**4. Induction of delayed phenotypes in DNA repair-deficient cells**

#### **4.1 Methods for detection of delayed phenotypes**

Since radiation-induced genomic instability is manifested as the expression of delayed effects in the progeny of surviving cells, most of the study isolated colonies formed by the cells surviving X-irradiation. The primary colonies were cloned 10 days after irradiation. The cells obtained from each colony have already passed 15 to 20 population doublings. Then, the primary clones were subjected to the secondary colony formation. After 10 day, the secondary clones were isolated, and the cells were at 30 to 35 population doubling levels after irradiation. Delayed reproductive death was examined by colony-forming ability. Colonies derived from the surviving cells often contain giant cells, which occupied an area in the colony several times greater than the rest of the cells. Colonies containing at least one giant cell were judged as giant cell-positive colony. Delayed chromosomal bridges were detected between two dividing daughter nuclei in the anaphase cells in the surviving colonies. Delayed chromosomal aberrations were examined in metaphase cells derived from the primary and the secondary clones. Both chromatid- and chromosome-type aberrations were analyzed, and total 200 metaphases were counted per each sample.

Delayed induction of DNA double strand breaks was determined by 53BP1 foci by immunofluorescence. 53BP1 is a protein, which was originally discovered as a p53-binding protein. Lately, it turns to be clear that 53BP1 is a critical component of DNA damage signalling pathway (Polo and Jackson, 2011). ATM-dependent DNA damage checkpoint plays a central role in protecting integrity of the genome (Kastan and Bartek, 2004, Lavin, 2008, Shiloh and Kastan, 2001, Shiloh, 2003). ATM, which forms dimer or oligomer in the control cells, dissociates into monomer through autophosphorylation at serine 1981 in response to ionizing radiation (Bakkenist and Kastan, 2003). Activated ATM transduces DNA damage signal through phosphorylation of down-stream factors, including histone H2AX, MDC1 and 53BP1 (Ciccia and Elledge, 2010, Kastan and Lim, 2000)(Figure 3), and DNA damage signal is amplified through the formation of ionizing radiation-induced foci

Involvement of Non-Homologous End-Joining in Radiation-Induced Genomic Instability 163

Fig. 4. Delayed reproductive death at 30-35 population doublings post-irradiation.

Fig. 5. Delayed giant cell formation at 30-35 population doublings post-irradiation.

Fig. 6. Delayed chromosome bridge (right) at 30-35 population doublings post-irradiation. In order to confirm that defective induction of these delayed phenotypes are caused by the simple Ku80-deficiency, the human Ku86 gene was introduced into xrs-5 cells. Expression of human KU80 protein was confirmed by western blotting, and sufficient amount of KU80 protein was detected in the complimented cells. Accordingly, it was confirmed that complementation of the defective Ku80 function in xrs-5 cells simultaneously restored delayed reproductive death, giant cell formation and delayed chromosomal bridge formation to the levels observed in CHO cells. Thus, the results clearly indicated that Ku80 dependent rejoining is involved in the manifestation of delayed phenotypes in the progenies

of X-ray-surviving cells.

(Bonner et al., 2008). Activated ATM also phosphorylates p53 and CHK2/Cds1, which result in an induction of apoptosis as well as cell cycle arrest. Furthermore, ATM phosphorylates MRE11, NBS1, and Artemis, by which DNA repair ability may be regulated. Previously, it was demonstrated that the number of DNA double-strand breaks and the number of foci are well correlated, and since then, ionizing radiation-induced foci have been treated as a trustable marker for DNA double-strand breaks (Bonner et al., 2008). Foci of phosphorylated histone H2AX at serine 139 were the first ones to be used as DNA damage marker (Paull et al., 2000). Subsequently, foci of phosphorylated ATM at serine 1981 were demonstrated to illustrate low level of DNA double-strand breaks (Suzuki et al., 2006). Compared to these foci, 53BP1 foci create larger discrete foci, which can be detected even in tissue specimens (Nakashima et al., 2008). Thus, 53BP1 foci have been treated as DNA damage marker.

Fig. 3. ATM-dependent DNA damage checkpoint signalling.

53BP1 foci were detected by a specific antibody against the human 53BP1 protein. The primary antibody was visualized by Alexa594-labelled anti-rabbit IgG antibody. The nuclei were counterstained with DAPI. The samples were examined with an Olympus fluorescence microscope. Digital images were captured by a cooled CCD camera, and the images were analyzed by image analysis software.

