May 23rd-24th, 2017
IAEA Consultancy Meeting
Hiroshima

Biological effects of low-dose radiation on cells
and their implications in cancer risk

Hidehiko Kawai
Department of Experimental Oncology
Research Institute for Radiation Biology and Medicine
Hiroshima University

Research aim
Acheiving understanding of>The molecular basis of radiation-induced
carcinogenesis>The biological effects of low-dose radiationand their implications in cancer risk

By performing a comprehensive study of thecellular responses in different types of cells
under chronic γ-irradiation

Age-related diseases andcellular responses to DNA damage
Endogenous DNA Damage
Age-related diseases
SSBs 3,000/cell/day
Aging phenotypes
DSBs 0.1~50/cell/day
DNA-damaging events 20,000~
Variousequivalent to about 3 Gy of γ-IR
Agingcellular
Stochasticwith/without
dysfunctions Deterministicmutations
M-phaseG2-phase

G1-phase

S-phaseTime

Maintaining genomic stability
or/and Homeostasis
Cellular responses
Respond or Not RespondBrakes
Prevent Cell Growth(Arrest, Death, Senescence)
DNA-damage

DNA-repair

Accelerators
Encourage Cell Growth, Cell Survival

Cellular responses to DNA damage
and genomic mutationsIntrinsic processes
Extrinsic factors Replication error
Replication stress
All cell types ofthe human body

A tissue stem cell

Mutations --Chronic genotoxic stress-->Cancer
via cell division
Tumor suppressor
mechanisms
Elimination of abnormal cells
Cell senescence, Cell death,
Cell differentiation,
Cancer immunity cycle, etc.

Age-related diseases andcellular responses to DNA damage
Endogenous DNA Damage
Age-related diseases
SSBs 3,000/cell/day
Aging phenotypes
DSBs 0.1~50/cell/day
DNA-damaging events 20,000~
Variousequivalent to about 3 Gy of γ-IRAgingcellularStochasticwith/without
M-phasemutations dysfunctions Deterministic

G2-phase
G1-phase
TimeS-phase
Chronic γ-irradiation+0.001~1.0 Gy/day
SSBs +0.85~850/cell/day
DSBs +0.04~40/cell/day

Maintaining genomic stability
or/and Homeostasis
Cellular responses
Respond or Not RespondBrakes
Prevent Cell Growth(Arrest, Death, Senescence)
DNA-damage

DNA-repair

Accelerators
Encourage Cell Growth, Cell Survival

Chronic γ-irradiation
over a wide range of dose-rates
Device: 137Cs Low-dose-rate γ-irradiator (1.11 TBq)
i. Background Condition (B. C.)
ii. 0.001 Gy/day (~0.001 mGy/min)iii. 0.01 Gy/day (0.007 mGy/min)
iv. 0.1 Gy/day (0.069 mGy/min)
v. 0.5 Gy/day (0.347 mGy/min)vi. 1 Gy/day (0.694 mGy/min)
Cells were cultured udner 137Cs γ-rays at various doserates in 37oC 5 % CO2 incubators

Hiroshima University,
Research Institute for Radiation Biology and Medicine

Chronic exposure to γ-radiation reveals
significant differences in radio-sensitivity
among different cell types
PLoS ONE 9(8): e104279. (2014)

Fibroblasts

Other cell lines

Chronicγ-irradiation(0.5 Gy/day)

Low dose-rate
γ-irradiator

B. C. 0.5

B. G. 0.5

B. G. 0.5

B. G. 0.5 (Gy/day)

B. C. 0.5

B. G. 0.5

B. G. 0.5

B. G. 0.5 (Gy/day)

10

1.00E+066

B. G.
10

1.00E+055

0.5 Gy/day
10

1.00E+044

0 1 2 3 4 5 6 7 8 9 1011

Culture Days

B. C. 0.01 0.1

0.5

1.0 (Gy/day)

PLoS ONE 9(8): e104279. (2014)

Number of Cells/dishHuman Fibroblasts

Number of Cells/dishNumber of Cells/dishCellular proliferation during γ-irradiation(at 0.5 Gy/day)
MCF-7
10

1.00E+077

B. G.

10

1.00E+066

0.5 Gy/day

10

1.00E+055

10

1.00E+044

0 1 2 3 4 5 6 7 8 9 1011

Culture Days

HCT-11610

1.00E+077

10

1.00E+066

B. G.
0.5 Gy/day

10

1.00E+055

10

1.00E+044

0 1 2 3 4 5 6 7 8 9 1011

Culture Days

Comparison of colony forming abilities
of different types of cells : acute versus
chronic γ-irradiation (Total dose : 5 Gy)

2.6%
3.3%
3.7%
9.4%
3.9%
3.7%
1.2%
3.3%

6.9%4.1% Fibroblasts
11.9%
64.3%
94.2% Other
91.4% celllines
93.3%
92.4%
PLoS ONE 9(8): e104279. (2014)

Fibroblasts are highly responsive to chronic genotoxic stressFibroblasts undergo senescence whenirradiated at a dose-rate of 1.0 Gy/day
Seeded at 100 cells/dishCultured for 10 days
at B. C., 0.01, 0.1, 0.5, 1.0 (Gy/day)

