VIII-Ⅱ-1. Project Research
Project 7

PR7

The Origin of Radiation Carcinogenesis is not DNA Damage

M. Watanabe
Laboratory of Radiation Biology, Department ofRadiation Life Science, Research Reactor Institute, Kyoto
University,
OBJECTIVES AND RESEARCH SUBJECTS:
It has been believed that the first target of radiation
carcinogenesis is DNA. However, this hypothesis is
not proved as a main route for carcinogenesis of low
dose radiation yet. After analyzing our results ofresearch of malignant cell transformation with lowdose radiation during the past 30 years, we came tostrongly believe that a radiation cancer-causing
primary target is not DNA itself. Recently, several
reports including our reports suggested that non-target
effects, such as bystander effect and delayed effect,
modify cell transformation frequency. From theseresults, we speculate that non-genomic damage plays
an important role in an initial process of cellularmalignant transformation. Especially, we speculatethat aneuploid may be closely related on the inductionof malignant cell transformation by radiation.
Therefore, the aim of this project is focused on
elucidation of process of aneuploid formation related
to carcinogenesis.
MAIN RESULTS OF THIS PROJECT:
As a result, we found that the intracellularoxidation degree, such as reactive oxidative radicals
and long lived radicals, was elevated by high density
culture and radiation exposure both in mammaliancells. Specially, long-lived radicals (LLRs) play animportant role of genetical effects of radiation. Theseradicals attack several proteins, such as telomere
related protein and centrosome, and destroy theirstructure. Telomere destabilization induces telomere
fusion and makes dicentric chromosome and reason ofchromosome instability. In fact, dicentric chromosome
is dominant aberration induced by low dose radiation.
Radiation induced radicals also attacked centrosome.
Centrosome destabilization induces nondisjunction
and raises the frequency of aneuploid. In early processof cell transformation, structural aberration ofchromosome does not occur, but aneuploid is seen inhigh frequency. Aneuploid is also induced by
deficiency in cell cycle checkpoint at G2-M. By
treatment of vitamin C prevent induction of LLRs,
aneuploid, and thymic lymphomas in B6C3F1 mice.
Interestingly, the thymic lymphomas in vitamine C
treated mice lacked point mutation of Ikaros,
suggesting a suppression of point mutation by VC
Low dose radiation activated repair capacity of
DNA damage in irradiated cells (A-7). Becauseradiation-induced genomic instability is induced insome fraction of the progeny of a single survived cell,
not a single gene mutation but some epigenetic
changes may be involved in the initiation of radiationinduced genomic instability. Oxidative stress andaltered chromatin structure have been proposed as themechanisms of perpetuation of radiation-induced genomic instability.
These results suggest a possibility that a main
target of radiation carcinogenesis is not DNA, but iscentrosome, which are the proteins to constitutechromosomal homeostasis maintenance mechanism(Fig1). If our results are right, “Mutation Theory ofCarcinogenesis” is to be wrong. I will suggest a new
hypothesis about radiation carcinogenesis, which wasnamed as "protein target theory" by this report.

Fig. 1. A new model of radiation carcinogenesis.

採択課題番号 23P7
DNA を標的としない放射線発がんの仕組みに関する研究（京大・原子炉）渡邉正己

プロジェクトPR7-1

Participation of Aneuploidy on Radiation-induced Cellular
Malignant Transformation

M.Watanabe, K.Watanabe, G. Kashino1, J. Kumagai2,
H. Nawata and K. Tano
Laboratory of Radiation Biology, Department of Radiation
Life Science, Research Reactor Institute, Kyoto University
1
Advanced Molecular Imaging Center, School of Medicine, Oita University
2
Graduate School of Engineer, Nagoya University

