**4. The DNA-damage response of human lymphocytes to indirect effect of ionizing radiation**

In addition the cellular effects arising as a direct response to ionizing radiation, in the last decade it has been suggested that extranuclear or extracellular targets can contribute to the genetic damage in non-irradiated (bystander) cells. The bystander effect (BE) is the biological response of non-irradiated cells induced by contact with irradiated cells. The contact with bystander factors may occur by direct cell–cell interaction or be mediated by the fluid surrounding the cells. It has been reported that the BE causes cell death, cell cycle arrest, apoptosis, changes in gene expression, and increases micronucleus induction, chromosomal aberrations, mutation frequency, and DNA damage in cells neighboring hit cells. In contrast to DNA damage induced by direct irradiation, bystander cell DNA damage is still poorly understood. Many data showed that early events of the radiation induced bystander effect are rapid calcium fluxes and generation of reactive oxygen species in bystander cells. Mitochondria seem to play a central role in bystander signaling: irradiated cell conditioned media can cause changes of mitochondrial distribution, loss of mitochondrial membrane potential, increases in ROS, and increase in apoptosis among the medium receptor cells, which can be blocked by treatments with antioxidants (Chen et al., 2008). Experiments carried out in hepatoma cell lines provide evidence that the BE can be modulated by the p53 status of irradiated cells and that a p53-dependent release of cytochrome-c from mitochondria may be involved in producing BE (He et al., 2011).

We investigated on the mechanisms of the medium-mediated bystander response induced by low doses of -rays in human tumoural lymphocytes (TK6 cells), a cell line growing in suspension, in which gap-junction communications are not involved in transferring bystander signals and only medium-mediated molecules may be responsible for BE induction. Cell cultures were irradiated and the culture medium discarded immediately after irradiation and replaced with a fresh one to eliminate ROS originating during irradiation. Irradiated cells were incubated for 6h in fresh medium, which, at the end of incubation time, is referred as conditioned medium (CM) and used to incubate nonirradiated TK6 cells for different times (2-48 h). In bystander cultures, cell mortality at the fixed incubation times ranged between 24 and 19%, very similar values to that of directly irradiated cells (28 and 20%). The mortality percentages for all incubation times were significantly higher with respect to that of the controls (0Gy and 0Gy CM). The survival fraction of directly 1Gy irradiated or CM incubated cells was determined by the clonogenic assay. The data show that both irradiated and bystander TK6 cells had a lower cloning efficiency than their respective controls. Figure 12 reports the results about cell mortality and survival (given as the ratio of the cloning efficiency of treated vs. untreated control cells) in TK6 cells exposed directly to IR or to CM. Apoptosis induction was tested by the presence of fragmented nuclei and apoptotic bodies at 2, 24 and 48h after 1Gy irradiation or CM incubation. The apoptotic index (A.I.) ranged between 7 and 9 % in irradiated cells and between 6 and 7.5 % in bystander cells, and was significantly higher than the relative controls at all times (Figure 13). The induction of apoptosis was also analyzed by the activation of caspase-3, the principal effector caspase, assayed by the cleavage of the peptide substrate DEVD-AFC, at 1, 2, 24 and 48h after irradiation or CM incubation. In bystander cells caspase-3 activation increased from 1.4- to 2.7-fold during the 48h of CM incubation, suggesting that bystander apoptosis increases after 48h. Bystander apoptosis in TK6 cells was sensitive to the inhibitor of caspase-8, the Z-IETD-fmk, added during CM treatment or

The DNA-Damage Response to Ionizing Radiation in Human Lymphocytes 19

post-irradiation incubation. The presence of the inhibitor significantly decreased the induction of apoptosis to the control level, but it did not significantly decrease the level of apoptosis in either irradiated or non-irradiated controls (Figure 14). These results suggest that caspase-8 activation is triggered by signaling molecules present in the conditioned medium. The addition of the ROS scavenger Cu-Zn superoxide dismutase and Nacetylcysteine to the conditioned medium allowed to investigate the involvement of oxidative stress in inducing bystander apoptosis. ROS scavengers did not significantly decrease the apoptotic index in CM cultures; by treating non-irradiated TK6 cells with medium irradiated without cells (IM), we evaluated the contribute of ROS produced by