Cultured
for an additional 10 days
at B. C. following Ch-IR

B.G. 0.01 0.1
0.5(Gy/day)

1.0PLoS ONE 9(8): e104279. (2014)

Fibroblasts undergo senescence whenirradiated at a dose-rate of 1.0 Gy/day

PLoS ONE 9(8): e104279. (2014)

The ATM/TP53/P21 pathway is essential forchronic γ-IR-induced cellular senescence

PLoS ONE 9(8): e104279. (2014)

Summary (I) Different cell types exhibit different response sensitivities tochronic γ-irradiation.
 Proliferating fibroblasts
chronic g-irradiation.

areparticularlysensitiveto A dose-rate threshold exists for the induction of senescencelike growth arrest (between 0.5 and 1.0 Gy/day).
 The ATM/TP53/P21 pathway plays a crucial role in responseto chronic γ-irradiation for the maintenance of genomic
integrity in fibroblasts.

Cellular responses to DNA damage
and genomic mutations
Intrinsic processes
Extrinsic factors Replication error
Replication stress
All cell types of
the human body

A tissue stem cell

Mutations --Chronic genotoxic stress-->Cancer
via cell division
Tumor suppressor
mechanisms
TP53-pathway
Elimination of abnormal cells

Fibroblasts

Cell Senescence, Cell death,
Cell differentiation,
Cancer immunity cycle, etc.

On-going research
Detection/Quantification of molecular changes
in fibroblasts under chronic g-irradiation>RNA sequencing
by Next generation sequencerRNA expression profiles
Bioinformatics

Next generation sequencing technologies
Transcriptome analysis of fibroblasts
following chronic γ-irradiation

non-IR,0.01, 0.1, 0.5, 1.0 Gy/day
For 4 days

NHDFs

Transcriptome analysis
Human fibroblasts
non-IR, 0.01, 0.1, 0.5, 1.0 Gy/day, for 4 days
Isolation of mRNA  RNA-seq by NGSRNA-Seq (RPKM ratio, 4 days Ch-IR)
0.1 Gy/day ｖｓ non-IR(Growth suppression)

1.0 Gy/day ｖｓ non-IR(Senescence)

DownRPKM mRNA expression non-IR

0.5 Gy/day ｖｓ non-IR(G1-arrest)

1.0 Gy/day ｖｓ 0.5 Gy/day(Senescence vs G1-arrest)On-going research
Detection/Quantification of molecular changes
in fibroblasts under chronic g-irradiation>RNA sequencing
by Next Generation SequencerRNA expression profiles
Bioinformatics
Identification of responsible genes and pathways
for cellular responses and cell-fate decisions>siRNA library screening
by High-throughput Imaging Systems
Cellular phenotype
Systems biology

High-Throughput siRNA Screening
Identification of the genes responsible forthe cellular responses to γ-irradiation
384-well platesiRNA libraryfor 21,584 genes（96well-plate258-plates）

Fibroblasts

siRNA
d0 d1

siRNA1-x+
siRNA-xCtrl-six 96 siRNA

siRNA-x siRNA-x 1 gene/4-wells++
TP53-si P21-si

No-IR

d4

0.1 Gy/dayd43) (Reversible) Growth arrest at G1-phase

0.5 Gy/day

d4

Non-IR d74) Senescence-like growth arrest

1.0 Gy/day

d4

Non-IR1) Un-irradiated condition
2) (Partial) Growth suppressiond7

High-Throughput siRNA Screening
siRNA library for 21,584 genes（96well-plate 258-plates）
Nuclear DNA: Hoechst
Anti-53BP1: Alexa 488
DNA damage, Repair
Anti-Ki67: Alexa 555
Proliferation

384-well plate

High-Throughput
Imaging Systems

Opera phenix™

High-Throughput siRNA Screening

Nuclear DNA

53BP1 fociKi67

Summary (II)Dose-rate
Damage&Repair
Cellular
responses&
Molecularresponses(Epi-)Genetic
Aberrations
BiologicalEffects

Endogenous DNA damage 20,000 events/cell/day
B. G. + 0.01
0.1
0.5
1.0
(Gy/day)4) RNA-seq

1) Cell-type specificity
2) Fibroblast G1-arrest5) Senescence

Cell proliferation3) TP53 pathway

High-density time- and dose-rate-dependent measurements of gene
expression profiles and cellular responses during chronic γ-irradiation

The complex effects of cellularresponses on stem cell division
Extrinsic factors Intrinsic processes

All types of cells
Stem cell

Differentiated cell

Microenvironment
Cancer immunity

Stochastic/Deterministic
Passive/Active
Division/No division
DivisionSenescence, Cell death,
Cancer risk
Differentiation, etc…

Individual
susceptibility
Genetic
background
Health condition
Age SexThe Research PerspectiveComprehensive studies of cell-type-, dose-rateand time- dependent cellular responses andsubsequent cell-fate decisions during chronic girradiation (especially in vivo, if it is possible) may
help us to better understand the relevant targets
for the biological effects of low-dose radiation.
The results from these studies may provide us
with more insights into individual susceptibility to
low-dose radiation and with the molecular basis
for epidemiological studies on radiation risks of
low-dose radiation exposure.