Photon Factory Activity Report 2017 #35 (2018)

BL-27B/2017G072

Repair Process of DNA Double Strand Breaks Induced by X-ray Bystander EffectMitsuaki OJIMA1*, Yohei KAWAMATA2, Shogo ONIZAWA2, Michiaki KAI1, Noriko USAMI3, andAtsushi ITO2
1
Oita University of Nursing and Health Sciences, Oita 840-1201, Japan
2
School of Engineering, Tokai Univ., Hiratsuka, Kanagawa 259-1292, Japan3
Photon Factory, KEK, Tsukuba, Ibaraki 305-0801, 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, neoplastic transformation, formation ofmicronuclei, and changes in gene expression [1-3]. In theprevious study, we investigated the repair kinetics of DSB
in non-irradiated primary normal human fibroblasts(MRC-5) co-cultured with 20 mGy-irradiated MRC-5.
After 48 h of co-culture, 81% of the initial numbers of DSB
remained in non-irradiated MRC-5 [4]. In addition, whenMRC-5 were irradiated with 1000 mGy after co-cultivatewith 20 mGy-irradiated MRC-5, we found that thenumbers of DSB significantly decreased compared with
1000 mGy-irradiated MRC-5 which were not experienced
co-cultivate with 20 mGy-irradiated MRC-5 (underpreparing submission). From these previous findings, wehypothesized that DSB resulting from the radiationinduced bystander effects might not be repaired, andunrepaired DSB by radiation-induced bystander effectmight contribute to induction of radioadaptive response.
In the present study, as a first step, we investigated therepair kinetics of DSB in MRC-5 which was irradiatedwith X-rays directly by X-ray microbeams.
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 Mylar sheet. They were then irradiatedwith 1 Gy of X-ray microbeam in various cell population(0.3 mm  0.3 mm, 0.9 mm  0.9 mm, 1.2 mm  3 mm).
Control samples were sham-irradiated. Subsequently, cells

were incubated for 24 h at 37°C under 5% CO2. Afterincubation, DSBs were detected by 53 binding protein 1(53BP1) immunofluorescent staining, and the numbers ofDSB were determined by assessing the number of 53BP1
foci.
3 Results and Discussion
The numbers of DSB in directly X-irradiated cell increased
depending on size of X-irradiated cell population (Fig. 1).
This result means that even if the cumulative dose ofradiation is the same per cell, with the increasing numberof directly X-irradiated cells, the DSB may easy to remain
in directly X-irradiated cells. This phenomenon may becaused by the interaction of directly X-irradiated cells andnon-irradiated bystander cells. In the next step, we will
investigate whether the number of directly X-irradiatedcell affect the induction of bystander effect.

Fig. 1. Number of DSBs in X-irradiated MRC-5
A: Control, B: 0.3 mm  0.3 mm, C: 0.9 mm  0.9 mm,
D: 1.2 mm  3 mm

References[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).
* ojima@oita-nhs.ac.jp