to measure cell cycle-specific expression of gammaH2AX. With the exception of xrs5 cells, gammaH2AX level was approximately 3 times lower in unirradiated G(1)-phase cells than S- and G(2)-phase cells, and the slope of the G(1)-phase dose-response curve was 2.8 times larger than the slope for S-phase cells. Cell cycle differences were confirmed using immunoblotting, indicating that reduced antibody accessibility in intact cells was not responsible for the reduced antibody binding in G(1)-phase cells. Early apoptotic cells could be easily identified on flow histograms as a population with 5-10-fold higher levels of gammaH2AX, although high expression was not maintained in apoptotic cells by 24 h. We conclude that expression of gammaH2AX is associated with DNA replication in unirradiated cells and that this reduces the sensitivity for detecting radiation-induced double-strand breaks in S- and G(2)-phase cells.     

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.     

A tumor suppressor function of Smurf2 associated with controlling chromatin landscape and genome stability through RNF20

In addition to allelic mutations, cancers are known to harbor alterations in their chromatin landscape. Here we show that genomic ablation of Smad ubiquitin regulatory factor 2 (Smurf2), a HECT-domain E3 ubiquitin ligase, results in dysregulation of both the DNA damage response and genomic stability, culminating in increased susceptibility to various types of cancers in aged mice. We show that Smurf2 regulates the monoubiquitination of histone H2B as well as the trimethylation of histone H3 at Lys4 and Lys79 by targeting ring finger protein 20 (RNF20) for proteasomal degradation in both mouse and human cells. We also show that Smurf2 and RNF20 are colocalized at the γ-H2AX foci of double-stranded DNA breaks in the nucleus. Thus, Smurf2 has a tumor suppression function that normally maintains genomic stability by controlling the epigenetic landscape of histone modifications through RNF20.     

Release of nucleosomes from nuclei by bleomycin-induced DNA strand scission

Mononucleosomes were released from both isolated mammalian (hog thyroid) and protozoan (Tetrahymena) nuclei by the bleomycin-induced DNA-strand breaking reaction. Trout sperm nuclei, on the other hand, were protected from the bleomycin-mediated DNA degradation. The mononucleosomes released from the bleomycin-treated nuclei contained the core histones H2A, H2B, H3, and H4; while HMG1 and HMG2 proteins, in addition to the core histones, were detected in the mononucleosomes obtained by micrococcal nuclease digestion of nuclei. HMGs, but not H1 histone, were dissociated into the supernatant by cleavage of chromatin DNA with bleomycin, whereas both HMGs and H1 were found in that fraction by digestion of nuclei with micrococcal nuclease. HMG1 and HMG2 were exclusively dissociated from chromatin with 1 mM bleomycin under the solvent condition where the DNA strand-breaking activity of the drug is repressed. These observations suggest the possibility that bleomycin preferentially binds to linker DNA regions not occupied by H1 histone in chromatin and exclusively dissociates HMG proteins and breaks the DNA strand. The results of the effects on bleomycin-induced DNA cleavage of nuclei of various drugs including polyamines, chelating agents, intercalating antibiotics such as mitomycin C or adriamycin, and radical scavengers are also presented.     

DNA-PK phosphorylates histone H2AX during apoptotic DNA fragmentation in mammalian cells

