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Zbigniew Darzynkiewicz - One of the best experts on this subject based on the ideXlab platform.
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cytometry of atm activation and Histone H2AX phosphorylation to estimate extent of dna damage induced by exogenous agents
Cytometry Part A, 2007Co-Authors: Xuan Huang, Frank Traganos, Dorota H Halicka, Hong Zhao, Toshiki Tanaka, Anthony P Albino, Zbigniew DarzynkiewiczAbstract:This review covers the topic of cytometric assessment of activation of Ataxia telangiectasia mutated (ATM) protein kinase and Histone H2AX phosphorylation on Ser139 in response to DNA damage, particularly the damage that involves formation of DNA double-strand breaks. Briefly described are molecular mechanisms associated with activation of ATM and the downstream events that lead to recruitment of DNA repair machinery, engagement of cell cycle checkpoints, and activation of apoptotic pathway. Examples of multiparameter analysis of ATM activation and H2AX phosphorylation vis-a-vis cell cycle phase position and induction of apoptosis that employ flow- and laser scanning-cytometry are provided. They include cells treated with a variety of exogenous genotoxic agents, such as ionizing and UV radiation, DNA topoisomerase I (topotecan) and II (mitoxantrone, etoposide) inhibitors, nitric oxide-releasing aspirin, DNA replication inhibitors (aphidicolin, hydroxyurea, thymidine), and complex environmental carcinogens such as present in tobacco smoke. Also presented is an approach to identify DNA replicating (BrdU incorporating) cells based on selective photolysis of DNA that triggers H2AX phosphorylation. Listed are strategies to distinguish ATM activation and H2AX phosphorylation induced by primary DNA damage by genotoxic agents from those effects triggered by DNA fragmentation that takes place during apoptosis. While we review most published data, recent new findings also are included. Examples of multivariate analysis of ATM activation and H2AX phosphorylation presented in this review illustrate the advantages of cytometric flow- and image-analysis of these events in terms of offering a sensitive and valuable tool in studies of factors that induce DNA damage and/or affect DNA repair and allow one to explore the linkage between DNA damage, cell cycle checkpoints and initiation of apoptosis. ' 2007 International Society for Analytical Cytology
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constitutive Histone H2AX phosphorylation and atm activation the reporters of dna damage by endogenous oxidants
Cell Cycle, 2006Co-Authors: Toshiki Tanaka, Xuan Huang, Frank Traganos, Dorota H Halicka, Zbigniew DarzynkiewiczAbstract:DNA in live cells undergoes continuous oxidative damage caused by metabolically generated endogenous as well as external oxidants and oxidant-inducers. The cumulative oxidative DNA damage is considered the key factor in aging and senescence while the effectiveness of anti-aging agents is often assessed by their ability to reduce such damage. Oxidative DNA damage also preconditions cells to neoplastic transformation. Sensitive reporters of DNA damage, particularly the induction of DNA double-strand breaks (DSBs), are activation of ATM, through its phosphorylation on Ser 1981, and phosphorylation of Histone H2AX on Ser 139; the phosphorylated form of H2AX has been named γH2AX. We review the observations that constitutive ATM activation (CAA) and H2AX phosphorylation (CHP) take place in normal cells as well in the cells of tumor lines untreated by exogenous genotoxic agents. We postulate that CAA and CHP, which have been measured by multiparameter cytometry in relation to the cell cycle phase, are triggered by oxidative DNA damage. This review also presents the findings on differences in CAA and CHP in various cell lines as well as on the effects of several agents and growth conditions that modulate the extent of these Histone and ATM modifications. Specifically, described are effects of the reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC), and the glutathione synthetase inhibitor buthionine sulfoximine (BSO) as well as suppression of cell metabolism by growth at higher cell density or in the presence of the glucose antimetabolite 2-deoxy-D-glucose. Collectively, the reviewed data indicate that multiparameter cytometric measurement of the level of CHP and/or CAA allows one to estimate the extent of ongoing oxidative DNA damage and to measure the DNA protective-effects of antioxidants or agents that reduce or amplify generation of endogenous ROS.
