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Zbigniew Darzynkiewicz - One of the best experts on this subject based on the ideXlab platform.

  • sequential phosphorylation of ser 10 on histone h3 and ser 139 on histone h2ax and atm activation during premature chromosome condensation relationship to Cell Cycle Phase and apoptosis
    Cytometry Part A, 2006
    Co-Authors: Xuan Huang, Frank Traganos, Toshiki Tanaka, Akira Kurose, Wei Dai, Zbigniew Darzynkiewicz
    Abstract:

    Background: Histone H1 and H3 phosphorylation associated with chromatin condensation during mitosis has been studied extensively. Less is known on histone modifications that occur during premature chromosome condensation (PCC). The aim of the present study was to reveal the status of histone H3 and H2AX phosphorylation on Ser-10 and Ser-139, respectively, as well as ATM activation through phosphorylation on Ser-1981, during PCC, and relate these events to Cell-Cycle Phase and to initiation of apoptosis. Materials and Methods: To induce PCC, A549 and HL-60 Cells were exposed to the phosphatase inhibitor calyculin A (Cal A). Phosphorylation of histone H3 and H2AX as well as ATM activation were detected immunocytochemically concurrent with analysis of Cellular DNA content and activation of caspase-3, a marker of apoptosis. The intensity of Cellular fluorescence was measured by flow- or laser scanning cytometry. Results: Induction of PCC led to rapid histone H3 phosphorylation, followed by activation of ATM and then H2AX phosphorylation in both, HL-60 and A549 Cells. All these events occurred sequentially, prior to caspase-3 activation, and affected Cells in all Phases of the Cell Cycle. ATM activation and H2AX phosphorylation was seen during mitosis of A549 but not HL-60 Cells. Conclusions: Because the Cal A-induced phosphorylation of histone H3 and H2AX, and of ATM, precede caspase-3 activation these modifications are pertinent to PCC and not to apoptosis-associated chromatin condensation. The sequence of histone H3 and H2AX phosphorylation and ATM activation during PCC is compatible with a role of ATM in mediating phosphorylation of H2AX but not H3. Mitosis in some Cell types may proceed without ATM activation and H2AX phosphorylation. © 2006 International Society for Analytical Cytology

  • cytometric assessment of histone h2ax phosphorylation a reporter of dna damage
    Methods of Molecular Biology, 2006
    Co-Authors: Xuan Huang, Zbigniew Darzynkiewicz
    Abstract:

    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.

  • cytometric assessment of dna damage in relation to Cell Cycle Phase and apoptosis
    Cell Proliferation, 2005
    Co-Authors: Xuan Huang, Frank Traganos, Dorota H Halicka, Toshiki Tanaka, Akira Kurose, Zbigniew Darzynkiewicz
    Abstract:

    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.

  • histone h2ax phosphorylation after Cell irradiation with uv b relationship to Cell Cycle Phase and induction of apoptosis
    Cell Cycle, 2005
    Co-Authors: Dorota H Halicka, Xuan Huang, Frank Traganos, Malcolm A King, Zbigniew Darzynkiewicz
    Abstract:

    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...

  • histone h2ax phosphorylation after Cell irradiation with uv b relationship to Cell Cycle Phase and induction of apoptosis
    Cell Cycle, 2005
    Co-Authors: Dorota H Halicka, Xuan Huang, Frank Traganos, Malcolm A King, Wei Dai, Zbigniew Darzynkiewicz
    Abstract:

    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.

Xuan Huang - One of the best experts on this subject based on the ideXlab platform.

  • sequential phosphorylation of ser 10 on histone h3 and ser 139 on histone h2ax and atm activation during premature chromosome condensation relationship to Cell Cycle Phase and apoptosis
    Cytometry Part A, 2006
    Co-Authors: Xuan Huang, Frank Traganos, Toshiki Tanaka, Akira Kurose, Wei Dai, Zbigniew Darzynkiewicz
    Abstract:

