**5. Conclusions**

20 Selected Topics in DNA Repair

formation of bystander factors that themselves generate ROS, leading to a self-sustaining system responsible for long-lasting effects (Yang 2005, Sokolov 2005, Kashino 2004, Lyng 2006). In irradiated TK6 cells both 53BP1 and NBS1p343 proteins co-localized with -H2AX

Fig. 15. Non-irradiated, irradiated and bystander TK6 cells were fixed and co-stained with anti- H2AX (green), anti-53BP1 and anti-NBS1-p343 (red), at 2 h from irradiation or CM incubation. The red and green images were merged and subjected to co-localization analysis. Arrows indicate γH2AX foci without co-localization of 53BP1and NBS1-proteins.

Nuclei were counterstained with DAPI.

foci, whereas in bystander cells co-localization was partial or absent (Figure 15).

The DNA-damage response pathway relies on the recruitment and modification of many different proteins that sense and signal the damage, activate transducer and effector proteins involved in cell cycle arrest, DNA repair and apoptosis. A correct DDR safeguards cells, whereas perturbations/defects in this pathway might contribute to the occurrence or to the acceleration of carcinogenesis. Our results have contributed to highlight cell response of human lymphocytes to DNA damage induced directly or indirectly by ionizing radiation. In particular, novel aspects of low- and high-LET radiation effects on human lymphocytes have been described, such as double strand break repair kinetics, mutational effects, micronuclei induction, apoptosis induction, cell cycle alterations, gene and microRNA expression changes. In addition, we have reported new findings about the cell response of human lymphocytes when ionizing radiation exposure occurred in microgravity, condition which has been experimentally simulated by the Rotating Wall Vessel. The results clearly indicate that modeled microgravity affects the cell response to radiation, thus contributing to increase the risk of radiation exposure during space missions. By considering that the levels of DNA repair genes were not significantly changed in MMG condition, we suppose that perturbations in the cell response to ionizing radiation are due to the altered activity of proteins playing an important role in DDR pathway. Evidences are accumulating on the strict dependence between efficiency in DNA repair and chromatin structural organization (Gontijo et al., 2003, Rübe et al., 2011). The elaborate higher-order organization of chromatin appears to be important in assembling the repair machinery, improving the accessibility of DNA lesions to repair complexes. Modifications of cell structure and perturbations of nuclear architecture induced by microgravity may affect the accessibility in chromatin to DNA repair machinery. The preliminary results obtained from miRNA-mRNA profiling

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represent new insights about the radio-responsiveness in MMG. They seem promising to clarify the role of miRNAs in DNA-damage response to radiation in microgravity, thus improving the scientific approach towards environmental exposure risk.

The studies on molecular mechanisms of bystander effect could have great implications in evaluating radiation risk of IR exposures, and also have the potential to reassess radiation damage models currently used in radiotherapy. The radiation-induced bystander effect was shown to occur in a number of experimental systems both *in vitro* and *in vivo* and it is supposed to be realized through several pathways of transmission of the stress signal: a direct cellular contact, interaction through gap-junctions and through the culture medium of the irradiated cells. In our experimental system the conditioned medium was the main way by which the irradiated cells communicate their stressed condition to the non-irradiated cells. ROS released by irradiated TK6 cells into the culture medium were short-lived and probably other soluble molecules are necessary to maintain high the level of cell mortality in bystander cells. Recent studies, investigating on the nature of such molecules, suggest that fragments of extracellular genomic DNA, probably released from the apoptotic irradiated cells in the culture medium, are able to induce the bystander effects (Ermakov et al., 2011). Such DNA fragments bind to the Toll-like receptors family, leading to a signaling mechanism whose outcome is the dynamic transformation of the cytoskeleton and alteration in the spatial localization of chromatin portions in the nucleus. Thus, in bystander cells, as for microgravity-incubated lymphocytes, modifications in the nuclear structural organization may affect the assembly of the DNA repair machinery.

### **6. Acknowledgment**

We gratefully acknowledge Dr. Cristiano De Pittà and Dr. Chiara Romualdi of the Department of Biology, University of Padova, for miRNA and mRNA expression profiling and statistical assistance. We acknowledge Dr. Vito Barbieri of the Department of Oncological and Surgical Sciences of Padova's University for cell irradiation with –rays and Roberto Cherubini of the INFN, Laboratori Nazionali di Legnaro, Padova, for cell irradiation with low-energy protons.