The phosphorylation of histone H2AX at serine 139 is one of the earliest responses of mammalian cells to ionizing radiation-induced DNA breaks. DNA breaks are also generated during the terminal stages of apoptosis when chromosomal DNA is cleaved into oligonucleosomal pieces. Apoptotic DNA fragmentation and the consequent chromatin condensation are important for efficient clearing of genomic DNA and nucleosomes and for protecting the organism from auto-immmunization and oncogenic transformation. In this study, we demonstrate that H2AX is phosphorylated during apoptotic DNA fragmentation in mouse, Chinese hamster ovary, and human cells. We have previously shown that ataxia telangiectasia mutated kinase (ATM) is primarily responsible for H2AX phosphorylation in murine cells in response to ionizing radiation. Interestingly, we find here that DNA-dependent protein kinase (DNA-PK) is solely responsible for H2AX phosphorylation during apoptosis while ATM is dispensable for the process. Moreover, the kinase activity of DNA-PKcs (catalytic subunit of DNA-PK) is specifically required for the induction of gammaH2AX. We further show that DNA-PKcs is robustly activated in apoptotic cells, as evidenced by autophosphorylation at serine 2056, before it is inactivated by cleavage. In contrast, ATM is degraded well before DNA fragmentation and gammaH2AX induction resulting in the predominance of DNA-PK during the later stages of apoptosis. Finally, we show that DNA-PKcs autophosphorylation and gammaH2AX induction occur only in apoptotic nuclei with characteristic chromatin condensation but not in non-apoptotic nuclei from the same culture establishing the most direct link between DNA fragmentation, DNA-PKcs activation, and H2AX phosphorylation. It is well established that DNA-PK is inactivated by cleavage late in apoptosis in order to forestall DNA repair. Our results demonstrate, for the first time, that DNA-PK is actually activated in late apoptotic cells and is able to initiate an early step in the DNA-damage response, namely H2AX phosphorylation, before it is inactivated by proteolysis.     

gamma-H2AX dephosphorylation by protein phosphatase 2A facilitates DNA double-strand break repair

Phosphorylated histone H2AX (gamma-H2AX) forms foci over large chromatin domains surrounding double-stranded DNA breaks (DSB). These foci recruit DSB repair proteins and dissolve during or after repair is completed. How gamma-H2AX is removed from chromatin remains unknown. Here, we show that protein phosphatase 2A (PP2A) is involved in removing gamma-H2AX foci. The PP2A catalytic subunit [PP2A(C)] and gamma-H2AX coimmunoprecipitate and colocalize in DNA damage foci and PP2A dephosphorylates gamma-H2AX in vitro. The recruitment of PP2A(C) to DNA damage foci is H2AX dependent. When PP2A(C) is inhibited or silenced by RNA interference, gamma-H2AX foci persist, DNA repair is inefficient, and cells are hypersensitive to DNA damage. The effect of PP2A on gamma-H2AX levels is independent of ATM, ATR, or DNA-PK activity.     

Oxidative Stress-Induced DNA Damage and Repair in Human Peripheral Blood Mononuclear Cells: Protective Role of Hemoglobin

Background

DNA repair is a cellular defence mechanism responding to DNA damage caused in large part by oxidative stress. There is a controversy with regard to the effect of red blood cells on DNA damage and cellular response.

Aim

To investigate the effect of red blood cells on H₂O₂-induced DNA damage and repair in human peripheral blood mononuclear cells.

Methods

DNA breaks were induced in peripheral blood mononuclear cells by H₂O₂ in the absence or presence of red blood cells, red blood cells hemolysate or hemoglobin. DNA repair was measured by ³H-thymidine uptake, % double-stranded DNA was measured by fluorometric assay of DNA unwinding. DNA damage was measured by the comet assay and by the detection of histone H2AX phosphorylation.

Results

Red blood cells and red blood cells hemolysate reduced DNA repair in a dose-dependent manner. Red blood cells hemolysate reduced % double-stranded DNA, DNA damage and phosphorylation of histone H2AX. Hemoglobin had the same effect as red blood cells hemolysate on % double-stranded DNA.

Conclusion

Red blood cells, via red blood cells hemolysate and hemoglobin, reduced the effect of oxidative stress on peripheral blood mononuclear cell DNA damage and phosphorylation of histone H2AX. Consequently, recruitment of DNA repair proteins diminished with reduction of DNA repair. This suggests that anemia predisposes to increased oxidative stress induced DNA damage, while a higher hemoglobin level provides protection against oxidative-stress-induced DNA damage.     

Mitochondria-independent morphological and biochemical apoptotic alterations promoted by the anti-tumor agent bleomycin in Saccharomyces cerevisiae.

