**3. Cellular effects of ionizing radiation in human lymphocytes cultured in microgravity condition**

The cellular response to ionizing radiation besides on genetic and physiological features of the biological systems, depends also on environmental conditions occurring during DNA repair. Space missions expose humans to an exogenous environment not encountered within our biosphere, in particular the contemporary presence of radiation and a condition of weightlessness called microgravity (10-4–10-6g). One of the important aspects of risk estimation during space flights, is whether the effects of radiation on astronauts are influenced by microgravity. The combination of microgravity and ionizing radiation has been demonstrated to have a synergistic action on human cells, both *in vivo* and *in vitro*. The effects of space environment experienced by astronauts include loss of calcium and minerals from bone, decreased skeletal muscle mass and depressed immune function (Longnecker et al., 2004). *Ex vivo* astronaut studies, in-flight cell cultures, and ground models of microgravity studies, have consistently demonstrated inhibition of lymphocyte proliferation and suppressed or altered cytokine secretion (Lewis et al., 1998; Grimm et al., 2002). Among the biological effects of the reduced gravity in human cell cultures, were described apoptosis induction, cytoskeletal alteration, cell growth inhibition and increased frequency of chromosome aberrations (Lewis et al., 1998; Grimm et al., 2002; Cubano et al., 2000; Sytkowski et al., 2001; Mosesso et al., 2001; Durante et al., 2003). Gene expression analyses

Fig. 3. Kinetics of -H2AX foci in PBL irradiated with -rays and low energy protons during the time after irradiation. A) Fraction of cells positive for -H2AX foci and B) mean number

The lower number of foci detected in peripheral lymphocytes could depend on the large amount of heterochromatin of resting cells, from which -H2AX foci are mostly excluded (Cowell et al., 2007) as well as on the small nuclear volume, where overlapping foci are difficult to detect separately. Thus, in accordance with the observations of Scherthan et al., (2008) we hypothesize that -H2AX foci detected very early after irradiation contained more than one DSB; later on, the number of foci decreased and probably each foci contained only one DSB. Furthermore , we found a size increase of -H2AX foci in cells irradiated with protons, as compared with gamma irradiations, probably as a consequence of DSBs clusters induced by high-LET radiation. Our results are in accordance with those in melanoma cells

**3. Cellular effects of ionizing radiation in human lymphocytes cultured in** 

The cellular response to ionizing radiation besides on genetic and physiological features of the biological systems, depends also on environmental conditions occurring during DNA repair. Space missions expose humans to an exogenous environment not encountered within our biosphere, in particular the contemporary presence of radiation and a condition of weightlessness called microgravity (10-4–10-6g). One of the important aspects of risk estimation during space flights, is whether the effects of radiation on astronauts are influenced by microgravity. The combination of microgravity and ionizing radiation has been demonstrated to have a synergistic action on human cells, both *in vivo* and *in vitro*. The effects of space environment experienced by astronauts include loss of calcium and minerals from bone, decreased skeletal muscle mass and depressed immune function (Longnecker et al., 2004). *Ex vivo* astronaut studies, in-flight cell cultures, and ground models of microgravity studies, have consistently demonstrated inhibition of lymphocyte proliferation and suppressed or altered cytokine secretion (Lewis et al., 1998; Grimm et al., 2002). Among the biological effects of the reduced gravity in human cell cultures, were described apoptosis induction, cytoskeletal alteration, cell growth inhibition and increased frequency of chromosome aberrations (Lewis et al., 1998; Grimm et al., 2002; Cubano et al., 2000; Sytkowski et al., 2001; Mosesso et al., 2001; Durante et al., 2003). Gene expression analyses

exposed to low- and high-LET radiation (Ibañez et al., 2009).

of -H2AX foci per nucleus.

**microgravity condition** 

on human cells grown in microgravity during space flights or in modeled microgravity (MMG) on Earth, report changes among genes involved in apoptosis induction, cell adhesion, cytoskeletal features and cell differentiation, even if large differences in culture conditions, cell types and methods to simulate microgravity were adopted in those experiments (Hammond et al., 2000; Lewis et al., 2001, Torigoe et al., 2001, Infanger et al., 2007). While the genotoxic effects of ionizing radiation have been intensely studied, the consequence of the reduced gravity together with radiation is still unclear. Therefore, it is of special importance to verify whether DDR is affected by the combined effects of IR and microgravity, in view of the prolonged permanence of man in future space missions. To analyze the possibility that a reduced gravitational force impairs the DDR pathway, increasing the risk of the exposure to conditions occurring during spaceflight, we studied the DDR to ionizing radiation in human PBL incubated in MMG and in parallel static conditions. Microgravity was simulated by culturing PBL in the Rotating Wall Vessel bioreactor (Synthecon, Cellon, Fig. 4) placed inside a humidified incubator, vertically rotating at 23 rpm.