Fig. 14. Apoptotic index in irradiated cells (1Gy), in irradiated cells incubated with caspase-8 inhibitor (1Gy + Z-IETD-fmk), in bystander cells incubated with caspase-8 inhibitor (CM 1Gy + Z-IETD-fmk), and with ROS scavengers SOD and NAC (CM 1Gy + scav.). Results are the means of 3-5 independent experiments ± S.E. Significant differences were observed in CM 1Gy vs CM 1Gy + Z-IETD-fmk at all times. **B**. Apoptotic index in cells incubated with irradiated medium (IM 1Gy), with irradiated medium in the presence of caspase-8 inhibitor (IM 1Gy + Z-IETD-fmk), and ROS scavengers (IM 1Gy + scav.). The values are the means of 3 independent experiments ± S.E. Significant differences were observed at 2 h of incubation

IM incubation for 2h increased the apoptotic index which was totally inhibited by ROS scavengers and little affected by incubation with the caspase-8 inhibitor, whereas at 24 and 48h no significant differences among samples incubated with IM were observed. DSBs induced by ionizing radiation can easily be detected by the extensive H2AX phosphorylation occurring near DNA lesions, forming foci that co-localize with several repair proteins (Fernandez-Capetillo et al., 2003). 85% of TK6 were -H2AX foci positive at 2h after irradiation with 1Gy, then this percentage decreased to the level of non-irradiated cells 24h later, fitting DNA repair kinetics. The incubation of cells with CM for 2h significantly increased the percentage of -H2AX foci positive cells (9-11%) but, when the CM was kept in contact with bystander cells for 24h the number of positive cells decreased to control levels, suggesting that DNA lesions induced at the beginning of CM incubation are repaired and no new damage accumulates later. Data from other human cells show that -H2AX foci induction in bystander cells persists in time, probably as a consequence of the

with IM 1 Gy vs IM 1Gy + Z-IETD-fmk (\**P*<0.05, \*\**P*<0.01, \*\*\**P*<0.001, *t*-test).

irradiation in inducing bystander apoptosis.

Fig. 12. Cell mortality determined by Trypan blue staining (A) and cloning efficiency (B) in non-irradiated control cells (0Gy), in irradiated cells (1Gy), in cells incubated with conditioned medium from non-irradiated cells (CM 0Gy) and in cells incubated with conditioned medium from irradiated cells (CM 1Gy). Results are the means of 3-5 independent experiments ± S.E. (\**P*< 0.05, \*\**P*<0.01, \*\*\**P*<0.001, *t* test).

Fig. 13. Apoptotic index (A) in irradiated and bystander cells. Results are the means ± S.E. of 5-8 independent experiments. Significant differences were observed in 1Gy and CM 1Gy vs 0Gy and CM 0Gy, respectively (\**P*<0.05, \*\**P*<0.01, \*\*\**P*<0.001, *t* test).

Fig. 12. Cell mortality determined by Trypan blue staining (A) and cloning efficiency (B) in

Fig. 13. Apoptotic index (A) in irradiated and bystander cells. Results are the means ± S.E. of 5-8 independent experiments. Significant differences were observed in 1Gy and CM 1Gy vs

non-irradiated control cells (0Gy), in irradiated cells (1Gy), in cells incubated with conditioned medium from non-irradiated cells (CM 0Gy) and in cells incubated with conditioned medium from irradiated cells (CM 1Gy). Results are the means of 3-5

independent experiments ± S.E. (\**P*< 0.05, \*\**P*<0.01, \*\*\**P*<0.001, *t* test).

0Gy and CM 0Gy, respectively (\**P*<0.05, \*\**P*<0.01, \*\*\**P*<0.001, *t* test).

post-irradiation incubation. The presence of the inhibitor significantly decreased the induction of apoptosis to the control level, but it did not significantly decrease the level of apoptosis in either irradiated or non-irradiated controls (Figure 14). These results suggest that caspase-8 activation is triggered by signaling molecules present in the conditioned medium. The addition of the ROS scavenger Cu-Zn superoxide dismutase and Nacetylcysteine to the conditioned medium allowed to investigate the involvement of oxidative stress in inducing bystander apoptosis. ROS scavengers did not significantly decrease the apoptotic index in CM cultures; by treating non-irradiated TK6 cells with medium irradiated without cells (IM), we evaluated the contribute of ROS produced by irradiation in inducing bystander apoptosis.

Fig. 14. Apoptotic index in irradiated cells (1Gy), in irradiated cells incubated with caspase-8 inhibitor (1Gy + Z-IETD-fmk), in bystander cells incubated with caspase-8 inhibitor (CM 1Gy + Z-IETD-fmk), and with ROS scavengers SOD and NAC (CM 1Gy + scav.). Results are the means of 3-5 independent experiments ± S.E. Significant differences were observed in CM 1Gy vs CM 1Gy + Z-IETD-fmk at all times. **B**. Apoptotic index in cells incubated with irradiated medium (IM 1Gy), with irradiated medium in the presence of caspase-8 inhibitor (IM 1Gy + Z-IETD-fmk), and ROS scavengers (IM 1Gy + scav.). The values are the means of 3 independent experiments ± S.E. Significant differences were observed at 2 h of incubation with IM 1 Gy vs IM 1Gy + Z-IETD-fmk (\**P*<0.05, \*\**P*<0.01, \*\*\**P*<0.001, *t*-test).