INTRODUCTION:
It has been believed that the first target of radiation
carcinogenesis is DNA. However, this is not proved forradiation carcinogenesis yet. We discovered that frequency of aneuploid cell was closely related to that ofradiation-induced cell transformation and natural celltransformation by high-density cultivation, but gene mutation was not [1-4]. Cell with p53 gene becomes
tetraploid, but does not get tumorigenicity. On the other
hand, cells without p53 gene function become a triploid
easily, and acquires tumorigenicity. Both radiation exposure and high-density cultivation elevated the level of
intracellular oxidative radicals [5-7]. These radicals induced centrosome destabilization and produced cells
carrying extra centrosome, which promote merotelic attachment of chromosome by altering spindle geometry.
Unresolved merotelic attachments can give rise to lagging chromosomes at anaphase. Aneuploidy was seen inhigh frequency in early process of cell transformation.
These results strongly suggest that a main target of carcinogenesis by low dose radiation is not DNA, but iscentrosome, which are the proteins to constitute chromosomal homeostasis maintenance mechanism. In addition, this route may be the same as that of natural carcinogenesis.
RESULTS AND DISCUSSION:
Cell with p53 gene becomes tetraploid, but does
not get tumorigenicity. On the other hand, cells without
p53 gene function become a triploid easily, and acquires
tumorigenicity. Both radiation exposure and high-density
cultivation elevated the level of intracellular oxidative
radicals and long-lived radicals in ME, SHE cells and HE
cells. These radicals attacked centrosome and induced
centrosome destabilization. Centrosome destabilization
promotes merotelic attachment of chromosome by altering spindle geometry. Unresolved merotelic attachments

can give rise to lagging chromosomes at anaphase. Thisis the main route of production of aneuploid cell. We
previously have shown that in early process of celltransformation, structural aberration of chromosome does
not occur, but aneuploid is seen in high frequency [2,4].
To study the effects of aneuploidy in normal human cells, we generated artificial cells of human primary
fibroblast having three chromosome 8 (trisomy 8 cells)
by using microcellmediatedchromosome transfer technique. In addition to decreased proliferation, the trisomy
8 cells lost contact inhibition and reproliferated after exhibiting senecence-like characteristics that are typical oftransformed cells [8]. Furthermore, the trisomy 8 cells
exhibited chromosome instability, and the overall gene
expression profile based on microarray analyses was significantly different from that of diploid human primary
fibroblasts. Our data suggest that aneuploidy, even a single chromosome gain, can be introduced into normal human cells and causes, in some cases, a partial cancer
phenotype due to a disruption in overall gene expression.
These results strongly suggest that a main target ofcarcinogenesis by low dose radiation is not DNA, but iscentrosome, which are the proteins to constitute chromosomal homeostasis maintenance mechanism.
REFERENCES:[1]Watanabe M, Horikawa M, Nikaido O., Radiat. Res.
98: 274-283, 1984[2]Watanabe M, Suzuki K, Kodama S., Cancer Res.
50(3): 760-765, 1990
[3]Watanabe M, Suzuki K., Mutat. Res. 249(1): 71-80,
1991[4]Suzuki K, Yasuda N, Suzuki F, Nikaido O, Watanabe
M., Int. J. Cancer 44 (6): 1057-1061, 1989
[5]Yoshii H, Watanabe M., J. Radiat. Res. 50: 127-137,
2009[6]Harada T, Kashino G, Suzuki K, Matsuda N, Kodama
S, Watanabe M, Int. J. Radiat. Biol. 379: 809-814,
2009[7]Roy K, Kodama S, Suzuki K, Fukase F, Watanabe M,
Radiat. Res. 154(6): 659-666, 2000
[8]Nawata H, Kashino G, Tano K, Daino K, Shimada Y,
Kugoh H, Oshimura M, Watanabe M. Pros ONE.
6(9): e25319, 2011

採択課題番号 23P7-1
放射線による染色体異数化と細胞がん化の関連性
プロジェクト（京大・原子炉）渡邉正己、渡邉喜美子、菓子野元郎、熊谷純、縄田寿克、田野恵三