Fig. 4. Rotating Wall Vessel Bioreactor (Synthecon).

The Rotating Wall Vessel was developed at the NASA Johnson Space Center (Houston, TX) to simulate, as accurately as possible, culture conditions predicted to occur during experiments in space. In the rotating system, the gravity is balanced by equal and opposite mechanical forces (centrifugal, Coriolis and shear components), and the gravitational vector is reduced to about 10−2 g. In these conditions, single cells are nearly always in suspension, rotating quasi-stationary with the fluid, in a low-shear culture environment (Unsworth 1998, Maccarone et al., 2003). Ground based (1 g) PBL cultures, both irradiated and non-irradiated, were kept at the same cell density in flasks inside a humidified incubator for 24 h.

#### **3.1 The DNA-damage response of human peripheral lymphocytes cultured in microgravity after γ-irradiation**

The DNA-damage response was investigated in human PBL irradiated *in vitro* with different doses of gamma rays and incubated for 24 h in 1 g or in modeled microgravity (MMG). While cell survival was only slight affected by MMG, the *HPRT* mutant frequency significantly increased in PBL incubated in MMG after irradiation compared with those

The DNA-Damage Response to Ionizing Radiation in Human Lymphocytes 11

that the level of H2AX phosphorylation was principally correlated to a delayed DSB

We then tested for the possibility that MMG incubation affects DNA damage response by altering the recruitment of the signaling proteins, 53BP1, NBS1-p343 and ATM-p1981, which co-localize with -H2AX foci to DSB sites (Fig. 6A). After irradiation ∼90% of cells became foci-positive for the three proteins in both gravity conditions (not shown). In contrast to - H2AX, the fraction of foci-positive cells persisted high up to 24 h after irradiation in 1g and no differences between the two culture conditions were detected. The number of foci/nucleus significantly decreased during post-irradiation incubation from 14–16 foci/nucleus at 30 min to 4–5 foci/nucleus at 24 h (Fig. 6B), without differences between samples in 1g and MMG. The discrepancies with the kinetics of -H2AX foci suggest that these proteins could represent the remaining scaffold structure used for DSB repair that persisted after the repair has been completed (Markova et al., 2007, van Veelen et al., 2005).

Fig. 6. Kinetics of 53BP1, ATM-p1981, NBS1-p343 foci in PBL irradiated with -rays and incubated in 1g. A) Co-localization with -H2AX foci to form the ionizing radiation-induced

We analyzed the DNA damage response to radiation also in human tumoral lymphocytes (TK6 cells, lymphoblastoid B cells) irradiated with rays (1, 2, 4 Gy) and incubated in 1g or in MMG during the repair time. In irradiated TK6 cells, we observed a higher survival in MMG than in 1g, and the difference was significant at 4Gy. In addition, in cells maintained in MMG rather than in 1g after γ-irradiation, higher frequency of HPRT mutants was observed at all irradiation doses, particularly at 4Gy (Figure 7A). Remarkably, at this dose, mutant frequency may often be underestimated, since cells with many and severe mutations are unable to repair DNA damage and die. Instead, in TK6 cells cultured in MMG after irradiation, mutant frequency increased with doses up to 4Gy (Figure 7A). The frequency of micronucleated cells was measured in both gravity conditions after irradiation. At the end of

**3.2 The DNA-damage response of human tumoral lymphocytes cultured in** 

resolution rather than apoptosis induction.

foci (IRIF). B) Mean number of foci per nucleus.

**microgravity after γ-irradiation** 

maintained in 1 g. Given the increase of *HPRT* mutants in MMG, we investigated whether the reduced gravity affected the progression of the rejoining of double strand breaks (DSBs) in human PBL irradiated with -rays and incubated in MMG or in 1g. The kinetics of - H2AX foci was monitored during the repair incubation, showing that DSBs rejoining was slower in MMG than in 1g at 6 and 24 h after irradiation. In addition, the mean number of - H2AX foci per nucleus was significantly higher in MMG than in 1g at the same time-points (Fig. 5).

Fig. 5. Kinetics of -H2AX foci in PBL irradiated with 5Gy of -rays and incubated in 1g or MMG during the repair time. A) Fraction of PBL positive for -H2AX foci. B) Mean number of -H2AX foci/nucleus (\*\*\**P*<0.001, *t*-test).