    Background: Histone H1 and H3 phosphorylation associated with chromatin condensation during mitosis has been studied extensively. Less is known on histone modifications that occur during premature chromosome condensation (PCC). The aim of the present study was to reveal the status of histone H3 and H2AX phosphorylation on Ser-10 and Ser-139, respectively, as well as ATM activation through phosphorylation on Ser-1981, during PCC, and relate these events to Cell-Cycle Phase and to initiation of apoptosis. Materials and Methods: To induce PCC, A549 and HL-60 Cells were exposed to the phosphatase inhibitor calyculin A (Cal A). Phosphorylation of histone H3 and H2AX as well as ATM activation were detected immunocytochemically concurrent with analysis of Cellular DNA content and activation of caspase-3, a marker of apoptosis. The intensity of Cellular fluorescence was measured by flow- or laser scanning cytometry. Results: Induction of PCC led to rapid histone H3 phosphorylation, followed by activation of ATM and then H2AX phosphorylation in both, HL-60 and A549 Cells. All these events occurred sequentially, prior to caspase-3 activation, and affected Cells in all Phases of the Cell Cycle. ATM activation and H2AX phosphorylation was seen during mitosis of A549 but not HL-60 Cells. Conclusions: Because the Cal A-induced phosphorylation of histone H3 and H2AX, and of ATM, precede caspase-3 activation these modifications are pertinent to PCC and not to apoptosis-associated chromatin condensation. The sequence of histone H3 and H2AX phosphorylation and ATM activation during PCC is compatible with a role of ATM in mediating phosphorylation of H2AX but not H3. Mitosis in some Cell types may proceed without ATM activation and H2AX phosphorylation. © 2006 International Society for Analytical Cytology

  • cytometric assessment of histone h2ax phosphorylation a reporter of dna damage
    Methods of Molecular Biology, 2006
    Co-Authors: Xuan Huang, Zbigniew Darzynkiewicz
    Abstract:

    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.

  • cytometric assessment of dna damage in relation to Cell Cycle Phase and apoptosis
    Cell Proliferation, 2005
    Co-Authors: Xuan Huang, Frank Traganos, Dorota H Halicka, Toshiki Tanaka, Akira Kurose, Zbigniew Darzynkiewicz
    Abstract:

    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.

  • histone h2ax phosphorylation after Cell irradiation with uv b relationship to Cell Cycle Phase and induction of apoptosis
    Cell Cycle, 2005
    Co-Authors: Dorota H Halicka, Xuan Huang, Frank Traganos, Malcolm A King, Zbigniew Darzynkiewicz
    Abstract:

    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...

  • histone h2ax phosphorylation after Cell irradiation with uv b relationship to Cell Cycle Phase and induction of apoptosis
    Cell Cycle, 2005
    Co-Authors: Dorota H Halicka, Xuan Huang, Frank Traganos, Malcolm A King, Wei Dai, Zbigniew Darzynkiewicz
    Abstract:

    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.

Frits A J Muskiet - One of the best experts on this subject based on the ideXlab platform.

  • in vivo manipulation of l1210 Cell Cycle Phase distribution with α difluoromethylornithine 4 amidinoindan 1 one 2 amidinohydrazone and n1 acetylspermine
    Biochimica et Biophysica Acta, 1998
    Co-Authors: B Dorhout, Harri Ferwerda, Aw Kingma, Elly De Hoog, Frits A J Muskiet
    Abstract:

    We investigated whether the in vivo growth inhibitory effect of the combination of 4-amidinoindan-1-one 2'-amidinohydrazone (CGP 48664A) and alpha-difluoromethylornithine (DFMO) is reversible by treatment with N1-acetylspermine (N1-acSp). DBA-2 mice were inoculated with 10(5) L1210 Cell i.p. on day 0. From day 1 they received 2.50 mg CGP 48664A/kg i.p. once daily and 500 mg DFMO/kg i.p. twice daily. On day 5 they received 3 x 2500 nmol N1-acSp i.p. with 15-min intervals. L1210 Cell numbers, S-Phase percentage and polyamine contents, and liver and spleen polyamine contents were monitored in the following 48 h. Four days treatment with CGP 48664A/DFMO reduced L1210 Cell numbers, S-Phase, and spermidine. N1-acSp treatment increased L1210 spermidine from < or = 8 h and percentage S-Phase from 12 h. Maxima for spermidine and S-Phase were reached at < or = 8 and 18 h, respectively. These were below levels of untreated controls. Decreases were noted from 12 and 18 h, respectively. N1-acSp was detectable in L1210 from 0-18 h. Liver spermidine was decreased by CGP 48664A/DFMO. After N1-acSp treatment, liver N1-acSp and N1-acSd increased from < or = 8 h, reached maxima at < or = 8 and 10 h, respectively, and were undetectable from 15 h. We conclude that the in vivo growth inhibitory effect of CGP 48664A/DFMO is reversible by N1-acSp treatment. The liver is probably involved in N1-acSp terminal catabolism. The effect of the polyamine depletion-repletion scheme on S-Phase Cell numbers may be much more profound than present estimates from 5-bromo-2'-deoxyuridine incorporation.