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effects of hydroxyurea and aphidicolin on phosphorylation of ataxia telangiectasia mutated on ser 1981 and Histone H2AX on ser 139 in relation to cell cycle phase and induction of apoptosis
Cytometry Part A, 2006Co-Authors: Akira Kurose, Xuan Huang, Frank Traganos, Toshiki Tanaka, Zbigniew DarzynkiewiczAbstract:Background: DNA replication stress often induces DNA damage. The antitumor drug hydroxyurea (HU), a potent inhibitor of ribonucleotide reductase that halts DNA replication through its effects on cellular deoxynucleotide pools, was shown to damage DNA inducing double-strand breaks (DSBs). Aphidicolin (APH), an inhibitor of α-like DNA polymerases, was also reported to cause DNA damage, but the evidence for induction of DSBs by APH is not straightforward. Histone H2AX is phosphorylated on Ser 139 in response to DSBs and one of the protein kinases that phosphorylate H2AX is ataxia telangiectasia mutated (ATM); activation of ATM is through its phosphorylation of Ser 1981. The present study was undertaken to reveal whether H2AX is phosphorylated in cells exposed to HU or APH and whether its phosphorylation is mediated by ATM. Materials and Methods: HL-60 cells were treated in cultures with 0.1–5.0 mM HU or 1–4 μM APH for up to 5 h. Activation of ATM and H2AX phosphorylation was detected immunocytochemically using Ab specific to Ser1981-ATM or Ser 139-H2AX epitopes, respectively, concurrent with measurement of cellular DNA content. Results: While exposure of cells to HU led to H2AX phosphorylation selectively during S phase and the cells progressing through the early portion of S (DI = 1.1–1.4) were more affected than late-S phase (DI = 1.6–1.9) cells, ATM was not activated by HU. In fact, the level of constitutive (“programmed”) ATM phosphorylation was distinctly suppressed, in all phases of the cell cycle, at 0.1–5.0 mM HU. Cells' exposure to APH also resulted in H2AX phosphorylation at Ser139 with no evidence of ATM activation, and as in the case of HU, the early-S cells were more affected than the late-S phase cells. The rise in frequency of apoptotic cells became apparent after 2 h of exposure to HU or APH, and all apoptotic cells had markedly elevated levels of both H2AX-Ser139 and ATM-Ser1981 phosphorylation. Conclusions: The lack of correlation between H2AX phosphorylation and ATM activation indicates that protein kinase(s) other than ATM (ATR and/or DNA-dependent protein kinase) are activated by DSBs induced by replication stress. Interestingly, HU inhibits the constitutive (“programmed”) level of ATM phosphorylation in untreated cells. However, DNA fragmentation during apoptosis activates ATM and dramatically increases level of H2AX phosphorylation. © 2006 International Society for Analytical Cytology
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atm activation and Histone H2AX phosphorylation as indicators of dna damage by dna topoisomerase i inhibitor topotecan and during apoptosis
Cell Proliferation, 2006Co-Authors: Xuan Huang, Toshiki Tanaka, Akira Kurose, Zbigniew DarzynkiewiczAbstract:. Damage that engenders DNA double-strand breaks (DSBs) activates ataxia telangiectasia mutated (ATM) kinase through its auto- or trans-phosphorylation on Ser1981 and activated ATM is one of the mediators of Histone H2AX phosphorylation on Ser139. The present study was designed to explore: (i) whether measurement of ATM activation combined with H2AX phosphorylation provides a more sensitive indicator of DSBs than each of these events alone, and (ii) to reveal possible involvement of ATM activation in H2AX phosphorylation during apoptosis. Activation of ATM and/or H2AX phosphorylation in HL-60 or Jurkat cells treated with topotecan (Tpt) was detected immunocytochemically in relation to cell cycle phase, by multiparameter cytometry. Exposure to Tpt led to concurrent phosphorylation of ATM and H2AX in S-phase cells, whereas G1 cells were unaffected. Immunofluorescence (IF) of the S-phase cells immunostained for ATM-S1981P and γH2AX combined was distinctly stronger compared to that of the cells stained for each of these proteins alone. However, because of the relatively high ATM-S1981P IF of G1 cells, the ratio of IF of S to G1 cells, that is, the factor that determines competence of the assay in distinction of cells with DSBs, was 2- to 3-fold lower for ATM-S1981P alone, or for ATM-S1981P and γH2AX IF combined, than for γH2AX alone. ATM activation concurrent with H2AX phosphorylation, likely triggered by induction of DSBs during DNA fragmentation, occurred during apoptosis. The data suggest that frequency of activated ATM and phosphorylated H2AX molecules, per apoptotic cell, is comparable.
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cytometric assessment of Histone H2AX phosphorylation a reporter of dna damage
Methods of Molecular Biology, 2006Co-Authors: Xuan Huang, Zbigniew DarzynkiewiczAbstract:DNA damage that leads to formation of DNA double-strand breaks (DSBs) induces phosphorylation of Histone H2AX on Ser-139 at sites flanking the breakage. Immunocytochemical detection of phosphorylated H2AX (denoted as γH2AX) thus provides a marker of DSBs. The method presented in this chapter describes the detection of γH2AX for revealing the presence of DSBs, combined with differential staining of cellular DNA for revealing the cell cycle phase. The detection of γH2AX is based on indirect immunofluorescence using secondary antibody tagged with fluorescein isothiocyanate (FITC) while DNA is counterstained with propidium iodide (PI). Intensity of cellular green (FITC) and red (PI) fluorescence is measured by flow cytometry and bivariate analysis of the data is used to correlate the presence of DSBs with the cell cycle phase.
Xuan Huang - One of the best experts on this subject based on the ideXlab platform.