### **4.2 Manifestation of delayed phenotypes**

Radiation-induced genomic instability needs to be examined at the same survival levels, so that 8 Gy and 10 Gy of X-rays were irradiated to CHO, while 2 and 4 Gy of X-rays were exposed to xrs-5 and xrs-6 cells, respectively. The primary clones were isolated, and they were subjected to the secondary colony formation. As shown in Figure 4, colony-forming ability is significantly reduced in CHO cells derived from surviving clones compared with those derived from the control clones. In contrast, no such difference is observed in both xrs-5 and xrs-6 cells, indicating that delayed reproductive death was completely absent in both xrs-5 and xrs-6 cells. In CHO cells, the frequency of giant cells and chromosomal bridges is also increased only in surviving clones, whereas, delayed induction of these phenotypes was completely abrogated in xrs-5 and xrs-6 cells (Figures 5 and 6).

(Bonner et al., 2008). Activated ATM also phosphorylates p53 and CHK2/Cds1, which result in an induction of apoptosis as well as cell cycle arrest. Furthermore, ATM phosphorylates MRE11, NBS1, and Artemis, by which DNA repair ability may be regulated. Previously, it was demonstrated that the number of DNA double-strand breaks and the number of foci are well correlated, and since then, ionizing radiation-induced foci have been treated as a trustable marker for DNA double-strand breaks (Bonner et al., 2008). Foci of phosphorylated histone H2AX at serine 139 were the first ones to be used as DNA damage marker (Paull et al., 2000). Subsequently, foci of phosphorylated ATM at serine 1981 were demonstrated to illustrate low level of DNA double-strand breaks (Suzuki et al., 2006). Compared to these foci, 53BP1 foci create larger discrete foci, which can be detected even in tissue specimens (Nakashima et al., 2008). Thus, 53BP1 foci have been treated as DNA damage marker.

Fig. 3. ATM-dependent DNA damage checkpoint signalling.

completely abrogated in xrs-5 and xrs-6 cells (Figures 5 and 6).

analyzed by image analysis software.

**4.2 Manifestation of delayed phenotypes** 

53BP1 foci were detected by a specific antibody against the human 53BP1 protein. The primary antibody was visualized by Alexa594-labelled anti-rabbit IgG antibody. The nuclei were counterstained with DAPI. The samples were examined with an Olympus fluorescence microscope. Digital images were captured by a cooled CCD camera, and the images were

Radiation-induced genomic instability needs to be examined at the same survival levels, so that 8 Gy and 10 Gy of X-rays were irradiated to CHO, while 2 and 4 Gy of X-rays were exposed to xrs-5 and xrs-6 cells, respectively. The primary clones were isolated, and they were subjected to the secondary colony formation. As shown in Figure 4, colony-forming ability is significantly reduced in CHO cells derived from surviving clones compared with those derived from the control clones. In contrast, no such difference is observed in both xrs-5 and xrs-6 cells, indicating that delayed reproductive death was completely absent in both xrs-5 and xrs-6 cells. In CHO cells, the frequency of giant cells and chromosomal bridges is also increased only in surviving clones, whereas, delayed induction of these phenotypes was

Fig. 4. Delayed reproductive death at 30-35 population doublings post-irradiation.

Fig. 5. Delayed giant cell formation at 30-35 population doublings post-irradiation.

Fig. 6. Delayed chromosome bridge (right) at 30-35 population doublings post-irradiation.

In order to confirm that defective induction of these delayed phenotypes are caused by the simple Ku80-deficiency, the human Ku86 gene was introduced into xrs-5 cells. Expression of human KU80 protein was confirmed by western blotting, and sufficient amount of KU80 protein was detected in the complimented cells. Accordingly, it was confirmed that complementation of the defective Ku80 function in xrs-5 cells simultaneously restored delayed reproductive death, giant cell formation and delayed chromosomal bridge formation to the levels observed in CHO cells. Thus, the results clearly indicated that Ku80 dependent rejoining is involved in the manifestation of delayed phenotypes in the progenies of X-ray-surviving cells.

Involvement of Non-Homologous End-Joining in Radiation-Induced Genomic Instability 165

Although the number of spontaneous foci per cell in the foci-positive cells is 1, we found multiple numbers of foci in not a small numbers of surviving cells. Therefore, it is possible that mis-rejoining of delayed DNA double-strand breaks results in delayed chromosomal instability. We tested this possibility by examining delayed induction of dicentric

Fig. 8. Delayed chromosomal instability at 30-35 population doublings post-irradiation.