  • in vivo effects of 4 amidinoindan l one 2 amidinohydrazone cgp 48664a and α difluoromethylornithine dfmo on l1210 growth Cell Cycle Phase distribution and polyamine contents
    International Journal of Cancer, 1995
    Co-Authors: B Dorhout, Harri Ferwerda, Elly De Hoog, Anneke W Kingma, Roelf Jakob Te Velde, Frits A J Muskiet
    Abstract:

    We studied the in vivo effects of 4-amidinoindan-1-one 2'-amidinohydrazone (CGP 48664A), alpha-difluoromethylornithine (DFMO) and a combination of CGP 48654A-DFMO on tumor growth, Cell-Cycle Phase distribution and polyamine contents. DBA-2, mice were inoculated i.p. with 10(5) L1210 Cells on day 0, treated i.p. on days 1-4 and killed on day 5. As compared to controls, CGP 48664A, DFMO and the CGP 48664A-DFMO combination reduced L1210 Cell numbers by 33, 43 and 85%, respectively. CGP 48664A did not affect Cell-Cycle Phase distribution. DFMO and the CGP 48664A-DFMO combination caused a moderate and a heavy accumulation in G(0)/G(1)- and G(2)/M-Phases, respectively. Compared with controls, the CGP 48664A-DFMO combination reduced putrescine, spermidine and total polyamines, but did not affect spermine. Compared with CGP 48664A, the CGP 48664A-DFMO combination caused lower putrescine and total polyamines, higher spermine, but no change in spermidine. Compared with DFMO, the CGP 48664A-DFMO combination caused higher putrescine and spermidine, lower spermine, but no change in total polyamine levels. We conclude that CGP 48664A potentiates the cystostatic effect of DFMO in vivo. The resulting growth inhibition is accompanied by an accumulation in G(0)/G(1)- and G(2)/M-Phases and a reduction of putrescine and spermidine. The data suggest that perturbed polyamine composition rather than reduced spermidine or total polyamine pool size causes a profound growth inhibition. (C) 1995 Wiley-Liss, Inc.

Frank Traganos - One of the best experts on this subject based on the ideXlab platform.

  • sequential phosphorylation of ser 10 on histone h3 and ser 139 on histone h2ax and atm activation during premature chromosome condensation relationship to Cell Cycle Phase and apoptosis
    Cytometry Part A, 2006
    Co-Authors: Xuan Huang, Frank Traganos, Toshiki Tanaka, Akira Kurose, Wei Dai, Zbigniew Darzynkiewicz
    Abstract:

    Background: Histone H1 and H3 phosphorylation associated with chromatin condensation during mitosis has been studied extensively. Less is known on histone modifications that occur during premature chromosome condensation (PCC). The aim of the present study was to reveal the status of histone H3 and H2AX phosphorylation on Ser-10 and Ser-139, respectively, as well as ATM activation through phosphorylation on Ser-1981, during PCC, and relate these events to Cell-Cycle Phase and to initiation of apoptosis. Materials and Methods: To induce PCC, A549 and HL-60 Cells were exposed to the phosphatase inhibitor calyculin A (Cal A). Phosphorylation of histone H3 and H2AX as well as ATM activation were detected immunocytochemically concurrent with analysis of Cellular DNA content and activation of caspase-3, a marker of apoptosis. The intensity of Cellular fluorescence was measured by flow- or laser scanning cytometry. Results: Induction of PCC led to rapid histone H3 phosphorylation, followed by activation of ATM and then H2AX phosphorylation in both, HL-60 and A549 Cells. All these events occurred sequentially, prior to caspase-3 activation, and affected Cells in all Phases of the Cell Cycle. ATM activation and H2AX phosphorylation was seen during mitosis of A549 but not HL-60 Cells. Conclusions: Because the Cal A-induced phosphorylation of histone H3 and H2AX, and of ATM, precede caspase-3 activation these modifications are pertinent to PCC and not to apoptosis-associated chromatin condensation. The sequence of histone H3 and H2AX phosphorylation and ATM activation during PCC is compatible with a role of ATM in mediating phosphorylation of H2AX but not H3. Mitosis in some Cell types may proceed without ATM activation and H2AX phosphorylation. © 2006 International Society for Analytical Cytology

  • cytometric assessment of dna damage in relation to Cell Cycle Phase and apoptosis
    Cell Proliferation, 2005
    Co-Authors: Xuan Huang, Frank Traganos, Dorota H Halicka, Toshiki Tanaka, Akira Kurose, Zbigniew Darzynkiewicz
    Abstract:

    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.