PR7-2The Change of Mitochondrial ROS by Radiation Induced Bystander Effect

G. Kashino, J. Kumagai1 and M. Watanabe2
Advanced Molecular Imaging Center, School of Medicine,
Oita University
1
Graduate School of Engineer, Oita University
2
Research Reactor Institute, Kyoto University
INTRODUCTION: The effects of ionizing radiation
have been examined in biological model. One of the impacts recently is that some signals are interacted between
targeted cells and non-targeted cells. This effect, called as“bystander effect” has impact on risk estimation in lower
dose range, because non-targeted cells may be leading tocancer. Some factors from irradiated cells are thought tobe effective in non-targeted cells. In this study, culture
medium with human cells in a culture flask was X- rayirradiated, and then the medium was transferred to otherculture flasks in which non-irradiated cells were plated. It
is expected that irradiated cells as donor cells releasesome soluble bystander factors into the medium, so thatthe non-irradiated cells as recipient cells may be affectedby the factors. The result showed that bystander factors
from irradiated cells modulate the membrane potential of
mitochondria in non-irradiated cells. This response isthought to be a trigger of following responses such as theinduction of reactive oxygen species (ROS), and genemutation.
EXPERIMENTS: Normal human cells (HE35, HE49,
NB1RGB) and tumor cell lines (HeLa, SF126) were usedin this study. Cells were cultured in MEM-alpha medium
supplemented with 10%FBS. Cells were maintained at
370C in a humidified atmosphere with 5% CO2. Cells

were seeded onto T25 flask one day prior to irradiation.
Immediately before irradiation, medium was changed
and cells were irradiated with 0.5 Gy of X-rays. Cells
were incubated for 24 hours following irradiation. The
culture medium was filtrated with 0.2m filter andtransferred to unirradiated cultured cells on T25.
Twenty-four hours after transfer, cells were treatedwith MitosoxRed staining dye to detect the mitochondrial supeoxide (O2-) level. After the 30 min treatment,
cells were washed with PBS and harvested. Then,suspended cells were analyzed by FACscan to detect
the fluorescent intensity of MitosoxRed. In the studyof radical scavenger treatment, 0.2 mM Ascorbic acidand 2% DMSO were used. Cells were treated withthese scavengers at the same time of medium transfer
of conditioned medium.

Fig.1 Relative ratio of fluorescent values for MitosoxRedin two human tumor cell lines (HeLa and SF126). Twenty-four hours after irradiation, conditioned medium weretransferred and then ROS levels were measured at 24 hafter transfer by MitosoxRed method. Ascorbic acid (0.2mM) was used for the purpose of relief of ROS in mitochondria.

In all normal human cells and humantumor cells, the mitochondrial ROS were detected by
MitosoxRed method, and levels were increased by thesecreted factors from 0.5Gy-irradiated cells. Therefore,
the bystander response (is meaning that reactions are
occurred from irradiated cells toward non-irradiatedcells) is occurred in both normal cells and tumor cells,
and mitochondira may be key organ in this response.
Also, these responses were suppressed by the treatment of ascorbic acid, but not DMSO. It is possiblethat ascorbic acid can be incorporated into mitochondria, therefore ascorbic acid is effective scavenger for
bystander responses. We hypothesized reactive oxygen
species (ROS) should be increased after the signal activation through secreted factor from irradiated cells.
We need to clarify the signal pathway. The increases of
ROS were thought to be leading to the production of“slow releasing long lived radicals” (SRLLRs), whichis thought to be a cause of mutagenesis. Our results
suggest that secreted factors from irradiated cells canlead to mutagenesis through the change of mitochondrial function inn both normal and tumor cells.
RESULTS:

採択課題番号 23P7-2
染色体異数化誘導へのバイスタンダー効果の影響（大分大・医）菓子野元郎（名大・院工）熊谷純（京大・原子炉）渡邉正己

プロジェクトPR7-3

SIRT2, a Mitotic Checkpoint Protein, as a Novel Target for Cancer Therapy:
SIRT2 Down-regulation in HeLa can Induce p53 Accumulation via p38 MAPK
Activation-dependent p300 Decrease, Eventually Leading to Apoptosis

