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Induction and Processing of the Radiation-Induced Gamma-H2AX Signal and Its Link to the Underlying Pattern of DSB: A Combined Experimental and Modelling Study.

Tommasino F, Friedrich T, Jakob B, Meyer B, Durante M, Scholz M - PLoS ONE (2015)

Bottom Line: The study was performed by quantitative flow cytometry measurements, since the use of foci counting would result in reasonable accuracy only in a limited dose range of a few Gy.Our results are found to be supportive for the basic assumptions on which GLOBLE is built.Apart from giving new insights into the H2AX phosphorylation process, experiments performed at high doses are of relevance in the context of radiation therapy, where hypo-fractionated schemes become increasingly popular.

View Article: PubMed Central - PubMed

Affiliation: GSI Helmholtzzentrum für Schwerionenforschung, Department of Biophysics, Darmstadt, Germany.

ABSTRACT
We present here an analysis of DSB induction and processing after irradiation with X-rays in an extended dose range based on the use of the γH2AX assay. The study was performed by quantitative flow cytometry measurements, since the use of foci counting would result in reasonable accuracy only in a limited dose range of a few Gy. The experimental data are complemented by a theoretical analysis based on the GLOBLE model. In fact, original aim of the study was to test GLOBLE predictions against new experimental data, in order to contribute to the validation of the model. Specifically, the γH2AX signal kinetics has been investigated up to 24 h after exposure to increasing photon doses between 2 and 500 Gy. The prolonged persistence of the signal at high doses strongly suggests dose dependence in DSB processing after low LET irradiation. Importantly, in the framework of our modelling analysis, this is related to a gradually increased fraction of DSB clustering at the micrometre scale. The parallel study of γH2AX dose response curves shows the onset of a pronounced saturation in two cell lines at a dose of about 20 Gy. This dose is much lower than expected according to model predictions based on the values usually adopted for the DSB induction yield (≈ 30 DSB/Gy) and for the γH2AX foci extension of approximately 2 Mbp around the DSB. We show and discuss how theoretical predictions and experimental findings can be in principle reconciled by combining an increased DSB induction yield with the assumption of a larger genomic extension for the single phosphorylated regions. As an alternative approach, we also considered in our model the possibility of a 3D spreading-mechanism of the H2AX phosphorylation around the induced DSB, and applied it to the analysis of both the aspects considered. Our results are found to be supportive for the basic assumptions on which GLOBLE is built. Apart from giving new insights into the H2AX phosphorylation process, experiments performed at high doses are of relevance in the context of radiation therapy, where hypo-fractionated schemes become increasingly popular.

No MeSH data available.


Related in: MedlinePlus

γH2AX fluorescence pattern at high photon doses.γH2AX fluorescence pattern (green) of AGD cells as observed at the confocal microscope 1 h after high doses of X-rays irradiation. The “sum slices” Z-projection obtained with the ImageJ software is shown after deconvolution. DNA was counterstained with DAPI (displayed in red). The insert shows a single slice of γH2AX in greyscale (deconvoluted) with a magnification factor of 1.5. Larger dark areas in the DNA staining which are free of γH2AX fluorescence represent nucleoli [68].
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pone.0129416.g001: γH2AX fluorescence pattern at high photon doses.γH2AX fluorescence pattern (green) of AGD cells as observed at the confocal microscope 1 h after high doses of X-rays irradiation. The “sum slices” Z-projection obtained with the ImageJ software is shown after deconvolution. DNA was counterstained with DAPI (displayed in red). The insert shows a single slice of γH2AX in greyscale (deconvoluted) with a magnification factor of 1.5. Larger dark areas in the DNA staining which are free of γH2AX fluorescence represent nucleoli [68].

Mentions: Two different irradiation setups were employed. Flow cytometry experiments were conducted by using an x-ray tube working at 250 kV and 16 mA. A dose rate of 7 Gy/min was employed. Due to the need to reach high doses, a different x-ray tube working at 35 kV and 80 mA was adopted to perform experiments at high doses. This allows reaching a dose rate of about 100 Gy/min. Despite the different voltages, according to literature data no considerable difference is expected among the DNA damage patterns induced by the two x-ray qualities employed [38]. In order to ensure a homogeneous irradiation field, a single petri dish was placed at a distance of few cm from the tube exit window. With the aim to limit photon absorption, the medium level in the dish was reduced to 0.5 ml during irradiation. After irradiation, the samples were put in the incubators at 37°C. In both cases, dosimetry was performed with a calibrated ionization chamber. The dosimetry was performed at different distances from the source and at different currents, aimed at avoiding saturation effects of the ionization chamber. In order to test the homogeneity of the irradiation field Gafchromic films were also employed. This confirmed that the dose is released homogeneously at the biological samples. This setup was adopted to perform microscopy experiments shown in Fig 1, as well as for the dose response curve in Fig 2 and for the study of the γH2AX kinetics at high doses (> 8 Gy) shown in Fig 3.