**5. Role of non-homologous end-joining on radiation-induced genomic** 

Radiation-induced genomic instability has been reported commonly in various cell systems including human and rodent cells (Little, 2003, Lorimore et al., 2003, Morgan et al., 1996, Suzuki et al., 2003). However, Chang and Little demonstrated that delayed reproductive death, one characteristic manifestation of radiation-induced genomic instability, was not observed in Ku80-deficient xrs5 cells (Chang and Little, 1992a). The authors suggested that the cellular processing of DNA double strand breaks during repair must play a role in delayed reproductive death. In fact, our current study confirmed their results and found that not only delayed cell death but also delayed induction of giant cells and chromosome bridge were absent in xrs-5 cells (Figures 5 and 6). Furthermore, other Ku80-deficient cell line, xrs6, also revealed deficiency in the induction of those delayed phenotypes. Thus, it becomes clear that Ku80-dependent non-homologous end-joining is involved in the

increased dicentric frequencies in surviving cells were observed.

**instability** 

We found that delayed formation of dicentric chromosomes was significantly increased in CHO cells. Whereas, it is absent in xrs5 and xrs6 cells, while the control levels of dicentric chromosome were comparable among those cells. To confirm whether defective induction of dicentric chromosomes is related to Ku80-deficiency, delayed chromosomal instability was examined in the complimented xrs5 cells. We observed that delayed induction of dicentric chromosomes was increased to the level observed in CHO cells. Delayed chromosomal instability was also examined in cells derived from DNA-PKcs-defective *scid* mouse (Figure 8). Cells were irradiated with equivalent 10% survival doses and delayed induction of dicentric chromosomes was analyzed 30-35 PDL post-irradiation. The frequency of dicentric chromosomes in the unirradiated *scid* was higher than that in the wild-type cells, and

**4.4 Mis-rejoining of delayed DNA double-strand breaks** 

chromosomes at 30-35 PDL post irradiation (Figure 8).

#### **4.3 Delayed induction of DNA double-strand breaks**

It is possible that delayed phenotypes are caused by delayed DNA double strand breaks and Ku80-dependent mis-rejoining. Delayed induction of DNA double strand breaks were examined by 53BP1 foci in cells at 30-35 PDL post-irradiation (Figure 7). Since the primary antibody against 53BP1 is visualized by the secondary antibody labeled with Alexa594, 53BP1 foci are detected as discrete large red dots within the blue nuclei. Upon X-irradiation, the foci of DNA damage checkpoint factors become detectable within a few minutes after exposure. At 1 to 2 hours after X-irradiation, the initial foci are detectable in all exposed cells. Many small foci, most of which diameter distributed between 0.4 and 1.0 micrometer, were observed. The number of foci is decreased thereafter, indicating DNA repair. While the number of the initial foci reduced significantly during the first 6 to 10 hours, some fraction of the initial foci is remained as residual foci. Diameter of these residual foci increased timedependently, and they are quite large in size after 24 hours (Yamauchi et al., 2008). The number of foci shows no significant change thereafter, however, the cells with large residual foci are diluted out, when the foci-negative cells dominate the population. Thus, the background foci is rarely detectable even in the cells surviving 10 Gy of X-rays. While the frequency of 53BP1 foci per cell in the control CHO cells was approximately 0.14 ± 0.07, it was increased to 0.41 ± 0.19 in 10 Gy-surviving cells. The frequency of 53BP1 foci in the unirradiated xrs5 cells were relatively higher (0.18 ± 0.08) compared to the CHO cells, and it was about 0.51 ± 0.27 in 4 Gy-surviving cells.

Previously, a few studies including ours have suggested that delayed DNA double strand breaks are induced several generations after the initial insult (Barber et al., 2006, Huang et al., 2007, Suzuki et al., 2003), which has been proven by examining the delayed induction of foci of DNA damage checkpoint factors, such as phosphorylated histone H2AX. While phosphorylated histone H2AX foci are frequently used as biochemical markers for DNA double strand breaks, the foci of other DNA damage checkpoint factors, such as phosphorylated ATM foci and 53BP1 foci, are colocalized with phosphorylated histone H2AX foci, and they could also be used as alternative markers for DNA damage. In the present study, we demonstrated that the frequency of 53BP1 foci was higher in the progenies of surviving cells compared to unirradiated cells. Thus, it is confirmed that delayed induction of DNA double strand breaks in the progeny of surviving cells associated with pleiotropic manifestation of radiation-induced genomic instability.