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cytometry of atm activation and Histone H2AX phosphorylation to estimate extent of dna damage induced by exogenous agents
Cytometry Part A, 2007Co-Authors: Xuan Huang, Frank Traganos, Dorota H Halicka, Hong Zhao, Toshiki Tanaka, Anthony P Albino, Zbigniew DarzynkiewiczAbstract:This review covers the topic of cytometric assessment of activation of Ataxia telangiectasia mutated (ATM) protein kinase and Histone H2AX phosphorylation on Ser139 in response to DNA damage, particularly the damage that involves formation of DNA double-strand breaks. Briefly described are molecular mechanisms associated with activation of ATM and the downstream events that lead to recruitment of DNA repair machinery, engagement of cell cycle checkpoints, and activation of apoptotic pathway. Examples of multiparameter analysis of ATM activation and H2AX phosphorylation vis-a-vis cell cycle phase position and induction of apoptosis that employ flow- and laser scanning-cytometry are provided. They include cells treated with a variety of exogenous genotoxic agents, such as ionizing and UV radiation, DNA topoisomerase I (topotecan) and II (mitoxantrone, etoposide) inhibitors, nitric oxide-releasing aspirin, DNA replication inhibitors (aphidicolin, hydroxyurea, thymidine), and complex environmental carcinogens such as present in tobacco smoke. Also presented is an approach to identify DNA replicating (BrdU incorporating) cells based on selective photolysis of DNA that triggers H2AX phosphorylation. Listed are strategies to distinguish ATM activation and H2AX phosphorylation induced by primary DNA damage by genotoxic agents from those effects triggered by DNA fragmentation that takes place during apoptosis. While we review most published data, recent new findings also are included. Examples of multivariate analysis of ATM activation and H2AX phosphorylation presented in this review illustrate the advantages of cytometric flow- and image-analysis of these events in terms of offering a sensitive and valuable tool in studies of factors that induce DNA damage and/or affect DNA repair and allow one to explore the linkage between DNA damage, cell cycle checkpoints and initiation of apoptosis. ' 2007 International Society for Analytical Cytology
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constitutive Histone H2AX phosphorylation and atm activation the reporters of dna damage by endogenous oxidants
Cell Cycle, 2006Co-Authors: Toshiki Tanaka, Xuan Huang, Frank Traganos, Dorota H Halicka, Zbigniew DarzynkiewiczAbstract:DNA in live cells undergoes continuous oxidative damage caused by metabolically generated endogenous as well as external oxidants and oxidant-inducers. The cumulative oxidative DNA damage is considered the key factor in aging and senescence while the effectiveness of anti-aging agents is often assessed by their ability to reduce such damage. Oxidative DNA damage also preconditions cells to neoplastic transformation. Sensitive reporters of DNA damage, particularly the induction of DNA double-strand breaks (DSBs), are activation of ATM, through its phosphorylation on Ser 1981, and phosphorylation of Histone H2AX on Ser 139; the phosphorylated form of H2AX has been named γH2AX. We review the observations that constitutive ATM activation (CAA) and H2AX phosphorylation (CHP) take place in normal cells as well in the cells of tumor lines untreated by exogenous genotoxic agents. We postulate that CAA and CHP, which have been measured by multiparameter cytometry in relation to the cell cycle phase, are triggered by oxidative DNA damage. This review also presents the findings on differences in CAA and CHP in various cell lines as well as on the effects of several agents and growth conditions that modulate the extent of these Histone and ATM modifications. Specifically, described are effects of the reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC), and the glutathione synthetase inhibitor buthionine sulfoximine (BSO) as well as suppression of cell metabolism by growth at higher cell density or in the presence of the glucose antimetabolite 2-deoxy-D-glucose. Collectively, the reviewed data indicate that multiparameter cytometric measurement of the level of CHP and/or CAA allows one to estimate the extent of ongoing oxidative DNA damage and to measure the DNA protective-effects of antioxidants or agents that reduce or amplify generation of endogenous ROS.
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effects of hydroxyurea and aphidicolin on phosphorylation of ataxia telangiectasia mutated on ser 1981 and Histone H2AX on ser 139 in relation to cell cycle phase and induction of apoptosis
Cytometry Part A, 2006Co-Authors: Akira Kurose, Xuan Huang, Frank Traganos, Toshiki Tanaka, Zbigniew DarzynkiewiczAbstract:Background: DNA replication stress often induces DNA damage. The antitumor drug hydroxyurea (HU), a potent inhibitor of ribonucleotide reductase that halts DNA replication through its effects on cellular deoxynucleotide pools, was shown to damage DNA inducing double-strand breaks (DSBs). Aphidicolin (APH), an inhibitor of α-like DNA polymerases, was also reported to cause DNA damage, but the evidence for induction of DSBs by APH is not straightforward. Histone H2AX is phosphorylated on Ser 139 in response to DSBs and one of the protein kinases that phosphorylate H2AX is ataxia telangiectasia mutated (ATM); activation of ATM is through its phosphorylation of Ser 1981. The present study was undertaken to reveal whether H2AX is phosphorylated in cells exposed to HU or APH and whether its phosphorylation is mediated by ATM. Materials and Methods: HL-60 cells were treated in cultures with 0.1–5.0 mM HU or 1–4 μM APH for up to 5 h. Activation of ATM and H2AX phosphorylation was detected immunocytochemically using Ab specific to Ser1981-ATM or Ser 139-H2AX epitopes, respectively, concurrent with measurement of cellular DNA content. Results: While