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Difference in Bystander Lethal Effect in Human Tumor
Cell Lines Depending on p53-gene Status Induced by
Carbon-ion Microbeams
M. Suzuki a) , T. Funayama b) , Y. Yokota b) , Y. Mutou b) , C. Tsuruoka a) ,
Y. Furusawa a) and Y. Kobayashi b)
a) Research Center for Charged Particle Therapy, NIRS,
b) Radiation-Applied Biology Division, QuBS, JAEA
Since 1994, a Phase I/II clinical study and cancer
radiotherapy have been begun using carbon-ion beams
generated with the Heavy Ion Medical Accelerator in Chiba
(HIMAC) at National Institute of Radiological Sciences 1) .
In the field of fundamental biological studies for high-LET
radiations, there are many reports regarding bystander
cellular effects after exposure to alpha particles derived from
238
Pu or He-ion microbeams. However, only limited sets
of studies have examined bystander effects after exposure to
different ion species heavier than helium. We have been
studying bystander lethal and mutagenic effects in normal
human fibroblasts irradiated with carbon-ion microbeams
using the 256(16 × 16)-cross-stripe irradiation method past 3
years. This year we examined the bystander lethal effect
using 1 normal human cell and 3 different human tumor cell
lines derived from different origins and considered the
relationship between bystander effect and p53-gene status.
Two different astrocytoma cell lines with wild- and
mutated-type p53 gene distributed by Institute for
Fermentation in Japan, amelanotic melanoma with
mutated-type p53 gene distributed by Health Science
Research Resources Bank in Japan and normal human skin
fibroblasts with wild-type p53 gene obtained from RIKEN
BioResource Center in Japan were used in this study.
Carbon-ion microbeams ( 12 C 5+ , 220 MeV) were generated
with the HZ1 port of TIARA. Approximately 8 × 105
exponentially growing the 4 different human cell types were
inoculated into each of microbeam dish, which was made of
acrylic resin ring with 36 mm diameter and attached
7.5 µm-thick polyimide film on the bottom of the ring,
2 days before the microbeam irradiation. In order to block
up cell-cell communication, half of the sample dishes were
treated with a specific inhibitor of gap-junction mediated
cell-cell communication (40 µM of gamma-isomer of
hexachloro- cyclohexane) one day before the irradiation.
Irradiation was carried out using the 256(16 × 16)-crossstripe
irradiation method described in the previous report 2) .
The linear energy transfer (LET) of carbon-ion microbeams
was estimated to be 103 keV/µm at the sample position.
Microbeams of 20 µm in diameter were irradiated in each
point with 8 delivered ions. Cell-killing effect was
detected using a colony formation assay as a reproductive
cell death.
Figure 1 showed cell-killing effect in 4 different human
cell types with different p53-gene status irradiated by
JAEA-Review 2010-065
carbon-ion microbeams. The surviving fraction in the cells
with wild-type p53 gene (C, D) in microbeam-irradiated
dishes (IR) was around 0.90, while almost 1.0 was detected
in microbeam-irradiated dishes with a specific inhibitor of
gap-junction mediated cell-cell communication (L+IR).
On the other hand, the surviving fraction in the cells with
mutated-type p53 gene (A, B) was the same between IR and
L+IR. The results show that bystander lethal effect was
observed in normal and tumor cells harboring wild-type p53
gene, but not in p53-mutated tumor cells. Moreover,
observed bystander effect was suppressed by treating with a
specific inhibitor of gap-junction mediated cell-cell
communication. There is evidence that both p53-mediated
cellular response and gap-junction-related bystander effect
are an important for carbon-ion induced bystander lethal
effect.
References
1) H. Tsuji et al., J. Radiat. Res. 48 (2007) 1.
2) M. Suzuki et al., JAEA Takasaki Ann. Rep. 2006 (2008)
107.
Surviving fraction
Surviving fraction
- 92 -
1.0
0.8
0.6
0.4
0.2
0
1.0
0.8
0.6
0.4
0.2
0
A
C
IR L+IR
IR L+IR
B
D
IR L+IR
IR L+IR
Fig. 1 Cell-killing effect in 4 human cell types with
different origin. A&B; Human tumor cell lines
harboring mutated-type p53 gene, C&D; Normal human
fibroblasts (C) and human tumor cell line (D) harboring
wild-type p53 gene. Cells were irradiated with
carbon-ion microbeams treated with (L+IR) / without
(IR) a specific inhibitor of gap-junction mediated
cell-cell communication. The results are the means and
standard errors from 3 independent beam times.