Induction and Processing of the Radiation-Induced Gamma-H2AX Signal and Its Link to the Underlying Pattern of DSB: A Combined Experimental and Modelling Study.

Tommasino F, Friedrich T, Jakob B, Meyer B, Durante M, Scholz M - PLoS ONE (2015)

γH2AX fluorescence pattern at high photon doses.γH2AX fluorescence pattern (green) of AGD cells as observed at the confocal microscope 1 h after high doses of X-rays irradiation. The “sum slices” Z-projection obtained with the ImageJ software is shown after deconvolution. DNA was counterstained with DAPI (displayed in red). The insert shows a single slice of γH2AX in greyscale (deconvoluted) with a magnification factor of 1.5. Larger dark areas in the DNA staining which are free of γH2AX fluorescence represent nucleoli [68].
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4465900&req=5

pone.0129416.g001: γH2AX fluorescence pattern at high photon doses.γH2AX fluorescence pattern (green) of AGD cells as observed at the confocal microscope 1 h after high doses of X-rays irradiation. The “sum slices” Z-projection obtained with the ImageJ software is shown after deconvolution. DNA was counterstained with DAPI (displayed in red). The insert shows a single slice of γH2AX in greyscale (deconvoluted) with a magnification factor of 1.5. Larger dark areas in the DNA staining which are free of γH2AX fluorescence represent nucleoli [68].
Mentions: Two different irradiation setups were employed. Flow cytometry experiments were conducted by using an x-ray tube working at 250 kV and 16 mA. A dose rate of 7 Gy/min was employed. Due to the need to reach high doses, a different x-ray tube working at 35 kV and 80 mA was adopted to perform experiments at high doses. This allows reaching a dose rate of about 100 Gy/min. Despite the different voltages, according to literature data no considerable difference is expected among the DNA damage patterns induced by the two x-ray qualities employed [38]. In order to ensure a homogeneous irradiation field, a single petri dish was placed at a distance of few cm from the tube exit window. With the aim to limit photon absorption, the medium level in the dish was reduced to 0.5 ml during irradiation. After irradiation, the samples were put in the incubators at 37°C. In both cases, dosimetry was performed with a calibrated ionization chamber. The dosimetry was performed at different distances from the source and at different currents, aimed at avoiding saturation effects of the ionization chamber. In order to test the homogeneity of the irradiation field Gafchromic films were also employed. This confirmed that the dose is released homogeneously at the biological samples. This setup was adopted to perform microscopy experiments shown in Fig 1, as well as for the dose response curve in Fig 2 and for the study of the γH2AX kinetics at high doses (> 8 Gy) shown in Fig 3.

Bottom Line: The study was performed by quantitative flow cytometry measurements, since the use of foci counting would result in reasonable accuracy only in a limited dose range of a few Gy.Our results are found to be supportive for the basic assumptions on which GLOBLE is built.Apart from giving new insights into the H2AX phosphorylation process, experiments performed at high doses are of relevance in the context of radiation therapy, where hypo-fractionated schemes become increasingly popular.

View Article: PubMed Central - PubMed

Affiliation: GSI Helmholtzzentrum für Schwerionenforschung, Department of Biophysics, Darmstadt, Germany.

ABSTRACT
We present here an analysis of DSB induction and processing after irradiation with X-rays in an extended dose range based on the use of the γH2AX assay. The study was performed by quantitative flow cytometry measurements, since the use of foci counting would result in reasonable accuracy only in a limited dose range of a few Gy. The experimental data are complemented by a theoretical analysis based on the GLOBLE model. In fact, original aim of the study was to test GLOBLE predictions against new experimental data, in order to contribute to the validation of the model. Specifically, the γH2AX signal kinetics has been investigated up to 24 h after exposure to increasing photon doses between 2 and 500 Gy. The prolonged persistence of the signal at high doses strongly suggests dose dependence in DSB processing after low LET irradiation. Importantly, in the framework of our modelling analysis, this is related to a gradually increased fraction of DSB clustering at the micrometre scale. The parallel study of γH2AX dose response curves shows the onset of a pronounced saturation in two cell lines at a dose of about 20 Gy. This dose is much lower than expected according to model predictions based on the values usually adopted for the DSB induction yield (≈ 30 DSB/Gy) and for the γH2AX foci extension of approximately 2 Mbp around the DSB. We show and discuss how theoretical predictions and experimental findings can be in principle reconciled by combining an increased DSB induction yield with the assumption of a larger genomic extension for the single phosphorylated regions. As an alternative approach, we also considered in our model the possibility of a 3D spreading-mechanism of the H2AX phosphorylation around the induced DSB, and applied it to the analysis of both the aspects considered. Our results are found to be supportive for the basic assumptions on which GLOBLE is built. Apart from giving new insights into the H2AX phosphorylation process, experiments performed at high doses are of relevance in the context of radiation therapy, where hypo-fractionated schemes become increasingly popular.

No MeSH data available.


Related in: MedlinePlus