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3-36 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.
3-37 JAEA-Review 2010-065 Heavy-ion Irradiation Induces Autophagy in Irradiated C2C12 Myoblasts and Their Bystander Cells M. Hino a) , N. Hamada b) , Y. Tajika a) , T. Funayama c) , Y. Morimura a) , T. Sakashita c) , Y. Yokota c) , K. Fukamoto d) , Y. Mutou c) , Y. Kobayashi c) and H. Yorifuji a) a) Department of Anatomy, Graduate School of Medicine, Gunma University, b) Radiation Safety Research Center, Nuclear Technology Research Laboratory, Central Research Institute of Electric Power Industry, c) Radiation-Applied Biology Division, QuBS, JAEA, d) Faculty of Textile Science and Technology, Shinshu University The C2C12 myoblasts were irradiated with 40 Ar (11.2 MeV/u) and 20 Ne (12.8 MeV/u) and the induction of autophagy was assayed by electron microscopy, immunofluorescence staining using LC-3 antibody and LysoTracker Red staining. These morphological observations showed that autophagy is induced in the irradiated C2C12 myoblasts. In addition to irradiated cells, bystander cells were also positive with LysoTracker Red staining. Altogether, these results suggest that heavy ion-irradiation induces autophagy not only in irradiated myoblasts but also in their bystander cells. 我々はこれまでに骨格筋単離筋線維の重粒子線照射による構造変化を電子顕微鏡を用い解析を行ってきた 1, 2) 。その結果照射領域の細胞膜では不規則な突起と陥凹、基底板の断片化が見られた。また照射領域の細胞質では筋原線維の配向の乱れ、筋小胞体の内腔の拡大が見られた。またオートファジー小体が多数観察された。オートファジーは真核生物に普遍的な現象で、細胞質の一部を新たに形成された膜系が取り囲み、隔離し分解する機構である。オートファジーの基本的な生理的機能は、栄養飢餓状態で自己の細胞質を分解することでアミノ酸などの栄養素を産生することとされている。一方で一定の領域の細胞質やミトコンドリア等のオルガネラをまとめて分解することができる機構であることから飢餓応答以外にも障害をうけた細胞質領域やオルガネラをバルクに除去することで細胞質のホメオスタシスを維持していると考えられている。今回我々は、重粒子線照射により誘導されるオートファジーについてマウス由来の骨格筋芽細胞 C2C12 株を用いてより詳細な検討を行った。 (1) C2C12 細胞に重粒子線 ( 40 Ar, 11.2 MeV/u; 20 Ne, 12.8 MeV/u)を照射した後 30 分までの各時点で細胞を固定し透過型電子顕微鏡による観察を行った。その結果照射細胞においてオートファジー小体が観察された。 (2) LC-3 はオートファジーの初期に隔離膜が細胞質を隔離する過程に必須の因子である。オートファジーが活性化した細胞では LC-3 の抗体染色像の増強が見られることからオートファジーの初期過程のマーカーとして用いられている。そこで C2C12 細胞に重粒子線を照射後 30 分の時点で固定し、抗 LC-3 抗体を用いて蛍光抗体染色を行った。重粒子線照射により蛍光強度の増強が見られた。 (3) オートファジー小体ではリソソームと融合することで内容物の分解が行われるため、成熟したオートファジー小体の内側は酸性である。C2C12 細胞を酸性オルガネラのマーカーである LysoTracker 試薬存在下で培養し、重粒子線を照射後 30 分の時点で細胞を固定し、オートファジー小体形成を観察した。重粒子線照射により LysoTracker 染色の蛍光強度の増強が見られた。 - 93 - 以上三点の結果は筋芽細胞 C2C12 において重粒子線照射がオートファジーを誘導することを示している。 (4)バイスタンダー効果は被ばくした細胞から周辺の直接は被ばくしなかった細胞へ遠隔的に被ばくの情報が伝えられる現象である。重粒子線によるオートファジー誘導がバイスタンダー細胞に引き起こされるか検討した(Fig. 1)。その結果、オートファジーの誘導はバイスタンダー細胞にも引き起こされることが明らかになった。また、このバイスタンダー効果は周辺の細胞と物理的に接触していない細胞でも観察されたことから液性因子の関与が示唆される。近年がんに対する放射線治療においてアポトーシスと並んでオートファジー性細胞死が注目を集めている。重粒子線によるオートファジー誘導の解明は重粒子線治療の効果を高める上でも有意義と考えられる。 Fig. 1 LysoTracker Red staining of irradiated and bystander cells. Cells were sham-irradiated or 2 Gy-irradiated with or without shielding. Cells were fixed at 30 min after irradiation. (a) Sham-irradiated controls. (b) 2 Gy-irradiated cells without shielding. (c) 2 Gy-irradiated cells in the irradiated area of partially shielded sample. (d) Non-irradiated bystander cells in the shielded area of 2 Gy-irradiated sample. References 1) M. Hino et al., Cell Struct. Funct. 32 (2007) 51. 2) M. Hino et al., Cell Struct. Funct. 34 (2009) 11.