  • histone h2ax phosphorylation after Cell irradiation with uv b relationship to Cell Cycle Phase and induction of apoptosis
    Cell Cycle, 2005
    Co-Authors: Dorota H Halicka, Xuan Huang, Frank Traganos, Malcolm A King, Zbigniew Darzynkiewicz
    Abstract:

    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...

  • histone h2ax phosphorylation after Cell irradiation with uv b relationship to Cell Cycle Phase and induction of apoptosis
    Cell Cycle, 2005
    Co-Authors: Dorota H Halicka, Xuan Huang, Frank Traganos, Malcolm A King, Wei Dai, Zbigniew Darzynkiewicz
    Abstract:

    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.

  • assessment of histone h2ax phosphorylation induced by dna topoisomerase i and ii inhibitors topotecan and mitoxantrone and by the dna cross linking agent cisplatin
    Cytometry Part A, 2004
    Co-Authors: Xuan Huang, Masaki Okafuji, Frank Traganos, Ed Luther, Elena Holden, Zbigniew Darzynkiewicz
    Abstract:

    BACKGROUND DNA double-strand breaks (DSBs) in chromatin, whether induced by radiation, antitumor drugs, or by apoptosis-associated (AA) DNA fragmentation, provide a signal for histone H2AX phosphorylation on Ser-139; the phosphorylated H2AX is denoted gammaH2AX. The intensity of immunofluorescence (IF) of gammaH2AX was reported to reveal the frequency of DSBs in chromatin induced by radiation or by DNA topoisomerase I (topo 1) and II (topo 2) inhibitors. The purpose of this study was to further characterize the drug-induced (DI) IF of gammaH2AX, and in particular to distinguish it from AA gammaH2AX IF triggered by DNA breaks that occur in the course of AA DNA fragmentation. METHODS HL-60 Cells in cultures were treated with topotecan (TPT), mitoxantrone (MTX), or with DNA cross-linking drug cisplatin (CP); using multiparameter flow and laser-scanning cytometry, induction of gammaH2AX was correlated with: 1) caspase-3 activation; 2) chromatin condensation, 3) Cell Cycle Phase, and 4) AA DNA fragmentation. The intensity of gammaH2AX IF was compensated for by an increase in histone/DNA content, which doubles during the Cell Cycle, and for the "programmed" H2AX phosphorylation, which occurs in untreated Cells. RESULTS In Cells treated with TPT or MTX, the increase in DI-gammaH2AX IF peaked at 1.5 or 2 h, and was maximal in S- or G(1)-Phase Cells, respectively, for each drug. In Cells treated with CP, compared with TPT, the gammaH2AX IF was less intense, peaked later (3 h) and showed no Cell Cycle-Phase specificity. In the presence of phosphatase inhibitor calyculin A, a continuous increase in the TPT-induced gammaH2AX IF was still seen past 1.5 h, and after 3 h gammaH2AX IF was 2.7- to 3.4-fold higher than in the absence of the inhibitor. The AA gammaH2AX IF was distinguished from the DI-gammaH2AX IF by: 1) its greater intensity; 2) its prevention by caspase inhibitor zVAD-FMK; and 3) the concurrent activation of caspase-3 in the same Cells. A decrease in AA gammaH2AX IF coinciding with AA chromatin condensation was seen in the late stages of apoptosis. CONCLUSIONS Multiparameter analysis of gammaH2AX IF, caspase-3 activation, Cellular DNA content, and chromatin condensation allowed us to distinguish the DI from AA H2AX phosphorylation and relate them to the Cell Cycle Phase and stage of apoptosis. With a comparable degree of ds DNA breaks, the Cells arrested at the G1 or G2/M checkpoint were less prone to undergo apoptosis than the Cells replicating DNA. H2AX phosphorylation seen in CP-treated Cells may be associated with DNA repair that involves nucleotide excision repair (NER) and nonhomologous end joining (NHEJ). When the primary drug-induced lesions do not involve ds DNA breaks, but ds DNA breaks are formed during DNA repair, as in the case of CP, analysis of H2AX phosphorylation may reflect extent of the repair process.