To verify whether the disappearance of -H2AX foci correlated with the rejoining of double strand breaks, we subjected irradiated lymphocytes to a non-radioactive PFGE assay (Gradzka et al., 2005). The fraction of DNA released (FR) from the plug after PFGE was considered a measure of DSB level. The kinetics of DSB removal in lymphocytes irradiated and incubated in 1g exhibits a typical fast initial component and a decreasing rate at longer repair intervals, in accordance with data from other authors (Stenerlow et al., 2000; Gradzka et al., 2005). Both the methods we used to quantify DNA fragmentation, reported a lower rate of DSB rejoining in lymphocytes incubated in MMG compared to those in 1g, in agreement with the kinetics of -H2AX foci. Our results provide evidences that MMG incubation during DNA repair delayed the rate of radiation-induced DSB rejoining, and increased, as a consequence, the genotoxic effects of ionizing radiation.

We then assessed whether MMG incubation affected IR-induced apoptosis. Human lymphocytes, irradiated and non-irradiated, were scored for the presence of fragmented nuclei and apoptotic bodies. Apoptotic index (A.I.) increased with time after irradiation and at 24 h it was significantly higher in PBL incubated in MMG compared to those in 1g (19.3% vs. 13.7% respectively, *P* < 0.001). Since DSBs can be induced, besides radiation, also by DNA fragmentation during early apoptosis, we measured caspase-3 activation at the same time-points by the cleavage of the peptide substrate DEVD-AFC. Caspase-3 activation was only slightly higher in PBL maintained in MMG than in 1g, in contrast to the high persistence of foci-positive cells (*P* < 0.01), and foci number/nucleus (*P* < 0.001), suggesting

maintained in 1 g. Given the increase of *HPRT* mutants in MMG, we investigated whether the reduced gravity affected the progression of the rejoining of double strand breaks (DSBs) in human PBL irradiated with -rays and incubated in MMG or in 1g. The kinetics of - H2AX foci was monitored during the repair incubation, showing that DSBs rejoining was slower in MMG than in 1g at 6 and 24 h after irradiation. In addition, the mean number of - H2AX foci per nucleus was significantly higher in MMG than in 1g at the same time-points

Fig. 5. Kinetics of -H2AX foci in PBL irradiated with 5Gy of -rays and incubated in 1g or MMG during the repair time. A) Fraction of PBL positive for -H2AX foci. B) Mean number

To verify whether the disappearance of -H2AX foci correlated with the rejoining of double strand breaks, we subjected irradiated lymphocytes to a non-radioactive PFGE assay (Gradzka et al., 2005). The fraction of DNA released (FR) from the plug after PFGE was considered a measure of DSB level. The kinetics of DSB removal in lymphocytes irradiated and incubated in 1g exhibits a typical fast initial component and a decreasing rate at longer repair intervals, in accordance with data from other authors (Stenerlow et al., 2000; Gradzka et al., 2005). Both the methods we used to quantify DNA fragmentation, reported a lower rate of DSB rejoining in lymphocytes incubated in MMG compared to those in 1g, in agreement with the kinetics of -H2AX foci. Our results provide evidences that MMG incubation during DNA repair delayed the rate of radiation-induced DSB rejoining, and

We then assessed whether MMG incubation affected IR-induced apoptosis. Human lymphocytes, irradiated and non-irradiated, were scored for the presence of fragmented nuclei and apoptotic bodies. Apoptotic index (A.I.) increased with time after irradiation and at 24 h it was significantly higher in PBL incubated in MMG compared to those in 1g (19.3% vs. 13.7% respectively, *P* < 0.001). Since DSBs can be induced, besides radiation, also by DNA fragmentation during early apoptosis, we measured caspase-3 activation at the same time-points by the cleavage of the peptide substrate DEVD-AFC. Caspase-3 activation was only slightly higher in PBL maintained in MMG than in 1g, in contrast to the high persistence of foci-positive cells (*P* < 0.01), and foci number/nucleus (*P* < 0.001), suggesting

increased, as a consequence, the genotoxic effects of ionizing radiation.

of -H2AX foci/nucleus (\*\*\**P*<0.001, *t*-test).

(Fig. 5).

that the level of H2AX phosphorylation was principally correlated to a delayed DSB resolution rather than apoptosis induction.