Bleomycin is a highly potent cytotoxic and genotoxic agent used in the chemotherapy of various types of tumors. It is a radiomimetic anticancer drug that produces single- and double-stranded DNA breaks in a catalytic way. Using Saccharomyces cerevisiae as a model system, we show that when a high amount of bleomycin molecules is internalized (100 micromol/L), morphological changes identical to those usually associated with apoptosis, i.e., a sub-G1 region peak, chromatin condensation, and very rapid DNA fragmentation into oligonucleosomal-sized fragments, are observed. The known bleomycin inhibitors cobalt and EDTA were able to prevent bleomycin nucleasic activity and thus apoptotic cell death. However, both oligomycin, a potent inhibitor of the mitochondrial F0F1-ATPase, and antimycin, a drug affecting mitochondria respiration, were unable to prevent the bleomycin-induced apoptotic-like cell death. These results suggest that high bleomycin concentrations induce an apoptosis-like mitochondria-independent cell death in yeast.     

Bleomycin-induced double-strand breaks in mitochondrial DNA of Drosophila cells are repaired

Mitochondrial DNA lesions cause numerous human diseases, and it is therefore important to identify the mechanisms whereby the mitochondrion repairs the damage. We have studied in cultured Drosophila cells the repair of bleomycin-induced double-strand breaks (DSBs) in mitochondrial DNA. Our results show that DSBs are repaired as rapidly and effectively in the mitochondria as in the nucleus. DNA repair is complete within 2h following bleomycin treatment, showing that Drosophila mitochondria have an effective system of DSB repair. The mechanism and mitochondrial proteins involved remain to be identified.     

The apoptotic ring: A novel entity with phosphorylated histones H2AX and H2B,and activated DNA damage response kinases

We recently showed that histone H2AX phosphorylated on serine 139 (γ-H2AX), a hallmark of DNA damage response (DDR), also forms early during apoptosis induced by death receptor activation. Here, we extend and discuss our findings on apoptotic γ-H2AX, which differs from the well-established DDR with nuclear foci. During apoptosis induced by death receptors agonists (TRAIL and FasL) and staurosporine, γ-H2AX is initiated in the nuclear periphery immediately inside the nuclear envelope while total H2AX remains distributed throughout the nucleus. This process is readily detectable by immunofluorescence microscopy and we refer to it as the “γ-H2AX ring”. It is conserved both in cancer and normal cells. The γ-H2AX ring contains the activated checkpoints kinases, ATM, Chk2 and DNA-PK; the latter being the main effector for the apoptotic γ-H2AX phosphorylation. Notably, we show here that the γ-H2AX ring coincides with phosphorylated H2B on serine 14 (PS14-H2B), another histone modification associated with apoptosis. The coordinated phosphorylations of H2AX and H2B suggest a previously unrecognized histone phosphorylation signature for apoptosis consisting of γ-H2AX together with PS14-H2B and possibly PY142-H2AX. This signature (“phospho-histone 2 code”) together with the γ-H2AX ring provides a new feature to monitor and study apoptosis.     

MST1 promotes apoptosis through phosphorylation of histone H2AX

MST1 (mammalian STE20-like kinase 1) is a serine/threonine kinase that is cleaved and activated by caspases during apoptosis. Overexpression of MST1 induces apoptotic morphological changes such as chromatin condensation, but the mechanism is not clear. Here we show that MST1 induces apoptotic chromatin condensation through its phosphorylation of histone H2AX at Ser-139. During etoposide-induced apoptosis in Jurkat cells, the cleavage of MST1 directly corresponded with strong H2AX phosphorylation. In vitro kinase assay results showed that MST1 strongly phosphorylates histone H2AX. Western blot and kinase assay results with a mutant S139A H2AX confirmed that MST1 phosphorylates H2AX at Ser-139. Direct binding of MST1 and H2AX can be detected when co-expressed in HEK293 cells and was also confirmed by an endogenous immunoprecipitation study. When overexpressed in HeLa cells, both the MST1 full-length protein and the MST1 kinase domain (MST1-NT), but not the kinase-negative mutant (MST1-NT-KN), could induce obvious endogenous histone H2AX phosphorylation. The caspase-3 inhibitor benzyloxycarbonyl-DEVD-fluoromethyl ketone (Z-DEVD-fmk) attenuates phosphorylation of H2AX by MST1 but cannot inhibit MST1-NT-induced histone H2AX phosphorylation, indicating that cleaved MST1 is responsible for H2AX phosphorylation during apoptosis. Histone H2AX phosphorylation and DNA fragmentation were suppressed in MST1 knockdown Jurkat cells after etoposide treatment. Taken together, our data indicated that H2AX is a substrate of MST1, which functions to induce apoptotic chromatin condensation and DNA fragmentation.     