Amelia J. Eisch - One of the best experts on this subject based on the ideXlab platform.

  • Chronic morphine induces premature mitosis of proliferating Cells in the adult mouse subgranular zone
    Journal of Neuroscience Research, 2004
    Co-Authors: Chitra D. Mandyam, Rebekah D. Norris, Amelia J. Eisch
    Abstract:

    The birth of Cells with neurogenic potential in the adult brain is assessed commonly by detection of exogenous S Phase markers, such as bromodeoxyuridine (BrdU). Analysis of other Phases of the Cell Cycle, however, can provide insight into how external factors, such as opiates, influence the cycling of newly born Cells. To this end, we examined the expression of two endogenous Cell Cycle markers in relation to BrdU: proliferating Cell nuclear antigen (PCNA) and phosphorylated histone H3 (pHisH3). Two hours after one intraperitoneal BrdU injection, BrdU-, PCNA-, and pHisH3-immunoreactive (IR) Cells exhibited similar distribution in the adult mouse subgranular zone (SGZ). Quantitative analysis within the SGZ revealed a relative abundance of Cells labeled for PCNA > BrdU >> pHisH3. Similar to our reports in rat SGZ, chronic morphine treatment decreased BrdU- and PCNA-IR Cells in mouse SGZ by 28 and 38%, respectively. We also show that pHisH3-IR Cells are influenced by chronic morphine to a greater extent (58% decrease) than are BrdU- or PCNA-IR Cells. Cell Cycle Phase analysis of SGZ BrdU-IR Cells using triple labeling for BrdU, PCNA, and pHisH3 revealed premature mitosis in chronic morphine-treated mice. These results suggest that morphine-treated mice have a shorter Gap2/mitosis (G2/M) Phase when compared to sham-treated mice. These findings demonstrate the power of using a combination of exogenous and endogenous Cell Cycle markers and nuclear morphology to track proliferating Cells through different Phases of the Cell Cycle and to reveal the regulation of Cell Cycle Phase by chronic morphine. © 2004 Wiley-Liss, Inc.

  • chronic morphine induces premature mitosis of proliferating Cells in the adult mouse subgranular zone
    Journal of Neuroscience Research, 2004
    Co-Authors: Chitra D. Mandyam, Rebekah D. Norris, Amelia J. Eisch
    Abstract:

    The birth of Cells with neurogenic potential in the adult brain is assessed commonly by detection of exogenous S Phase markers, such as bromodeoxyuridine (BrdU). Analysis of other Phases of the Cell Cycle, however, can provide insight into how external factors, such as opiates, influence the cycling of newly born Cells. To this end, we examined the expression of two endogenous Cell Cycle markers in relation to BrdU: proliferating Cell nuclear antigen (PCNA) and phosphorylated histone H3 (pHisH3). Two hours after one intraperitoneal BrdU injection, BrdU-, PCNA-, and pHisH3-immunoreactive (IR) Cells exhibited similar distribution in the adult mouse subgranular zone (SGZ). Quantitative analysis within the SGZ revealed a relative abundance of Cells labeled for PCNA > BrdU >> pHisH3. Similar to our reports in rat SGZ, chronic morphine treatment decreased BrdU- and PCNA-IR Cells in mouse SGZ by 28 and 38%, respectively. We also show that pHisH3-IR Cells are influenced by chronic morphine to a greater extent (58% decrease) than are BrdU- or PCNA-IR Cells. Cell Cycle Phase analysis of SGZ BrdU-IR Cells using triple labeling for BrdU, PCNA, and pHisH3 revealed premature mitosis in chronic morphine-treated mice. These results suggest that morphine-treated mice have a shorter Gap2/mitosis (G(2)/M) Phase when compared to sham-treated mice. These findings demonstrate the power of using a combination of exogenous and endogenous Cell Cycle markers and nuclear morphology to track proliferating Cells through different Phases of the Cell Cycle and to reveal the regulation of Cell Cycle Phase by chronic morphine.