exposure of cells to HU led to H2AX phosphorylation selectively during S phase and the cells progressing through the early portion of S (DI = 1.1–1.4) were more affected than late-S phase (DI = 1.6–1.9) cells, ATM was not activated by HU. In fact, the level of constitutive (“programmed”) ATM phosphorylation was distinctly suppressed, in all phases of the cell cycle, at 0.1–5.0 mM HU. Cells' exposure to APH also resulted in H2AX phosphorylation at Ser139 with no evidence of ATM activation, and as in the case of HU, the early-S cells were more affected than the late-S phase cells. The rise in frequency of apoptotic cells became apparent after 2 h of exposure to HU or APH, and all apoptotic cells had markedly elevated levels of both H2AX-Ser139 and ATM-Ser1981 phosphorylation. Conclusions: The lack of correlation between H2AX phosphorylation and ATM activation indicates that protein kinase(s) other than ATM (ATR and/or DNA-dependent protein kinase) are activated by DSBs induced by replication stress. Interestingly, HU inhibits the constitutive (“programmed”) level of ATM phosphorylation in untreated cells. However, DNA fragmentation during apoptosis activates ATM and dramatically increases level of H2AX phosphorylation. © 2006 International Society for Analytical Cytology
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atm activation and Histone H2AX phosphorylation as indicators of dna damage by dna topoisomerase i inhibitor topotecan and during apoptosis
Cell Proliferation, 2006Co-Authors: Xuan Huang, Toshiki Tanaka, Akira Kurose, Zbigniew DarzynkiewiczAbstract:. Damage that engenders DNA double-strand breaks (DSBs) activates ataxia telangiectasia mutated (ATM) kinase through its auto- or trans-phosphorylation on Ser1981 and activated ATM is one of the mediators of Histone H2AX phosphorylation on Ser139. The present study was designed to explore: (i) whether measurement of ATM activation combined with H2AX phosphorylation provides a more sensitive indicator of DSBs than each of these events alone, and (ii) to reveal possible involvement of ATM activation in H2AX phosphorylation during apoptosis. Activation of ATM and/or H2AX phosphorylation in HL-60 or Jurkat cells treated with topotecan (Tpt) was detected immunocytochemically in relation to cell cycle phase, by multiparameter cytometry. Exposure to Tpt led to concurrent phosphorylation of ATM and H2AX in S-phase cells, whereas G1 cells were unaffected. Immunofluorescence (IF) of the S-phase cells immunostained for ATM-S1981P and γH2AX combined was distinctly stronger compared to that of the cells stained for each of these proteins alone. However, because of the relatively high ATM-S1981P IF of G1 cells, the ratio of IF of S to G1 cells, that is, the factor that determines competence of the assay in distinction of cells with DSBs, was 2- to 3-fold lower for ATM-S1981P alone, or for ATM-S1981P and γH2AX IF combined, than for γH2AX alone. ATM activation concurrent with H2AX phosphorylation, likely triggered by induction of DSBs during DNA fragmentation, occurred during apoptosis. The data suggest that frequency of activated ATM and phosphorylated H2AX molecules, per apoptotic cell, is comparable.
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cytometric assessment of Histone H2AX phosphorylation a reporter of dna damage
Methods of Molecular Biology, 2006Co-Authors: Xuan Huang, Zbigniew DarzynkiewiczAbstract:DNA damage that leads to formation of DNA double-strand breaks (DSBs) induces phosphorylation of Histone H2AX on Ser-139 at sites flanking the breakage. Immunocytochemical detection of phosphorylated H2AX (denoted as γH2AX) thus provides a marker of DSBs. The method presented in this chapter describes the detection of γH2AX for revealing the presence of DSBs, combined with differential staining of cellular DNA for revealing the cell cycle phase. The detection of γH2AX is based on indirect immunofluorescence using secondary antibody tagged with fluorescein isothiocyanate (FITC) while DNA is counterstained with propidium iodide (PI). Intensity of cellular green (FITC) and red (PI) fluorescence is measured by flow cytometry and bivariate analysis of the data is used to correlate the presence of DSBs with the cell cycle phase.
Frank Traganos - One of the best experts on this subject based on the ideXlab platform.
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cytometry of atm activation and Histone H2AX phosphorylation to estimate extent of dna damage induced by exogenous agents
Cytometry Part A, 2007Co-Authors: Xuan Huang, Frank Traganos, Dorota H Halicka, Hong Zhao, Toshiki Tanaka, Anthony P Albino, Zbigniew DarzynkiewiczAbstract:This review covers the topic of cytometric assessment of activation of Ataxia telangiectasia mutated (ATM) protein kinase and Histone H2AX phosphorylation on Ser139 in response to DNA damage, particularly the damage that involves formation of DNA double-strand breaks. Briefly described are molecular mechanisms associated with activation of ATM and the downstream events that lead to recruitment of DNA repair machinery, engagement of cell cycle checkpoints, and activation of apoptotic pathway. Examples of multiparameter analysis of ATM activation and H2AX phosphorylation vis-a-vis cell cycle phase position and induction of apoptosis that employ flow- and laser scanning-cytometry are provided. They include cells treated with a variety of exogenous genotoxic agents, such as ionizing and UV radiation, DNA topoisomerase I (topotecan) and II (mitoxantrone, etoposide) inhibitors, nitric oxide-releasing aspirin, DNA replication inhibitors (aphidicolin, hydroxyurea, thymidine), and complex environmental carcinogens such as present in tobacco smoke. Also presented is an approach to identify DNA replicating (BrdU incorporating) cells based on selective photolysis of DNA that triggers H2AX phosphorylation. Listed are strategies to distinguish ATM activation and H2AX phosphorylation induced by primary DNA damage by genotoxic agents from those effects triggered by DNA fragmentation that takes place during apoptosis. While we review most published data, recent new findings also are included. Examples of multivariate analysis of ATM activation and H2AX phosphorylation presented in this review illustrate the advantages of cytometric flow- and image-analysis of these events in terms of offering a sensitive and valuable tool in studies of factors that induce DNA damage and/or affect DNA repair and allow one to explore the linkage between DNA damage, cell cycle checkpoints and initiation of apoptosis. ' 2007 International Society for Analytical Cytology