Y. Li and T. Inoue
Division of Human Genome Science, Department of Molecular and Cellular Biology, School of Life Science,
Faculty of Medicine, Tottori University
INTRODUCTION: The yeast silent formation regulator protein-2 (sir2) is a NAD+-dependent protein
deacetylase, the mammalian homolog of sir2 is the sirtuin
family containing 7 members, SIRT1-SIRT7. Thesesirtuins are involved in gene silencing, cell cycle control,
apoptosis, and even energy homeostasis. In our previous
study mentioned above, we observed that SIRT2
downregulation confers resistance to mitotic cell deathfrom the spindle checkpoint in the presence of microtubule inhibitors in HCT116 cells, a mitotic checkpoint-proficient cancer cell line used for studying checkpoints. However, in some cancer cell lines, we observedthat siRNA-mediated SIRT2 knockdown causes massive
cell death even in the absence of microtubule inhibitors.
This raises the possibility that SIRT2 inhibition may be atarget for the killing of cancer.
EXPERIMENTS: We sought to obtain evidences forthe molecular mechanism through which SIRT2 inhibition is involved in tumor cytotoxicity in the present study.
Several human cell lines were transfected with siRNA of
SIRT2 or negative control siRNA. Transfection of siRNA
to SIRT2 resulted in significantly decreased cell numbers
in HeLa, hiMSC, HT1080, 293T, and CC1cell lines, but
not in normal cells such as TIG-1 cells. The mostprominent case was HeLa cells, while thus, we used
HeLa cells for further study to delineate the mechanism
by which SIRT2 downregulation leads to suppression ofcolony formation.

採択課題番号 23P7-3（鳥取大・医）李 艶沢（鳥取大・医）井上敏昭

RESULTS: The apoptosis was caused by p53 accumulation, which is mediated by p38 MAPK activation-dependent degradation of p300 and the subsequent
MDM2 degradation. Consistent with the observation, thesensitivity of SIRT2 siRNA among cancer cell lines reflects the p53 status.
Sirtuin inhibitors are emerging as antitumor drugs andthis function has been ascribed to the inhibition of SIRT1,
the most well-characterized sirtuin that deacetylases p53
to promote cell survival and also binds to other proteinsin response to genotoxic stress. The present study suggests that SIRT2 can be a novel molecular target for cancer therapy and provides a molecular basis for the efficacy of SIRT2 for future cancer therapy.

REFERENCES:[1] Y. Li, H. Matsumori, Y. Nakayama, M. Osaki, H.
Kojima, A. Kurimasa, H. Ito, S. Mori, M. Katoh, M.
Oshimura, and T. Inoue. Genes Cells. 16 (2011) 34-45.

新規 M 期チェックポイント蛋白 SIRT2 の中心体における機能の解明

プロジェクトPR7-4Chromosome Instability in Radiation-induced Mouse Lymphomas: Study II

A. Nakata, M. Yoshida, M. Akiyama, T. Nishimura, M.
Watanabe1, S. Kakinuma and Y. Shimada
Experimental Radiobiology for Children’s Health Research Group, National Institute of Radiological Sciences
1
Laboratory of Radiation B iology, Research Reactor
Institute, Kyoto University, Kyoto University
INTRODUCTION:
Irradiation with heavy-ion beams is one of theeffective cancer radiotherapy because of its good conformity to tumor shape, high relative biological effectiveness (RBE), smaller independency of oxygen concentration and, thereby, high local control rates. In order todevelop more efficient and safer protocol with less side
effects, it is important to know the acute and late biological responses of both cancer cells and normal tissues toheavy ion beams. Generally, heavy ions give large relative biological effectiveness (RBEs) for cell killing andinduction of mutations and chromosome aberrations.
The high frequency of the intrachromomal and complex-type chromosomal exchange is reported characteristic for the cells treated with carbon ions than for thosetreated with X-rays [1]. Chromosome instability, which
can be seen in the progeny of irradiated cells after several
cell divisions, is also manifested by carbon ions more
efficiently than X rays [2]. Several studies documented
experimental data on the induction of cancers by heavy
ions using in vitro and in vivo model [3-6].
Mouse thymic lymphomas (TLs) can be reproducibly
induced by radiation, and have been used for the characterization of genes and chromosomes involved in the development of radiation lymphomagenesis. Cytogenetic
studies have reported interstitial deletion of chromosome
11, translocation with deletion of the distal region inchromosome 12 and trisomy of chromosome 15 in radiation-induced lymphomas [7] . The former two chromosome aberrations are consistent with frequent loss ofheterozygosity on chromosomes 11 and 12, where tumor
suppressor genes, Ikaros and Bcl11b, are mapped, respectively [8-10]. On the other hand, trisomy for chromosome 15 was most frequently identified in both spontaneous and carcinogen-induced lymphomas [7].
Age is an important factor that influences the carcinogenic effect of radiation. Generally, the younger the ageof subject is, the larger the cancer risk is. It is wellknown that childhood thyroid cancers have frequent
RET-PTC conversion whereas adult type thyroid cancers
have B-RAF point mutation. However, information on
the distinct molecular changes in cancers after early life

