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

164 Selected Topics in DNA Repair

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

Fig. 7. Delayed induction of 53BP1 foci in CHO and xrs-5 cells at 30-35 population doublings

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.

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

was about 0.51 ± 0.27 in 4 Gy-surviving cells.

post-irradiation.

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

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

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 increased dicentric frequencies in surviving cells were observed.