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constitutive Histone H2AX phosphorylation and atm activation the reporters of dna damage by endogenous oxidants
Cell Cycle, 2006Co-Authors: Toshiki Tanaka, Xuan Huang, Frank Traganos, Dorota H Halicka, Zbigniew DarzynkiewiczAbstract:DNA in live cells undergoes continuous oxidative damage caused by metabolically generated endogenous as well as external oxidants and oxidant-inducers. The cumulative oxidative DNA damage is considered the key factor in aging and senescence while the effectiveness of anti-aging agents is often assessed by their ability to reduce such damage. Oxidative DNA damage also preconditions cells to neoplastic transformation. Sensitive reporters of DNA damage, particularly the induction of DNA double-strand breaks (DSBs), are activation of ATM, through its phosphorylation on Ser 1981, and phosphorylation of Histone H2AX on Ser 139; the phosphorylated form of H2AX has been named γH2AX. We review the observations that constitutive ATM activation (CAA) and H2AX phosphorylation (CHP) take place in normal cells as well in the cells of tumor lines untreated by exogenous genotoxic agents. We postulate that CAA and CHP, which have been measured by multiparameter cytometry in relation to the cell cycle phase, are triggered by oxidative DNA damage. This review also presents the findings on differences in CAA and CHP in various cell lines as well as on the effects of several agents and growth conditions that modulate the extent of these Histone and ATM modifications. Specifically, described are effects of the reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC), and the glutathione synthetase inhibitor buthionine sulfoximine (BSO) as well as suppression of cell metabolism by growth at higher cell density or in the presence of the glucose antimetabolite 2-deoxy-D-glucose. Collectively, the reviewed data indicate that multiparameter cytometric measurement of the level of CHP and/or CAA allows one to estimate the extent of ongoing oxidative DNA damage and to measure the DNA protective-effects of antioxidants or agents that reduce or amplify generation of endogenous ROS.
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effects of hydroxyurea and aphidicolin on phosphorylation of ataxia telangiectasia mutated on ser 1981 and Histone H2AX on ser 139 in relation to cell cycle phase and induction of apoptosis
Cytometry Part A, 2006Co-Authors: Akira Kurose, Xuan Huang, Frank Traganos, Toshiki Tanaka, Zbigniew DarzynkiewiczAbstract:Background: DNA replication stress often induces DNA damage. The antitumor drug hydroxyurea (HU), a potent inhibitor of ribonucleotide reductase that halts DNA replication through its effects on cellular deoxynucleotide pools, was shown to damage DNA inducing double-strand breaks (DSBs). Aphidicolin (APH), an inhibitor of α-like DNA polymerases, was also reported to cause DNA damage, but the evidence for induction of DSBs by APH is not straightforward. Histone H2AX is phosphorylated on Ser 139 in response to DSBs and one of the protein kinases that phosphorylate H2AX is ataxia telangiectasia mutated (ATM); activation of ATM is through its phosphorylation of Ser 1981. The present study was undertaken to reveal whether H2AX is phosphorylated in cells exposed to HU or APH and whether its phosphorylation is mediated by ATM. Materials and Methods: HL-60 cells were treated in cultures with 0.1–5.0 mM HU or 1–4 μM APH for up to 5 h. Activation of ATM and H2AX phosphorylation was detected immunocytochemically using Ab specific to Ser1981-ATM or Ser 139-H2AX epitopes, respectively, concurrent with measurement of cellular DNA content. Results: While exposure of cells to HU led to H2AX phosphorylation selectively during S phase and the cells progressing through the early portion of S (DI = 1.1–1.4) were more affected than late-S phase (DI = 1.6–1.9) cells, ATM was not activated by HU. In fact, the level of constitutive (“programmed”) ATM phosphorylation was distinctly suppressed, in all phases of the cell cycle, at 0.1–5.0 mM HU. Cells' exposure to APH also resulted in H2AX phosphorylation at Ser139 with no evidence of ATM activation, and as in the case of HU, the early-S cells were more affected than the late-S phase cells. The rise in frequency of apoptotic cells became apparent after 2 h of exposure to HU or APH, and all apoptotic cells had markedly elevated levels of both H2AX-Ser139 and ATM-Ser1981 phosphorylation. Conclusions: The lack of correlation between H2AX phosphorylation and ATM activation indicates that protein kinase(s) other than ATM (ATR and/or DNA-dependent protein kinase) are activated by DSBs induced by replication stress. Interestingly, HU inhibits the constitutive (“programmed”) level of ATM phosphorylation in untreated cells. However, DNA fragmentation during apoptosis activates ATM and dramatically increases level of H2AX phosphorylation. © 2006 International Society for Analytical Cytology
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Histone H2AX phosphorylation after cell irradiation with uv b relationship to cell cycle phase and induction of apoptosis
Cell Cycle, 2005Co-Authors: Dorota H Halicka, Xuan Huang, Frank Traganos, Malcolm A King, Zbigniew DarzynkiewiczAbstract: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...