IM incubation for 2h increased the apoptotic index which was totally inhibited by ROS scavengers and little affected by incubation with the caspase-8 inhibitor, whereas at 24 and 48h no significant differences among samples incubated with IM were observed. DSBs induced by ionizing radiation can easily be detected by the extensive H2AX phosphorylation occurring near DNA lesions, forming foci that co-localize with several repair proteins (Fernandez-Capetillo et al., 2003). 85% of TK6 were -H2AX foci positive at 2h after irradiation with 1Gy, then this percentage decreased to the level of non-irradiated cells 24h later, fitting DNA repair kinetics. The incubation of cells with CM for 2h significantly increased the percentage of -H2AX foci positive cells (9-11%) but, when the CM was kept in contact with bystander cells for 24h the number of positive cells decreased to control levels, suggesting that DNA lesions induced at the beginning of CM incubation are repaired and no new damage accumulates later. Data from other human cells show that -H2AX foci induction in bystander cells persists in time, probably as a consequence of the

The DNA-Damage Response to Ionizing Radiation in Human Lymphocytes 21

We suggest that the short-lived ROS released in the medium by irradiated cells are responsible for DNA lesions, unlike double strand breaks, which activate H2AX phosphorylation but do not require the 53BP1 and NBS1p343 proteins to be repaired. It is possible that in our experiments DNA damage induced by CM treatment consisted of a few DSBs, the repair of which requires the recruitment of 53BP1 and NBS1p343 proteins and mainly in other types of DNA lesions, in which repair occurs without these proteins. Recent studies suggest that there are important differences between the DNA damage response in directly irradiated cells and non-targeted cells via bystander signals. The DNA damage in bystander cells seems to persist for a prolonged time (Burdak-Rothkamm et al., 2007), differently from DNA damage induced directly by irradiation which is repaired completely within few hours depending on radiation dose. Studies carried out in p53 wild-type (TK6), p53 null (NH32),and p53 mutant (WTK1) lymphoblastoid cells using siRNA to knockdown DNA PKcs demonstrated the central role of non-homologous end-joining in processing bystander damage, in contrast to the role of homologous recombination which seems to be essential only in inducing sister chromatid exchanges in bystander cells (Zhang et al., 2008). ATM- and Rad3-related (ATR) protein kinases have a central role into DNA damage signaling in bystander cells, with ATM activation occurring downstream of ATR. DNA-PK is not essential in mortality inducing in bystander cells neither for bystander γH2AX foci induction (Burdak-Rothkamm et al., 2007). These differences between bystander and direct DNA damage response offer new potential targets for repair inhibitors, with the aim to

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

protect bystander normal tissues during cancer radiotherapy.

**5. Conclusions** 

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

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.

We suggest that the short-lived ROS released in the medium by irradiated cells are responsible for DNA lesions, unlike double strand breaks, which activate H2AX phosphorylation but do not require the 53BP1 and NBS1p343 proteins to be repaired. It is possible that in our experiments DNA damage induced by CM treatment consisted of a few DSBs, the repair of which requires the recruitment of 53BP1 and NBS1p343 proteins and mainly in other types of DNA lesions, in which repair occurs without these proteins. Recent studies suggest that there are important differences between the DNA damage response in directly irradiated cells and non-targeted cells via bystander signals. The DNA damage in bystander cells seems to persist for a prolonged time (Burdak-Rothkamm et al., 2007), differently from DNA damage induced directly by irradiation which is repaired completely within few hours depending on radiation dose. Studies carried out in p53 wild-type (TK6), p53 null (NH32),and p53 mutant (WTK1) lymphoblastoid cells using siRNA to knockdown DNA PKcs demonstrated the central role of non-homologous end-joining in processing bystander damage, in contrast to the role of homologous recombination which seems to be essential only in inducing sister chromatid exchanges in bystander cells (Zhang et al., 2008). ATM- and Rad3-related (ATR) protein kinases have a central role into DNA damage signaling in bystander cells, with ATM activation occurring downstream of ATR. DNA-PK is not essential in mortality inducing in bystander cells neither for bystander γH2AX foci induction (Burdak-Rothkamm et al., 2007). These differences between bystander and direct DNA damage response offer new potential targets for repair inhibitors, with the aim to protect bystander normal tissues during cancer radiotherapy.
