Photon Factory Activity Report 2019 #37 (2020)

BL-27B/2019G030

The Response of Cells to DNA Damage and Cell Proliferation
Depends on the Size of the X-irradiated Cell Population
Mitsuaki OJIMA1*, Atsushi ITO2, Keiji SUZUKI3, Noriko USAMI4, Maki OHARA4,and Michiaki KAI1
1
Oita University of Nursing and Health Sciences, Oita 840-1201, Japan
2
School of Engineering, Tokai Univ., Hiratsuka, Kanagawa 259-1292, Japan3
Department of Radiation Medical Sciences, Atomic Bomb Disease Institute, Nagasaki University,
1-12-4 Sakamoto, Nagasaki 852-8523, Japan
4
Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research
Organization, 1-1 Oho, Tsukuba, 305-0801, Japan
1 Introduction
After the Fukushima nuclear accident, internal exposuredue to insoluble radioactive cesium (Cs) deposited in thelungs has become a problem [1, 2]. Although the healtheffects of internal exposure from radioactive Cs is very
important issue from the perspective of radiation
protection [3, 4], the biological mechanism has not beenclarified.
X-ray microbeam is very effective to reproduce thesituation of local exposure in internal exposure. In thisstudy, we investigated the response of normal human cell
populations irradiated with different beam sizes to DNA
damage and cell proliferation.
2 Experiment
Cell culture: Primary normal human fibroblasts fromthe lung, MRC-5 (European Collection of Cell Cultures),were cultured on a cover glass with a grid line (GC1300,Matsunami Glass Industry Co., Ltd.) in MEM
supplemented with 10% fetal bovine serum and penicillinstreptomycin at 37°C in a humidified incubator with 5%
CO2 to prepare a cell population of about 5×104 cells in
530 mm2. The irradiation field can be specified by using
the cover glass with a grid line.
X-ray microbeam irradiation: X-ray microbeam wasdelivered by an X-ray microbeam generator at BL-27B
with 5.3 keV. Dose rate was 20 R/s.
The response of cell population to DNA damages: Inorder to analyze the difference in cell population response
depending on the X-irradiation size to DNA damage, thesize of 0.02 mm2, 0.09 mm2, 0.81 mm2, and 1.89 mm2 on
the cell population were irradiated with X-ray of 1 Gy.
Subsequently, cells were incubated for 4~48 h at 37°C
under 5% CO2. After incubation, DSBs were detected by
53 binding protein 1 (53BP1) immunofluorescent staining,
and the numbers of DSB per cell were analyzed .
The response of cell population to cell proliferation:
In order to analyze the difference in cell proliferation
depending on the X-irradiation size, the size of 0.02 mm2,0.09 mm2, 0.81 mm2, and 1.89 mm2 in the cell population
were irradiated with X-ray of 1 Gy. Subsequently, cells
were incubated for 24~48 h at 37°C under 5% CO2. Afterincubation, cell proliferations were detected by Ki67

immunofluorescent staining, and the frequencies of Ki67
positive cells were analyzed.
3 Results and Discussion
Table1. The response of cell population to DNA damages
Control
0.02 mm2
0.09 mm20.81 mm2
1.89 mm2

No. of 53BP1 per cell ± SD
4h
24h
48h
0.57± 0.088
0.69 ± 0.14
0.47 ± 0.039
3.68 ± 0.64❇ 0.51 ± 0.043
0.44 ± 0.087
3.30 ± 0.19❇ 0.44 ± 0.075
5.53 ± 2.29❇ 0.85 ± 0.19
0.67 ± 0.087❇
6.49 ± 0.72❇ 0.80 ± 0.17
0.88 ± 0.20❇
❇ P<0.05, T-test

Table2. The response of cell population to cell proliferation

Control
0.02 mm2
0.09 mm20.81 mm2
1.89 mm2

Frequencies ofKi67 positive cell ± SD
24h
48h
0.72 ± 0.068
0.29 ± 0.031
0.61 ± 0.029
0.60 ± 0.038
0.19 ± 0.0058❇
❇
0.35 ± 0.11
0.17 ± 0.095❇
❇
0.37 ± 0.058
0.14 ± 0.038❇
❇ P<0.05, T-test

I. An X-irradiated cell populations size-dependent
reduction in the number of DNA damages wasobserved at < 0.81 mm2 (Table.1). In addition, there
was a tendency that DNA damage remained for a longtime in 0.81 mm2 and 1.89 mm2.
II. When the size of X-irradiated cell population waslarger than 0.09 mm2, the frequency of cell proliferation
decreased at 24 hours after X-irradiation (Table. 2).
These results indicate that when the X-irradiation
size was small, X-irradiated cells received some signal(rescue signal) from surrounding nonirradiated cells,
and DNA damage were rapidly repaired or cells with
DNA damage were eliminated.
References[1] Chino, M. et al., J. Nucl. Sci. Technol. 48 (2011).
[2] Satou, Y. et al., Anthropocene 14 (2016).
[3] Sakama, M. et al., Springer, Tokyo (2016).
[4] Ishikawa, T. et al., J. Radiol. Prot. 38 (2018).
* ojima@oita-nhs.ac.jp