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Histone H2AX phosphorylation after cell irradiation with uv b relationship to cell cycle phase and induction of apoptosis
Cell Cycle, 2005Co-Authors: Dorota H Halicka, Xuan Huang, Frank Traganos, Malcolm A King, Wei Dai, Zbigniew DarzynkiewiczAbstract:Damage to DNA that engenders double-strand breaks (DSBs) triggers phosphorylation of Histone H2AX on Ser-139. Expression of phosphorylated H2AX (gammaH2AX) can be revealed immunocytochemically; the intensity of gammaH2AX 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 gammaH2AX following exposure of HeLa and HL-60 cells to a wide range of doses of UV-B light (6.1 J/m(2)-3.45 kJ/m(2)) 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 (G(1), G(2) 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 gammaH2AX in G(1), G(2) 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 gammaH2AX IF than that initially induced by UV; predominantly S-phase cells underwent apoptosis. While the suppression of DNA replication, by 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 gammaH2AX. Furthermore, aphidicolin itself induced gammaH2AX in early-S phase cells. The induction of gammaH2AX 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 gammaH2AX in G(1), G(2) 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.
Toshiki Tanaka - One of the best experts on this subject based on the ideXlab platform.
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cytometry of atm activation and Histone H2AX phosphorylation to estimate extent of dna damage induced by exogenous agents
Cytometry Part A, 2007Co-Authors: Xuan Huang, Frank Traganos, Dorota H Halicka, Hong Zhao, Toshiki Tanaka, Anthony P Albino, Zbigniew DarzynkiewiczAbstract:This review covers the topic of cytometric assessment of activation of Ataxia telangiectasia mutated (ATM) protein kinase and Histone H2AX phosphorylation on Ser139 in response to DNA damage, particularly the damage that involves formation of DNA double-strand breaks. Briefly described are molecular mechanisms associated with activation of ATM and the downstream events that lead to recruitment of DNA repair machinery, engagement of cell cycle checkpoints, and activation of apoptotic pathway. Examples of multiparameter analysis of ATM activation and H2AX phosphorylation vis-a-vis cell cycle phase position and induction of apoptosis that employ flow- and laser scanning-cytometry are provided. They include cells treated with a variety of exogenous genotoxic agents, such as ionizing and UV radiation, DNA topoisomerase I (topotecan) and II (mitoxantrone, etoposide) inhibitors, nitric oxide-releasing aspirin, DNA replication inhibitors (aphidicolin, hydroxyurea, thymidine), and complex environmental carcinogens such as present in tobacco smoke. Also presented is an approach to identify DNA replicating (BrdU incorporating) cells based on selective photolysis of DNA that triggers H2AX phosphorylation. Listed are strategies to distinguish ATM activation and H2AX phosphorylation induced by primary DNA damage by genotoxic agents from those effects triggered by DNA fragmentation that takes place during apoptosis. While we review most published data, recent new findings also are included. Examples of multivariate analysis of ATM activation and H2AX phosphorylation presented in this review illustrate the advantages of cytometric flow- and image-analysis of these events in terms of offering a sensitive and valuable tool in studies of factors that induce DNA damage and/or affect DNA repair and allow one to explore the linkage between DNA damage, cell cycle checkpoints and initiation of apoptosis. ' 2007 International Society for Analytical Cytology
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constitutive Histone H2AX phosphorylation and atm activation the reporters of dna damage by endogenous oxidants
Cell Cycle, 2006Co-Authors: Toshiki Tanaka, Xuan Huang, Frank Traganos, Dorota H Halicka, Zbigniew DarzynkiewiczAbstract:DNA in live cells undergoes continuous oxidative damage caused by metabolically generated endogenous as well as external oxidants and oxidant-inducers. The cumulative oxidative DNA damage is considered the key factor in aging and senescence while the effectiveness of anti-aging agents is often assessed by their ability to reduce such damage. Oxidative DNA damage also preconditions cells to neoplastic transformation. Sensitive reporters of DNA damage, particularly the induction of DNA double-strand breaks (DSBs), are activation of ATM, through its phosphorylation on Ser 1981, and phosphorylation of Histone H2AX on Ser 139; the phosphorylated form of H2AX has been named γH2AX. We review the observations that constitutive ATM activation (CAA) and H2AX phosphorylation (CHP) take place in normal cells as well in the cells of tumor lines untreated by exogenous genotoxic agents. We postulate that CAA and CHP, which have been measured by multiparameter cytometry in relation to the cell cycle phase, are triggered by oxidative DNA damage. This review also presents the findings on differences in CAA and CHP in various cell lines as well as on the effects of several agents and growth conditions that modulate the extent of these Histone and ATM modifications. Specifically, described are effects of the reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC), and the glutathione synthetase inhibitor buthionine sulfoximine (BSO) as well as suppression of cell metabolism by growth at higher cell density or in the presence of the glucose antimetabolite 2-deoxy-D-glucose. Collectively, the reviewed data indicate that multiparameter cytometric measurement of the level of CHP and/or CAA allows one to estimate the extent of ongoing oxidative DNA damage and to measure the DNA protective-effects of antioxidants or agents that reduce or amplify generation of endogenous ROS.