採択課題番号 23P7-5

exposure and those after adulthood exposure are still insufficient.
In order to identify the age-dependent genetic events
during lymphomagenesis by heavy-ion carbon beam irradiation, karyotypes was compared in TLs induced bycarbon beams in B6C3F1 mice, which were irradiated ateither one week of age or four weeks of age.
EXPERIMENTS:
We used the B6C3F1 mice at the age of one or fourweeks of age (Charles River Japan Inc., Yokohama).
TLs were induced by whole-body irradiation with carbon
ions (1.2 or 1.6 Gy) once a week for four consecutive
weeks.
Chromosomes from the enlarged thymus
were prepared for analysis by short-term culture or direct
methods. To confirm chromosome loss, painting and
BAC FISH techniques were used in several cases.
RESULTS:We first found that the incidence of aneuploidy, especially trisomy of chromosome 15, was high for carbon
induced TLs in both age group. It is of note that smalldeletion in centrometic region and structural change inchromosome X was more characteristic to TLs from mice
irradiated at one week of age. Karyotype and array
CGH analysis indicated high frequency of loss of Bcl11b
in TL form mice irradiated at one week of age (6/7; 86%),
compared to adult TLs (4/8; 50%). These results indicate that there are age-associated chromosome aberrations in TLs induced by carbon ions.
REFERENCES:[1] M. Durante et al., Adv. Space Res. 22 (1998)
1653-1662[2] H. Hofman-Huther et al., Radiat. Res. 166 (2006)
858-869.
[3] E.L. Alpen et al., Radiat. Res. 136 (1993) 382-391.
[4] K. Ando et al., J. Radiat. Res. (Tokyo) 46 (2005)
185-190.
[5] T. Imaoka et al., Int. J. Radiat. Oncol. Biol. Phys. 69(2007) 194-203.
[6] ML. Yamamoto et al., Radiat Res. 175 (2011)231-239.
[7] M.A. Yoshida et al., Cancer Genet. Cytogenet. 179(2007) 1-10.
[8] Y. Shimada et al., Radiat. Res. 154 (2000) 293-300.
[9] S. Kakinuma et al., Radiat. Res. 157 (2002) 331-340[10] T. Takabatake et al., Radiat. Res. 169 (2008)
426-436.

インビボ発がんにおける染色体異数化（放医研・発達期）島田義也、（京大・原子炉）菓子野元郎、渡邉正己

プロジェクトPR7-5 Involvement of Quality Control of Organela in Cancer Development and Progression
K. Tano, E. Inoue1 and M. Watanabe
Reaserch Reactor Institute, Kyoto University
1
Graduate School of Pharmaceutical Sciences, Tohoku
University
INTRODUCTION: Peroxiredoxins (Prdxs) are a familyof peroxidases currently known to possess six mammalian isoforms. Although their roles in cellular redox regulation and antioxidant protection are distinct, all of themconvert hydrogen peroxide to water and oxygen.
Among PRDX family proteins, PRDX1 is present in thecytoplasm and PRDX2 is present in the mitochondria.
Both of them work as a molecular chaperone to reduce anintermolecular disulfide bond within other proteins besides metabolizing hydrogen peroxide. To investigate theroles of PRDX1and 2 to maintain normal intracellularoxygen environment, in this study we have generated