We then tested for the possibility that MMG incubation affects DNA damage response by altering the recruitment of the signaling proteins, 53BP1, NBS1-p343 and ATM-p1981, which co-localize with -H2AX foci to DSB sites (Fig. 6A). After irradiation ∼90% of cells became foci-positive for the three proteins in both gravity conditions (not shown). In contrast to - H2AX, the fraction of foci-positive cells persisted high up to 24 h after irradiation in 1g and no differences between the two culture conditions were detected. The number of foci/nucleus significantly decreased during post-irradiation incubation from 14–16 foci/nucleus at 30 min to 4–5 foci/nucleus at 24 h (Fig. 6B), without differences between samples in 1g and MMG. The discrepancies with the kinetics of -H2AX foci suggest that these proteins could represent the remaining scaffold structure used for DSB repair that persisted after the repair has been completed (Markova et al., 2007, van Veelen et al., 2005).

Fig. 6. Kinetics of 53BP1, ATM-p1981, NBS1-p343 foci in PBL irradiated with -rays and incubated in 1g. A) Co-localization with -H2AX foci to form the ionizing radiation-induced foci (IRIF). B) Mean number of foci per nucleus.

#### **3.2 The DNA-damage response of human tumoral lymphocytes cultured in microgravity after γ-irradiation**

We analyzed the DNA damage response to radiation also in human tumoral lymphocytes (TK6 cells, lymphoblastoid B cells) irradiated with rays (1, 2, 4 Gy) and incubated in 1g or in MMG during the repair time. In irradiated TK6 cells, we observed a higher survival in MMG than in 1g, and the difference was significant at 4Gy. In addition, in cells maintained in MMG rather than in 1g after γ-irradiation, higher frequency of HPRT mutants was observed at all irradiation doses, particularly at 4Gy (Figure 7A). Remarkably, at this dose, mutant frequency may often be underestimated, since cells with many and severe mutations are unable to repair DNA damage and die. Instead, in TK6 cells cultured in MMG after irradiation, mutant frequency increased with doses up to 4Gy (Figure 7A). The frequency of micronucleated cells was measured in both gravity conditions after irradiation. At the end of

The DNA-Damage Response to Ionizing Radiation in Human Lymphocytes 13

Fig. 8. Cell cycle distribution of irradiated and non-irradiated TK6 cells at the end of 24h

al., 1998; Hughes-Fulford 2001; Kita et al., 2000), which in turn may explain the results reported here. It remains to be determined if one upstream or several downstream genes belonging to the pathway of the radiation response are involved in the effects induced by

**3.3 Gene expression changes in human lymphocytes cultured in microgravity during** 

Gene expression changes represent an early bio-indicator of radiation exposure. Given the increase of *HPRT* mutants observed in human lymphocytes incubated in modeled microgravity, we investigated whether this gravity condition can alter the transcription of 14 genes representative of the main DNA repair pathways. The genes analyzed are representative of the major DNA repair pathways: four genes (*Ku70*, *Ku80*, *DNA-ligase IV*, *XRCC4*) are involved in non-homologous end joining processes (NHEJ), three genes (*BRCA1*, *BRCA2*, *RAD51*) in homologous recombination (HR), four genes (*XRCC1*, *PCNA*,*GADD45A*, *p21Cip1/Waf1*) in base excision repair (BER) and two genes (*DDB2*, *XPC*) in nucleotide excision repair (NER). *DNA-ligase I*, involved in both BER and NER repair pathways, was analyzed too. Analyses were carried out in three pools of three donor, each by quantitative real time PCR. Results show that almost all BER and NER genes were up-regulated in irradiated PBL, whereas the expression of HR and NHEJ genes was only slightly or not affected by radiation (Fig.9). Incubation in modeled microgravity after irradiation did not significantly change the expression of genes involved in DNA repair, suggesting that transcriptional impairment was not responsible for the increase of mutant frequency observed in irradiated cells incubated in microgravity in comparison to the static 1 g condition. These findings in agreement with previous studies on gene expression of non-irradiated space flown and RWV cultured cells, showing that DNA repair genes were unaffected by low-gravity whereas intracellular signaling, growth regulatory, cytoskeletal and tumor suppressor genes were altered (Lewis et

incubation in 1g or in MMG.

**the DNA-damage response to radiation** 

al., 2001; Hammond et al., 2000; Pardo et al., 2005).

MMG incubation.

post-irradiation incubation (24 h time-point), the percentage of micronuclei (MN) was significantly higher in both non-irradiated and in irradiated cells incubated in MMG compared with 1g (Fig.7B). Eighteen hours later (42 h from irradiation), the percentage of MN in cultures incubated in MMG was higher than in 1g only at 2Gy γ-ray dose. At 48 h time-point, MN frequencies observed in 1g or MMG were comparable. As expected, MN significantly increased after irradiation in both gravity conditions with respect to nonirradiated cells; a significant difference was still observed at 48 h after irradiation at both 1 and 2Gy. The significant increase of micronucleated cells in MMG suggested that MMG itself was able to induce chromosome damage.

