Photon Factory Activity Report 2018 #36 (2019)

BL-27B/2017G072

Repair Process of DNA Double Strand Breaks Induced by X-ray Bystander EffectMitsuaki OJIMA1*, Maki OHARA2, Michiaki KAI1, Noriko USAMI2, and Atsushi ITO3
1
Oita University of Nursing and Health Sciences, Oita 840-1201, Japan2
Photon Factory, KEK, Tsukuba, Ibaraki 305-0801, Japan3
School of Engineering, Tokai Univ., Hiratsuka, Kanagawa 259-1292, Japan
1 Introduction
Recent evidence suggests that damage signals may betransmitted from irradiated to non-irradiated cells in apopulation, leading to the induction of genetic changes that
include gene mutations in bystander cells that received noradiation exposure. This phenomenon, the radiationinduced bystander effect, has been observed in mainly
fibroblast and epithelial cells by assay for various
endpoints, including DSB, chromosome aberrations, cellkilling, formation of micronuclei, and changes in gene
expression [1-3]. In the previous study, we investigated therepair kinetics of DSB in non-irradiated primary normal
human fibroblasts (MRC-5) co-cultured with 20 mGyirradiated MRC-5. After 48 h of co-culture, 81% of theinitial numbers of DSB remained in non-irradiated MRC5 [4]. This results suggested that the bystander effects canincrease chances of genetic changes in non-irradiated cells,
and may enhance the risk of radiation carcinogenesis in
the low dose region.
On another hand, it has been accumulating evidence thatdetrimental effects, such as DSB and cell death, in directly
irradiated cells are reduced upon receiving feedback
signals from non-irradiated bystander cells or from themedium previously conditioning non-irradiated bystander
cells. [5]. This phenomenon is called radiation-induced
rescue effect and may constitute one of the protective
effects of low dose radiation.
Both the radiation-induced bystander effect and theradiation-induced rescue effects represent importantphenomena that have a significant impact on novel
biological responses induced by low dose radiation.
However, these mechanisms remain largely unknown.
In this study, we analyzed the repair kinetics of DSB in
MRC-5 which was irradiated with X-rays directly by Xray microbeams to clarify the actual situation of radiationinduced rescue effects.
2 Materials and Methods
Cell culture. Primary normal human fibroblasts fromthe lung, MRC-5 (European Collection of Cell Cultures),were grown on a sterilized cover glass in MEM
supplemented with 10% fetal bovine serum and penicillinstreptomycin at 37°C in a humidified incubator with 5%
CO2. All experiments were performed using non-dividing
confluent cell cultures, the confluent state was kept for atleast 24 h before experiment, in order to eliminate disparatecell-cycle phase radio-sensitivities.

X-ray microbeam irradiation. X-ray microbeam weredelivered by an X-ray microbeam generator in Photon
Factory with 5.3 keV. Dose rate was 20 R/s.
Repair kinetics of DSBs in directly X-irradiated
MRC-5 by X-ray microbeam. Cover glass with confluent
cells was put on a Mirer sheet. They were then irradiatedwith 1 Gy of X-ray microbeam in various cell population(0.09 mm2, 0.81 mm2, 1.89 mm2). Control samples weresham-irradiated. Subsequently, cells were incubated for 24
h at 37°C under 5% CO2. After incubation, DSBs weredetected by 53 binding protein 1 (53BP1)
immunofluorescent staining, and the numbers of DSB
were determined by assessing the number of 53BP1 foci.
3 Results and Discussion
Depending on the size of X-irradiated cell population, theboth the numbers of DSB in directly X-irradiated cellincreased and the DSBs in directly X-irradiated cell
persisted after X-irradiation. This result means that even if
the cumulative dose of radiation is the same per cell, with
the increasing number of directly X-irradiated cells, theDSB may easy to remain in directly X-irradiated cells. Thisphenomenon may be caused by the interaction of directly
X-irradiated cells and non-irradiated bystander cells.

Fig. 1: Number of DSBs in X-irradiated MRC-5●：0.09 mm2, ▲：0.81 mm2, ■：1.89 mm2References[1] H. Nagasawa et al., Radiat Res. 152, 552 (1999).
[2] H. Zhou et al., PNAS. 97, 2099 (2000).
[3] S.G. Swant et al., Radiat Res. 155, 397 (2001).
[4] M. Ojima et al., Radiat Res. 175, 90 (2011).
[5] S. Chen et al., Mutat Res. 706, 59 (2011).
* ojima@oita-nhs.ac.jp