conditional PRDX1-/- DT40 cells in which human
PRDX1 trans-gene was expressed under control of thetetracycline inducible promoter. PDRX1 depleted cells

showed lethal phenotype and the depletion of PRDX1
resulted in the accumulation of intracellular oxidative
stress. The lethality and increased of oxidative stress of
PRDX1 depleted cells was completely suppressed by
2-mercaptethanol. Our data, in contrast to SOD1 or
SOD2, suggested that PRDX1 has roles not only as antioxidant but also as coupled thiol-redox-dependent pathway in cells.
EXPERIMENTS: Chicken DT40 used in this study
were cultured in RPMI 1640 supplemented with 10%
fetal bovine serum, 1% chicken serum, and kanamycin at
39°C under 5% CO2. Conditional PRDX1-knock-out
DT40 cells were established and the expression of transgenic human PRDX1 is suppressed by treatment with 100
ng/ ml doxycycline (Dox). To assay the effect of various
antioxidants for cell growth, cells cultured in the presenceor absence of Dox for the indicated periods were treatedwith 200 µM L-ascorbic acid phosphate magnesium salt,
5 mM N-acetyl-L-cystein (NAC), 50µM 6-hydroxy-2,5,7,8-tetramethylchroman- 2-carboxylic acid (Trolox), 250µM 1,2-Dihydroxy-3,5-benzenedisulfonic acid (Tiron,) or
50µM 2-mercaptoethano (EtSH,). Generation of growth
curves has been described previously [1,2]. Intracellularoxidative stress was measured based on the intracellularperoxide-dependent oxidation of DCFH-DA (Molecular
Probes, USA).
RESULTS: Our previous work showed that SOD1 wasessential for cell viability and SOD2 was not essential,
but was required for normal cell growth [1,2]. Our intriguing observation from previous work is that lethality
observed in SOD1-/- cells was completely rescued in thepresence of ascorbic acid, L-ascorbic acid phosphate

Fig. 1. PDRX1 depleted
cells showed lethal phenotype. In contrast to
SOD1 or SOD2 depleted
cells, 2-mercaptethanolcompletely suppressedlethality of PRDX1 depleted cells.

Fig. 2. Intracellular ROS levels were increased upon depletion of Prdx1. 2-mercaptethanol completely compensatedwith regard to the increase intracellular ROS levels.

agnesium salt [2]. PDRX1 depleted cells also showedlethal phenotype. In contrast to lethality SOD1 depleted
cells, lethality of PRDX depleted cells was completely
suppressed by 2-mercaptethanol (Fig. 1). Because
PRDX1 is exclusively localized in cytoplasm, it was assumed that increased levels of overall oxidative levels
would be elevated followed by depletion of PRDX1. To
confirm so, the amounts of hydrogen peroxide in cytoplasm were determined by staining PRDX1 depleted cellswith DCFH. DCFH is cell-permeable fluorescence dye
reacting to a broad spectrum of cellular reactive oxygen
species reflected by intra-cellular oxidative stress. As
shown in Fig. 2, PRDX1-depletion caused an increase ofoxidative stress. In the presence of 2-mercaptethanol, it
was hardly detected the increase of oxidative stress in
PRDX1depleted cells. In contrast to SOD1 depleted cells,
an elevation of sister chromatid exchange (SCE) frequency was not seen in PRDX1 depleted cells.
The results revealed that PRDX1 was essential for viability and that depletion of PRDX1did not increase DNA
damages, suggesting that hydrogen peroxide is accumulated cytoplasm in PRDX1depleted cells might not impact on genome. Our data suggested that PRDX1 hasroles for coupled thiol-redox-dependent pathway in addition to roles of antioxidant in cells.
REFERENCES:[1] S. Takeda et al., Biochem. Biophys. Res. Commun.
379: 233-238 (2009).
[2] E. Inoue et al., J. Nucleic Acids, pii: 795946 (2010)

採択課題番号 23P7-7 細胞内オルガネラの質的管理機構不全の発癌過程への関与（京大・原子炉）田野恵三、渡邉正己（東北大・薬）井上絵里

プロジェクト