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effects of hydroxyurea and aphidicolin on phosphorylation of ataxia telangiectasia mutated on ser 1981 and Histone H2AX on ser 139 in relation to cell cycle phase and induction of apoptosis
Cytometry Part A, 2006Co-Authors: Akira Kurose, Xuan Huang, Frank Traganos, Toshiki Tanaka, Zbigniew DarzynkiewiczAbstract:Background: DNA replication stress often induces DNA damage. The antitumor drug hydroxyurea (HU), a potent inhibitor of ribonucleotide reductase that halts DNA replication through its effects on cellular deoxynucleotide pools, was shown to damage DNA inducing double-strand breaks (DSBs). Aphidicolin (APH), an inhibitor of α-like DNA polymerases, was also reported to cause DNA damage, but the evidence for induction of DSBs by APH is not straightforward. Histone H2AX is phosphorylated on Ser 139 in response to DSBs and one of the protein kinases that phosphorylate H2AX is ataxia telangiectasia mutated (ATM); activation of ATM is through its phosphorylation of Ser 1981. The present study was undertaken to reveal whether H2AX is phosphorylated in cells exposed to HU or APH and whether its phosphorylation is mediated by ATM. Materials and Methods: HL-60 cells were treated in cultures with 0.1–5.0 mM HU or 1–4 μM APH for up to 5 h. Activation of ATM and H2AX phosphorylation was detected immunocytochemically using Ab specific to Ser1981-ATM or Ser 139-H2AX epitopes, respectively, concurrent with measurement of cellular DNA content. Results: While exposure of cells to HU led to H2AX phosphorylation selectively during S phase and the cells progressing through the early portion of S (DI = 1.1–1.4) were more affected than late-S phase (DI = 1.6–1.9) cells, ATM was not activated by HU. In fact, the level of constitutive (“programmed”) ATM phosphorylation was distinctly suppressed, in all phases of the cell cycle, at 0.1–5.0 mM HU. Cells' exposure to APH also resulted in H2AX phosphorylation at Ser139 with no evidence of ATM activation, and as in the case of HU, the early-S cells were more affected than the late-S phase cells. The rise in frequency of apoptotic cells became apparent after 2 h of exposure to HU or APH, and all apoptotic cells had markedly elevated levels of both H2AX-Ser139 and ATM-Ser1981 phosphorylation. Conclusions: The lack of correlation between H2AX phosphorylation and ATM activation indicates that protein kinase(s) other than ATM (ATR and/or DNA-dependent protein kinase) are activated by DSBs induced by replication stress. Interestingly, HU inhibits the constitutive (“programmed”) level of ATM phosphorylation in untreated cells. However, DNA fragmentation during apoptosis activates ATM and dramatically increases level of H2AX phosphorylation. © 2006 International Society for Analytical Cytology
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atm activation and Histone H2AX phosphorylation as indicators of dna damage by dna topoisomerase i inhibitor topotecan and during apoptosis
Cell Proliferation, 2006Co-Authors: Xuan Huang, Toshiki Tanaka, Akira Kurose, Zbigniew DarzynkiewiczAbstract:. Damage that engenders DNA double-strand breaks (DSBs) activates ataxia telangiectasia mutated (ATM) kinase through its auto- or trans-phosphorylation on Ser1981 and activated ATM is one of the mediators of Histone H2AX phosphorylation on Ser139. The present study was designed to explore: (i) whether measurement of ATM activation combined with H2AX phosphorylation provides a more sensitive indicator of DSBs than each of these events alone, and (ii) to reveal possible involvement of ATM activation in H2AX phosphorylation during apoptosis. Activation of ATM and/or H2AX phosphorylation in HL-60 or Jurkat cells treated with topotecan (Tpt) was detected immunocytochemically in relation to cell cycle phase, by multiparameter cytometry. Exposure to Tpt led to concurrent phosphorylation of ATM and H2AX in S-phase cells, whereas G1 cells were unaffected. Immunofluorescence (IF) of the S-phase cells immunostained for ATM-S1981P and γH2AX combined was distinctly stronger compared to that of the cells stained for each of these proteins alone. However, because of the relatively high ATM-S1981P IF of G1 cells, the ratio of IF of S to G1 cells, that is, the factor that determines competence of the assay in distinction of cells with DSBs, was 2- to 3-fold lower for ATM-S1981P alone, or for ATM-S1981P and γH2AX IF combined, than for γH2AX alone. ATM activation concurrent with H2AX phosphorylation, likely triggered by induction of DSBs during DNA fragmentation, occurred during apoptosis. The data suggest that frequency of activated ATM and phosphorylated H2AX molecules, per apoptotic cell, is comparable.
William M Bonner - One of the best experts on this subject based on the ideXlab platform.
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H2AX Haploinsufficiency Modifies Genomic Stability and Tumor Susceptibility
Cell, 2003Co-Authors: Arkady Celeste, Olga A Sedelnikova, William M Bonner, Michael J Difilippantonio, Duane R Pilch, Simone Difilippantonio, Oscar Fernandez-capetillo, Michael Eckhaus, Thomas Ried, André NussenzweigAbstract:Abstract Histone H2AX becomes phosphorylated in chromatin domains flanking sites of DNA double-strand breakage associated with γ-irradiation, meiotic recombination, DNA replication, and antigen receptor rearrangements. Here, we show that loss of a single H2AX allele compromises genomic integrity and enhances the susceptibility to cancer in the absence of p53. In comparison with heterozygotes, tumors arise earlier in the H2AX homozygous null background, and H2AX −/− p53 −/− lymphomas harbor an increased frequency of clonal nonreciprocal translocations and amplifications. These include complex rearrangements that juxtapose the c- myc oncogene to antigen receptor loci. Restoration of the H2AX null allele with wild-type H2AX restores genomic stability and radiation resistance, but this effect is abolished by substitution of the conserved serine phosphorylation sites in H2AX with alanine or glutamic acid residues. Our results establish H2AX as genomic caretaker that requires the function of both gene alleles for optimal protection against tumorigenesis.
