Use of the γ-H2AX assay to investigate DNA repair dynamics following multiple radiation exposures.

Radiation therapy is one of the most common and effective strategies used to treat cancer. The irradiation is usually performed with a fractionated scheme, where the dose required to kill tumour cells is given in several sessions, spaced by specific time intervals, to allow healthy tissue recovery. In this work, we examined the DNA repair dynamics of cells exposed to radiation delivered in fractions, by assessing the response of histone-2AX (H2AX) phosphorylation (γ-H2AX), a marker of DNA double strand breaks. γ-H2AX foci induction and disappearance were monitored following split dose irradiation experiments in which time interval between exposure and dose were varied. Experimental data have been coupled to an analytical theoretical model, in order to quantify key parameters involved in the foci induction process. Induction of γ-H2AX foci was found to be affected by the initial radiation exposure with a smaller number of foci induced by subsequent exposures. This was compared to chromatin relaxation and cell survival. The time needed for full recovery of γ-H2AX foci induction was quantified (12 hours) and the 1:1 relationship between radiation induced DNA double strand breaks and foci numbers was critically assessed in the multiple irradiation scenarios.

Investigation of the mechanisms underpinning IL-6 cytokine release in bystander responses: the roles of radiation dose, radiation quality and specific ROS/RNS scavengers.

PURPOSE: To investigate the mechanisms regulating the pathways of the bystander transmission in vitro, focusing on the radiation-perturbed signalling (via Interleukine 6, IL-6) of the irradiated cells after exposure to low doses of different radiation types. MATERIALS AND METHODS: An integrated 'systems radiation biology' approach was adopted. Experimentally the level of the secreted cytokine from human fibroblasts was detected with ELISA (Enzyme-Linked ImmunoSorbent Assay) method and subsequently the data were analyzed and coupled with a phenomenological model based on differential equations to evaluate the single-cell release mechanisms. RESULTS: The data confirmed the important effect of radiation on the IL-6 pathway, clearly showing a crucial role of the ROS (Reactive Oxygen Species) in transducing the effect of initial radiation exposure and the subsequent long-term release of IL-6. Furthermore, a systematic investigation of radiation dose/radiation quality dependence seems to indicate an increasing efficiency of high LET (Linear Energy Transfer) irradiation in the release of the cytokine. Basic hypotheses were tested, on the correlation between direct radiobiological damage and signal release and on the radiation target for this endpoint (secretion of IL-6). CONCLUSIONS: The results demonstrate the role of reactive oxygen and nitrogen species in the signaling pathways of IL-6. Furthermore the systems radiation biology approach here adopted, allowed us to test and verify hypotheses on the behavior of the single cell in the release of cytokine, after the exposure to different doses and different qualities of ionizing radiation.