Fig. 7. A) Mutant frequency at the HPRT locus of irradiated and non-irradiated TK6 cells incubated for 24h in 1g or in modeled microgravity. B) Micronucleus frequencies (%) in irradiated and non-irradiated TK6 cells incubated in 1g of MMG for the first 24h after irradiation and then cultured in 1g up to 48 h. \**P*<0.05; \*\**P*<0.01; \*\*\**P*<0.001 (*G* test).

The effect of MMG incubation on cell cycle alteration induced by γ−ray exposure was assessed by flow cytometry analysis. Figure 8 shows the cell cycle distribution of TK6 cells at various time-points from irradiation and incubation in MMG or 1g by representative DNA histograms.-ray irradiation induced an increase in G2/M-phase cells and a reduction in S-phase cells, both in TK6 maintained in 1g and MMG after irradiation. At the end of MMG or 1g incubation (24 h time-point), the percentages of cells in G1-phase were higher in cultures irradiated with 2-4 Gy and incubated in MMG compared with cells maintained in 1g. Moreover, the G2/M block after irradiation was less evident in MMG than in 1g condition. Also radiation-induced apoptosis was affected in TK6 cells by MMG incubation. Induction of apoptosis was significantly lower in irradiated TK6 cells incubated in MMG compared with cells irradiated with the same dose and incubated in 1g. The differences were more pronounced in cells analyzed at long post-incubation times (72 h time-point).

The observed decrease of apoptotic response in MMG incubated cultures could allow severely damaged cells, which in 1g condition should be eliminated by selection, to survive, with negative consequences on genomic integrity. Alterations in cell response to ionizing radiation due to MMG incubation during the DNA repair period may be caused by the reduced activity of some proteins, which play a crucial role in damage signaling. Previous data have shown that absence or reduction of gravity can alter gene expression (Walther et

post-irradiation incubation (24 h time-point), the percentage of micronuclei (MN) was significantly higher in both non-irradiated and in irradiated cells incubated in MMG compared with 1g (Fig.7B). Eighteen hours later (42 h from irradiation), the percentage of MN in cultures incubated in MMG was higher than in 1g only at 2Gy γ-ray dose. At 48 h time-point, MN frequencies observed in 1g or MMG were comparable. As expected, MN significantly increased after irradiation in both gravity conditions with respect to nonirradiated cells; a significant difference was still observed at 48 h after irradiation at both 1 and 2Gy. The significant increase of micronucleated cells in MMG suggested that MMG

Fig. 7. A) Mutant frequency at the HPRT locus of irradiated and non-irradiated TK6 cells incubated for 24h in 1g or in modeled microgravity. B) Micronucleus frequencies (%) in irradiated and non-irradiated TK6 cells incubated in 1g of MMG for the first 24h after irradiation and then cultured in 1g up to 48 h. \**P*<0.05; \*\**P*<0.01; \*\*\**P*<0.001 (*G* test).

The effect of MMG incubation on cell cycle alteration induced by γ−ray exposure was assessed by flow cytometry analysis. Figure 8 shows the cell cycle distribution of TK6 cells at various time-points from irradiation and incubation in MMG or 1g by representative DNA histograms.-ray irradiation induced an increase in G2/M-phase cells and a reduction in S-phase cells, both in TK6 maintained in 1g and MMG after irradiation. At the end of MMG or 1g incubation (24 h time-point), the percentages of cells in G1-phase were higher in cultures irradiated with 2-4 Gy and incubated in MMG compared with cells maintained in 1g. Moreover, the G2/M block after irradiation was less evident in MMG than in 1g condition. Also radiation-induced apoptosis was affected in TK6 cells by MMG incubation. Induction of apoptosis was significantly lower in irradiated TK6 cells incubated in MMG compared with cells irradiated with the same dose and incubated in 1g. The differences were more pronounced in cells analyzed at long post-incubation times (72 h time-point). The observed decrease of apoptotic response in MMG incubated cultures could allow severely damaged cells, which in 1g condition should be eliminated by selection, to survive, with negative consequences on genomic integrity. Alterations in cell response to ionizing radiation due to MMG incubation during the DNA repair period may be caused by the reduced activity of some proteins, which play a crucial role in damage signaling. Previous data have shown that absence or reduction of gravity can alter gene expression (Walther et

itself was able to induce chromosome damage.

