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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

Predicted two-dimensional H2AX phosphorylation patterns with and without the 3D spreading mechanism.Example of XY projections of hit domains only (A) and of all phosphorylated domains obtained with the random expansion algorithm (B); the exposure to 1 Gy of X-rays photon radiation was simulated. The cell nucleus is represented in light grey in the background. The colour scale indicates the multiplicity of hit domains in the column of interest.
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pone.0129416.g009: Predicted two-dimensional H2AX phosphorylation patterns with and without the 3D spreading mechanism.Example of XY projections of hit domains only (A) and of all phosphorylated domains obtained with the random expansion algorithm (B); the exposure to 1 Gy of X-rays photon radiation was simulated. The cell nucleus is represented in light grey in the background. The colour scale indicates the multiplicity of hit domains in the column of interest.

Mentions: Concerning the relevance of the 3D spreading mechanisms, we extracted from the model calculations the Z projections of hit domains only and of all phosphorylated domains, as obtained when the random expansion algorithm is considered. The comparison between the two projections indicates that the overlap of neighbouring foci could affect the scoring of single foci. An example picture where the exposure to 1 Gy of photon radiation was simulated is shown in Fig 9. In panel A we can observe how the hit domains are distributed. This allows realizing that already at low doses it is possible that projected hit domains are close to each other, and that overlapping can take place in the xy plane as well as in z direction. By measuring the xy projection at the microscope, this second aspect would automatically translate in losing some DSB when scoring the foci. However, the problem can be partially overcome by using appropriate algorithms for the analysis which also increase the sensitivity in longitudinal direction. At the same time, in panel B we can observe how the fluorescence pattern would be in a manner confused by the 3D spreading of the H2AX phosphorylation, resulting in not well defined boundaries between single foci, in a distribution of foci sizes and in different local intensities.


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)

Predicted two-dimensional H2AX phosphorylation patterns with and without the 3D spreading mechanism.Example of XY projections of hit domains only (A) and of all phosphorylated domains obtained with the random expansion algorithm (B); the exposure to 1 Gy of X-rays photon radiation was simulated. The cell nucleus is represented in light grey in the background. The colour scale indicates the multiplicity of hit domains in the column of interest.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0129416.g009: Predicted two-dimensional H2AX phosphorylation patterns with and without the 3D spreading mechanism.Example of XY projections of hit domains only (A) and of all phosphorylated domains obtained with the random expansion algorithm (B); the exposure to 1 Gy of X-rays photon radiation was simulated. The cell nucleus is represented in light grey in the background. The colour scale indicates the multiplicity of hit domains in the column of interest.
Mentions: Concerning the relevance of the 3D spreading mechanisms, we extracted from the model calculations the Z projections of hit domains only and of all phosphorylated domains, as obtained when the random expansion algorithm is considered. The comparison between the two projections indicates that the overlap of neighbouring foci could affect the scoring of single foci. An example picture where the exposure to 1 Gy of photon radiation was simulated is shown in Fig 9. In panel A we can observe how the hit domains are distributed. This allows realizing that already at low doses it is possible that projected hit domains are close to each other, and that overlapping can take place in the xy plane as well as in z direction. By measuring the xy projection at the microscope, this second aspect would automatically translate in losing some DSB when scoring the foci. However, the problem can be partially overcome by using appropriate algorithms for the analysis which also increase the sensitivity in longitudinal direction. At the same time, in panel B we can observe how the fluorescence pattern would be in a manner confused by the 3D spreading of the H2AX phosphorylation, resulting in not well defined boundaries between single foci, in a distribution of foci sizes and in different local intensities.

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