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

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

Myc Is Required for Activation of the ATM-Dependent Checkpoints in Response to DNA Damage

Background: The MYC protein controls cellular functions such as differentiation, proliferation, and apoptosis. In response to genotoxic agents, cells overexpressing MYC undergo apoptosis. However, the MYC-regulated effectors acting upstream of the mitochondrial apoptotic pathway are still unknown. Principal Findings: In this study, we demonstrate that expression of Myc is required to activate the Ataxia telangiectasia mutated (ATM)-dependent DNA damage checkpoint responses in rat cell lines exposed to ionizing radiation (IR) or the bacterial cytolethal distending toxin (CDT). Phosphorylation of the ATM kinase and its downstream effectors, such as histone H2AX, were impaired in the myc null cell line HO15.19, compared to the myc positive TGR-1 and HOmyc3 cells. Nuclear foci formation of the Nijmegen Breakage Syndrome (Nbs) 1 protein, essential for efficient ATM activation, was also reduced in absence of myc. Knock down of the endogenous levels of MYC by siRNA in the human cell line HCT116 resulted in decreased ATM and CHK2 phosphorylation in response to irradiation. Conversely, cell death induced by UV irradiation, known to activate the ATR-dependent checkpoint, was similar in all the cell lines, independently of the myc status. Conclusion: These data demonstrate that MYC contributes to the activation of the ATM-dependent checkpoint responses, leading to cell death in response to specific genotoxic stimuli.Swedish Cancer SocietySwedish Research Counci

<i>myc</i> deletion impairs Nbs1 foci formation in response to DNA damage.

<p>TGR-1, the <i>myc</i> reconstituted HOmyc3, and the <i>myc</i> null HO15.19 cells were exposed to bacterial lysates (1∶2000) expressing the mutant (CTR) or the wild type form (CDT) of <i>H. hepaticus</i> CDT, or irradiated (IR, 20 Gy) and further incubated in complete medium for the indicated periods of time. The Nbs1 protein was detected by indirect immunostaining, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008924#s2" target="_blank">Material and Methods</a>. The figure shows the sub-cellular distribution of Nbs1 at 2h post-treatment.</p

<i>myc</i> deletion does not alter cell death kinetics upon UV irradiation.

<p>TGR-1, the <i>myc</i> reconstituted cells HOmyc3, and the <i>myc</i> null HO15.19 cells were left untreated or exposed to UV irradiation and further incubated in complete medium for the indicated periods of time. <b>A</b>) Analysis of the cell cycle distribution assessed by PI staining and flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008924#s2" target="_blank">Material and Methods</a>. <b>B</b>) Phase contrast micrographs of the cells taken at the indicated time points (Magnification 40×). One out of three independent experiments is shown.</p

Knock down of MYC expression decreases activation of ATM, CHK2 and p53.

<p><b>A</b>) HCT116 cells were transfected either with the control GFP-specific (CTR) or MYC specific siRNA for 48h. The expression of MYC was assessed by immunoprecipitation and western-blot analysis. One out of three independent experiments is shown. <b>B</b>) HCT116 cells, transfected either with the GFP-specific (CTR) or MYC specific siRNA were left untreated or exposed to IR (20 Gy), and further incubated in complete medium for the indicated periods of time. The levels of phosphorylation of ATM, CHK2 and p53 were assessed by western blot analysis. Actin and total ATM were used as an internal loading control. One out of three independent experiments is shown.</p

<i>myc</i> deletion prevents p53 stabilization in response to DNA damage.

<p><b>A</b>) TGR-1 and the <i>myc</i> reconstituted HOmyc3 cells were left untreated or exposed to IR (20 Gy) for the indicated periods of time. The levels of p53 were assessed by western blot analysis. <b>B</b>) TGR-1, the <i>myc</i> reconstituted HOmyc3 and the HO15.19 cells were left untreated or exposed to IR (20 Gy) for the indicated periods of time. The levels of p53 were assessed by western blot analysis. Actin was used as an internal loading control. One out of three independent experiments is shown.</p

<i>myc</i> deletion delays cell death upon irradiation.

<p><b>A</b>) The levels of the endogenous Myc protein were assessed by immunoprecipitation followed by western blot analysis in TGR-1, the <i>myc</i> reconstituted cells HOmyc3, and the <i>myc</i> null HO15.19 cells using α-Myc antibodies. Expression of actin in total cell lysates was used as control. <b>B</b>) TGR-1, the <i>myc</i> reconstituted cells HOmyc3, and the <i>myc</i> null HO15.19 cells were left untreated or irradiated (20Gy) and further incubated in complete medium for the indicated periods of time. Analysis of the cell cycle distribution was assessed by PI staining and flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008924#s2" target="_blank">Material and Methods</a>. One out of four independent experiments is shown.</p

Activation of ATM and ATM-dependent responses upon induction of DNA damage is Myc dependent.

<p>TGR-1, HOmyc3, and HO15.19 cells were left untreated or exposed to IR (20Gy), and further incubated in complete medium for 2h. The levels of phospho-ATM and phospho-H2AX (γH2AX) were assessed by western blot analysis. Actin and total ATM were used as internal loading controls. One of three experiments is shown.</p