Fig. 8. Cell cycle distribution of irradiated and non-irradiated TK6 cells at the end of 24h incubation in 1g or in MMG.

al., 1998; Hughes-Fulford 2001; Kita et al., 2000), which in turn may explain the results reported here. It remains to be determined if one upstream or several downstream genes belonging to the pathway of the radiation response are involved in the effects induced by MMG incubation.

#### **3.3 Gene expression changes in human lymphocytes cultured in microgravity during the DNA-damage response to radiation**

Gene expression changes represent an early bio-indicator of radiation exposure. Given the increase of *HPRT* mutants observed in human lymphocytes incubated in modeled microgravity, we investigated whether this gravity condition can alter the transcription of 14 genes representative of the main DNA repair pathways. The genes analyzed are representative of the major DNA repair pathways: four genes (*Ku70*, *Ku80*, *DNA-ligase IV*, *XRCC4*) are involved in non-homologous end joining processes (NHEJ), three genes (*BRCA1*, *BRCA2*, *RAD51*) in homologous recombination (HR), four genes (*XRCC1*, *PCNA*,*GADD45A*, *p21Cip1/Waf1*) in base excision repair (BER) and two genes (*DDB2*, *XPC*) in nucleotide excision repair (NER). *DNA-ligase I*, involved in both BER and NER repair pathways, was analyzed too. Analyses were carried out in three pools of three donor, each by quantitative real time PCR. Results show that almost all BER and NER genes were up-regulated in irradiated PBL, whereas the expression of HR and NHEJ genes was only slightly or not affected by radiation (Fig.9). Incubation in modeled microgravity after irradiation did not significantly change the expression of genes involved in DNA repair, suggesting that transcriptional impairment was not responsible for the increase of mutant frequency observed in irradiated cells incubated in microgravity in comparison to the static 1 g condition. These findings in agreement with previous studies on gene expression of non-irradiated space flown and RWV cultured cells, showing that DNA repair genes were unaffected by low-gravity whereas intracellular signaling, growth regulatory, cytoskeletal and tumor suppressor genes were altered (Lewis et al., 2001; Hammond et al., 2000; Pardo et al., 2005).

The DNA-Damage Response to Ionizing Radiation in Human Lymphocytes 15

dendrogram relative to some miRNAs differentially expressed following ionizing radiation

Fig. 10. A) Dendrogram showing several miRNAs differentially expressed in human PBL at 4 and 24h after irradiation with 0.2Gy. Range of expression value is determined as the log2 ratio of irradiated/non-irradiated sample. Down-regulated and up-regulated miRNAs correspond to green and red boxes, respectively. B) Fraction of radio-responsive miRNAs (%) in human PBL irradiated with 0.2 and 2Gy and incubated for 4 and 24h- in 1g or in

MiRNA expression profile was carried out at 4h and 24h after irradiation with 0.2Gy and 2Gy and incubation in 1g and MMG and compared to that of non-irradiated PBL maintained in parallel conditions. Results showed that in both gravity conditions the miRNA expression profile was dose-specific, as indicated by the low percentage of common miRNA responsive to both doses; moreover, the effects of the higher dose predominated at the late time point. Interestingly, MMG tended to decrease the number of radio-responsive

To predict the target genes of differentially expressed miRNAs we first performed a computational analyses using PITA algorithm available on line (Kertesz et al., 2007). However, all available software for target prediction are characterized by a large fraction of false positives, thus to identify the most likely targets, we have integrated mRNA and miRNA expression data, obtained on the same lymphocyte samples, using MAGIA (MiRNA And Genes Integrated Analysis) web tool (Sales et al., 2010). We used a non-parametric index (Spearman correlation coefficient), the most indicated statistical coefficient for a small number of measures, to estimate the degree of anti-correlation (e.g. up-regulated miRNA and corresponding down-regulated mRNA target) between any putative pairs of miRNA and mRNA (Xin et al., 2009; Wang and Li 2009). The anti-correlated transcripts were then classified according to DAVID (Database for Annotation, Visualization and Integrated Discovery) web tool (Huang et al. 2009), to determine which Gene Ontology (GO) terms were significantly enriched in our set of genes. Results of G0 analysis of anti-correlated

miRNAs respect to 1g condition, in particular at 24h after irradiation (Figure 10B).

in human PBL.

modeled microgravity (MMG).

Fig. 9. Expression ratios in PBL of pools B–D incubated in 1 g and modeled microgravity after X-irradiation. (A) *R* values of BER and NER genes in 1g; (A) *R* values of BER and NER in MMG; (B) *R* values of HR and NHEJ genes in 1g; (B) *R* values of HR and NHEJ genes in MMG.