Cytometric assessment of DNA damage in relation to cell cycle phase and apoptosis

Reviewed are the methods aimed to detect DNA damage in individual cells, estimate its extent and relate it to cell cycle phase and induction of apoptosis. They include the assays that reveal DNA fragmentation during apoptosis, as well as DNA damage induced by genotoxic agents. DNA fragmentation that occurs in the course of apoptosis is detected by selective extraction of degraded DNA. DNA in chromatin of apoptotic cells shows also increased propensity to undergo denaturation. The most common assay of DNA fragmentation relies on labelling DNA strand breaks with fluorochrome-tagged deoxynucleotides. The induction of double-strand DNA breaks (DSBs) by genotoxic agents provides a signal for histone H2AX phosphorylation on Ser139; the phosphorylated H2AX is named gammaH2AX. Also, ATM-kinase is activated through its autophosphorylation on Ser1981. Immunocytochemical detection of gammaH2AX and/or ATM-Ser1981(P) are sensitive probes to reveal induction of DSBs. When used concurrently with analysis of cellular DNA content and caspase-3 activation, they allow one to correlate the extent of DNA damage with the cell cycle phase and with activation of the apoptotic pathway. The presented data reveal cell cycle phase-specific patterns of H2AX phosphorylation and ATM autophosphorylation in response to induction of DSBs by ionizing radiation, topoisomerase I and II inhibitors and carcinogens. Detection of DNA damage in tumour cells during radio- or chemotherapy may provide an early marker predictive of response to treatment.     

Phosphorylation of H2AX histone as indirect evidence for double-stranded DNA breaks related to the exchange of nuclear proteins and chromatin remodeling in Chara vulgaris spermiogenesis

Phosphorylation of H2AX histone results not only from DNA damage (caused by ionizing radiation, UV or chemical substances, e.g. hydroxyurea), but also regularly takes place during spermiogenesis, enabling correct chromatin remodeling. Immunocytochemical analysis using antibodies against H2AX histone phosphorylated at serine 139 indirectly revealed endogenous double-stranded DNA breaks in Chara vulgaris spermatids in mid-spermiogenesis (stages V, VI and VII), when protamine-type proteins appear in the nucleus. Fluorescent foci were not observed in early (stages I-IV) and late (VIII-X) spermiogenesis, after replacement of histones by protamine-type proteins was finished. A similar phenomenon exists in animals. Determination of the localization of fluorescent foci and the ultrastructure of nuclei led to the hypothesis that DNA breaks at stage V, when condensed chromatin adheres to the nuclear envelope. This is transformed into a net-like structure during stage VI, probably allowing chromosome repositioning to specific regions in the mature spermatozoid. However, at stages VI and VII, DNA breaks are necessary for transformation of the nucleosomal structure into a fibrillar and finally the extremely condensed status of sleeping genes at stage X.     

Megabase chromatin domains involved in DNA double-strand breaks in vivo.

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 gamma-H2AX. An antibody prepared to the unique region of human gamma-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 gamma-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, gamma-H2AX foci form at these sites. In mitotic cells from cultures exposed to nonlethal amounts of ionizing radiation, gamma-H2AX foci form band-like structures on chromosome arms and on the end of broken arms. These results offer direct visual confirmation that gamma-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.