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Histone H2AX phosphorylation is dispensable for the initial recognition of dna breaks
Nature Cell Biology, 2003Co-Authors: Arkady Celeste, William M Bonner, Oscar Fernandezcapetillo, Michael J Kruhlak, Duane R Pilch, David W Staudt, Robert F Bonner, André NussenzweigAbstract:Histone H2AX is rapidly phosphorylated in the chromatin micro-environment surrounding a DNA double-strand break (DSB). Although H2AX deficiency is not detrimental to life, H2AX is required for the accumulation of numerous essential proteins into irradiation induced foci (IRIF). However, the relationship between IRIF formation, H2AX phosphorylation (γ-H2AX) and the detection of DNA damage is unclear. Here, we show that the migration of repair and signalling proteins to DSBs is not abrogated in H2AX−/− cells, or in H2AX-deficient cells that have been reconstituted with H2AX mutants that eliminate phosphorylation. Despite their initial recruitment to DSBs, numerous factors, including Nbs1, 53BP1 and Brca1, subsequently fail to form IRIF. We propose that γ-H2AX does not constitute the primary signal required for the redistribution of repair complexes to damaged chromatin, but may function to concentrate proteins in the vicinity of DNA lesions. The differential requirements for factor recruitment to DSBs and sequestration into IRIF may explain why essential regulatory pathways controlling the ability of cells to respond to DNA damage are not abolished in the absence of H2AX.
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H2AX is required for chromatin remodeling and inactivation of sex chromosomes in male mouse meiosis
Developmental Cell, 2003Co-Authors: Oscar Fernandezcapetillo, William M Bonner, Arkady Celeste, Peter J Romanienko, Daniel R Cameriniotero, Paul S Burgoyne, Shantha K Mahadevaiah, Katia Manova, André NussenzweigAbstract:During meiotic prophase in male mammals, the X and Y chromosomes condense to form a macrochromatin body, termed the sex, or XY, body, within which X- and Y-linked genes are transcriptionally repressed. The molecular basis and biological function of both sex body formation and meiotic sex chromosome inactivation (MSCI) are unknown. A phosphorylated form of H2AX, a Histone H2A variant implicated in DNA repair, accumulates in the sex body in a manner independent of meiotic recombination-associated double-strand breaks. Here we show that the X and Y chromosomes of Histone H2AX-deficient spermatocytes fail to condense to form a sex body, do not initiate MSCI, and exhibit severe defects in meiotic pairing. Moreover, other sex body proteins, including macroH2A1.2 and XMR, do not preferentially localize with the sex chromosomes in the absence of H2AX. Thus, H2AX is required for the chromatin remodeling and associated silencing in male meiosis.
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quantitative detection of 125idu induced dna double strand breaks with γ H2AX antibody
Radiation Research, 2002Co-Authors: Olga A Sedelnikova, Emmy P Rogakou, Igor G Panyutin, William M BonnerAbstract:Abstract Sedelnikova, O. A., Rogakou, E. P., Panyutin, I. G. and Bonner, W. M. Quantitative Detection of 125IdU-Induced DNA Double-Strand Breaks with γ-H2AX Antibody. Radiat. Res. 158, 486–492 (2002). 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 (γ-H2AX) demonstrates that γ-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 125I, 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 125IdU and processed immunocytochemically to determine the number of γ-H2AX foci. The numbers of 125IdU disintegrations per cell were measured by exposing...
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a critical role for Histone H2AX in recruitment of repair factors to nuclear foci after dna damage
Current Biology, 2000Co-Authors: Tanya T Paull, Martin Gellert, Emmy P Rogakou, Vikky Yamazaki, Cordula U Kirchgessner, William M BonnerAbstract:Abstract Background: The response of eukaryotic cells to double-strand breaks in genomic DNA includes the sequestration of many factors into nuclear foci. Recently it has been reported that a member of the Histone H2A family, H2AX, becomes extensively phosphorylated within 1–3 minutes of DNA damage and forms foci at break sites. Results: In this work, we examine the role of H2AX phosphorylation in focus formation by several repair-related complexes, and investigate what factors may be involved in initiating this response. Using two different methods to create DNA double-strand breaks in human cells, we found that the repair factors Rad50 and Rad51 each colocalized with phosphorylated H2AX (γ-H2AX) foci after DNA damage. The product of the tumor suppressor gene BRCA1 also colocalized with γ-H2AX and was recruited to these sites before Rad50 or Rad51. Exposure of cells to the fungal inhibitor wortmannin eliminated focus formation by all repair factors examined, suggesting a role for the phosphoinositide (PI)-3 family of protein kinases in mediating this response. Wortmannin treatment was effective only when it was added early enough to prevent γ-H2AX formation, indicating that γ-H2AX is necessary for the recruitment of other factors to the sites of DNA damage. DNA repair-deficient cells exhibit a substantially reduced ability to increase the phosphorylation of H2AX in response to ionizing radiation, consistent with a role for γ-H2AX in DNA repair. Conclusions: The pattern of γ-H2AX foci that is established within a few minutes of DNA damage accounts for the patterns of Rad50, Rad51, and Brca1 foci seen much later during recovery from damage. The evidence presented strongly supports a role for the γ-H2AX and the PI-3 protein kinase family in focus formation at sites of double-strand breaks and suggests the possibility of a change in chromatin structure accompanying double-strand break repair.