Recently, a new class of important gene modulators has been discovered: microRNAs. They are a large family of small non-coding RNAs of 18-24 nucleotides that negatively regulate gene expression levels by binding to microRNA-binding elements in the 3' untranslatedregion (3'UTR) of target mRNAs thereby triggering decreased protein translation mainly through mRNA degradation (Guo et al., 2010). A single miRNA may have broad effects on gene expression networks, such as regulating cell lineage specificity, cellular functions or stress response. By considering the complexity of the DNA-damage response (DDR), addressed to maintain genome integrity through cell cycle arrest, DNA repair and/or apoptosis, it is expected that miRNAs have an important role in this cellular process. Whilst miRNA-mediated DDR has been studied after UV radiation and hypoxic stress (Pothof et al., 2009; Crosby et al., 2009) that of radiation combined with microgravity has not been studied yet and should give important information about risk assessment in space environment. MicroRNAs profiling were carried out by using the platform "Human miRNA Microarray kit (V2)" (Agilent), according to the Agilent miRNA protocol. For mRNA expression profile we used the "Whole Human Genome Oligo Microarray" (Agilent), consisting of ~41.000 (60-mer) oligonucleotide probes, which span conserved exons across the transcripts of the targeted full-length genes. Identification of differentially expressed genes and miRNAs was performed with one and two class Significance Analysis of Microarray (SAM) program (Tusher et al., 2001) with default settings. Figure 10A shows a

Fig. 9. Expression ratios in PBL of pools B–D incubated in 1 g and modeled microgravity after X-irradiation. (A) *R* values of BER and NER genes in 1g; (A) *R* values of BER and NER in MMG; (B) *R* values of HR and NHEJ genes in 1g; (B) *R* values of HR and NHEJ genes in

Recently, a new class of important gene modulators has been discovered: microRNAs. They are a large family of small non-coding RNAs of 18-24 nucleotides that negatively regulate gene expression levels by binding to microRNA-binding elements in the 3' untranslatedregion (3'UTR) of target mRNAs thereby triggering decreased protein translation mainly through mRNA degradation (Guo et al., 2010). A single miRNA may have broad effects on gene expression networks, such as regulating cell lineage specificity, cellular functions or stress response. By considering the complexity of the DNA-damage response (DDR), addressed to maintain genome integrity through cell cycle arrest, DNA repair and/or apoptosis, it is expected that miRNAs have an important role in this cellular process. Whilst miRNA-mediated DDR has been studied after UV radiation and hypoxic stress (Pothof et al., 2009; Crosby et al., 2009) that of radiation combined with microgravity has not been studied yet and should give important information about risk assessment in space environment. MicroRNAs profiling were carried out by using the platform "Human miRNA Microarray kit (V2)" (Agilent), according to the Agilent miRNA protocol. For mRNA expression profile we used the "Whole Human Genome Oligo Microarray" (Agilent), consisting of ~41.000 (60-mer) oligonucleotide probes, which span conserved exons across the transcripts of the targeted full-length genes. Identification of differentially expressed genes and miRNAs was performed with one and two class Significance Analysis of Microarray (SAM) program (Tusher et al., 2001) with default settings. Figure 10A shows a

MMG.

dendrogram relative to some miRNAs differentially expressed following ionizing radiation in human PBL.

Fig. 10. A) Dendrogram showing several miRNAs differentially expressed in human PBL at 4 and 24h after irradiation with 0.2Gy. Range of expression value is determined as the log2 ratio of irradiated/non-irradiated sample. Down-regulated and up-regulated miRNAs correspond to green and red boxes, respectively. B) Fraction of radio-responsive miRNAs (%) in human PBL irradiated with 0.2 and 2Gy and incubated for 4 and 24h- in 1g or in modeled microgravity (MMG).

MiRNA expression profile was carried out at 4h and 24h after irradiation with 0.2Gy and 2Gy and incubation in 1g and MMG and compared to that of non-irradiated PBL maintained in parallel conditions. Results showed that in both gravity conditions the miRNA expression profile was dose-specific, as indicated by the low percentage of common miRNA responsive to both doses; moreover, the effects of the higher dose predominated at the late time point. Interestingly, MMG tended to decrease the number of radio-responsive miRNAs respect to 1g condition, in particular at 24h after irradiation (Figure 10B).

To predict the target genes of differentially expressed miRNAs we first performed a computational analyses using PITA algorithm available on line (Kertesz et al., 2007). However, all available software for target prediction are characterized by a large fraction of false positives, thus to identify the most likely targets, we have integrated mRNA and miRNA expression data, obtained on the same lymphocyte samples, using MAGIA (MiRNA And Genes Integrated Analysis) web tool (Sales et al., 2010). We used a non-parametric index (Spearman correlation coefficient), the most indicated statistical coefficient for a small number of measures, to estimate the degree of anti-correlation (e.g. up-regulated miRNA and corresponding down-regulated mRNA target) between any putative pairs of miRNA and mRNA (Xin et al., 2009; Wang and Li 2009). The anti-correlated transcripts were then classified according to DAVID (Database for Annotation, Visualization and Integrated Discovery) web tool (Huang et al. 2009), to determine which Gene Ontology (GO) terms were significantly enriched in our set of genes. Results of G0 analysis of anti-correlated

The DNA-Damage Response to Ionizing Radiation in Human Lymphocytes 17

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

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

**ionizing radiation** 

genes showed that in MMG-incubated PBL were not enriched the categories of response to stress, to DNA damage and to apoptosis. miRNA-mRNA anti-correlations of DDR pathway were visualized by using Cytoscape software package (Shannon et al., 2003; Cline et al., 2007) (Figure 11). The results showed that, in most cases, the same mRNA was targeted by different miRNA species according to the different condition of gravity.

Future research is addressed to validate several of the anti-correlations highlighted with our analyses as important in DDR pathway. In particular, we will perform a functional assay to demonstrate the regulatory effect of a particular miRNA on its putative target mRNA. The luciferase assay represents the most efficient approach to evaluate the activity of a miRNA on its anti-correlated mRNA. This assay allows to demonstrate the activity of a miRNA on its anti-correlated mRNA by the quantification of the luminescent signal derived from the luciferase reporter enzyme. Cells are co-transfected with a reporter vector containing the firefly luciferase gene together with the 3'UTR target sequence predicted for that miRNA and the miRNA precursor (pre-miRNA) or inhibitor (anti-miRNA), which respectively mimics and inhibits the endogenous miRNA. The binding of pre-miRNA to the complementary target sequence will cause the repression of luciferase gene expression, whereas the binding of anti-miRNA to the endogenous miRNA will induce the expression of luciferase gene. The quantification of the luminescent signal derived from the luciferase reporter enzyme thus allows to demonstrate the activity of a miRNA on its putative target mRNA. In addition to the luciferase assay, it would be interesting to study the role of selected miRNAs in DDR pathway by a biological approach. Usually, several end points such as cell survival, DNA repair, cell cycle progression and apoptosis induction are analyzed in cells over-or under-expressing the miRNA of interest.

Fig. 11. Example of visualization of inversely correlated miRNA-mRNA relationships in irradiated human PBL. Circles represent transcripts and triangles miRNAs, shown with the color corresponding to the expression value.

genes showed that in MMG-incubated PBL were not enriched the categories of response to stress, to DNA damage and to apoptosis. miRNA-mRNA anti-correlations of DDR pathway were visualized by using Cytoscape software package (Shannon et al., 2003; Cline et al., 2007) (Figure 11). The results showed that, in most cases, the same mRNA was targeted by

Future research is addressed to validate several of the anti-correlations highlighted with our analyses as important in DDR pathway. In particular, we will perform a functional assay to demonstrate the regulatory effect of a particular miRNA on its putative target mRNA. The luciferase assay represents the most efficient approach to evaluate the activity of a miRNA on its anti-correlated mRNA. This assay allows to demonstrate the activity of a miRNA on its anti-correlated mRNA by the quantification of the luminescent signal derived from the luciferase reporter enzyme. Cells are co-transfected with a reporter vector containing the firefly luciferase gene together with the 3'UTR target sequence predicted for that miRNA and the miRNA precursor (pre-miRNA) or inhibitor (anti-miRNA), which respectively mimics and inhibits the endogenous miRNA. The binding of pre-miRNA to the complementary target sequence will cause the repression of luciferase gene expression, whereas the binding of anti-miRNA to the endogenous miRNA will induce the expression of luciferase gene. The quantification of the luminescent signal derived from the luciferase reporter enzyme thus allows to demonstrate the activity of a miRNA on its putative target mRNA. In addition to the luciferase assay, it would be interesting to study the role of selected miRNAs in DDR pathway by a biological approach. Usually, several end points such as cell survival, DNA repair, cell cycle progression and apoptosis induction are

different miRNA species according to the different condition of gravity.

analyzed in cells over-or under-expressing the miRNA of interest.

Fig. 11. Example of visualization of inversely correlated miRNA-mRNA relationships in irradiated human PBL. Circles represent transcripts and triangles miRNAs, shown with the

color corresponding to the expression value.