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JAEA-Review 2010-065 JAEA Takasaki
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JAEA-Review 2010-065 PREFACE This r
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JAEA-Review 2010-065 and pulsed hea
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JAEA-Review 2010-065 1-23 Studies o
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JAEA-Review 2010-065 3-24 Lethal Ef
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JAEA-Review 2010-065 4-07 Fabricati
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JAEA-Review 2010-065 5. Status of I
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JAEA-Review 2010-065 1-13 Alpha-rad
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1-01 Radiation Resistance of InGaP/
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1-03 Evaluation of Soft Error Rates
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1-05 Heavy-ion Induced Current in S
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Total Ionizing Dose Tolerance of Si
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1-09 Evaluation of Single Event Eff
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Hydrogen Diffusion in a-Si:H Thin F
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1-13 Alpha-radiolysis of Organic Ex
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Study on Stability of Cs·Sr Solven
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Irradiation Effect of Gamma-Rays on
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1-19 Development of Radiation Resis
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1-21 Alpha-Ray Irradiation Damage o
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1-23 Studies on Microstructure and
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1-25 Effect of Groundwater Radiolys
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1-27 Simulation of Neutron Damage M
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1-29 Irradiation Hardening in Ion-i
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1-31 Preparation of Anion-Exchange
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1-33 Radiation-Induced Graft Polyme
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JAEA-Review 2010-065 2. Environment
Page 58 and 59: 2-02 Development of Zwitterionic Mo
Page 60 and 61: Modification of Hydroxypropyl Cellu
Page 62 and 63: The Recovery of Precious Metals Usi
Page 64 and 65: 2-08 ESR Study on Carboxymethyl Chi
Page 67 and 68: JAEA-Review 2010-065 3. Biotechnolo
Page 69 and 70: JAEA-Review 2010-065 3-28 Electron
Page 71: JAEA-Review 2010-065 3-51 PET Studi
Page 74 and 75: 3-02 Mutagenic effects of He ion pa
Page 76 and 77: 3-04 Functional Analysis of Flavono
Page 78 and 79: 3-06 Generation New Ornamental Plan
Page 80 and 81: 3-08 Stability of Flower-colour Mut
Page 82 and 83: 3-10 JAEA-Review 2010-065 Effects o
Page 84 and 85: 3-12 Producing New Gene Resources i
Page 86 and 87: 3-14 Production of Soybean Mutants
Page 88 and 89: Assessment of Irradiation Treatment
Page 90 and 91: 3-18 Effect of Different LET Radiat
Page 92 and 93: 3-20 JAEA-Review 2010-065 Fungicide
Page 94 and 95: 3-22 JAEA-Review 2010-065 FACS-base
Page 96 and 97: 3-24 Lethal Effects of Different LE
Page 98 and 99: 3-26 JAEA-Review 2010-065 Ion Beam
Page 100 and 101: 3-28 Electron Spin Relaxation Behav
Page 102 and 103: 3-30 Target Irradiation of Individu
Page 104 and 105: 3-32 Carbon-ion Microbeam Induces B
Page 106 and 107: 3-34 Biological Effects of Carbon I
Page 110 and 111: 3-38 Analysis of Lethal Effect Medi
Page 112 and 113: 3-40 Irradiation with Carbon Ion Be
Page 114 and 115: 3-42 Expression of Two Gelsolins in
Page 116 and 117: 3-44 Analysis of Bystander Cell Sig
Page 118 and 119: 3-46 Quantitative Evaluation of Ric
Page 120 and 121: 3-48 Visualization of 107 Cd Accumu
Page 122 and 123: 3-50 Uniformity Measurement of Newl
Page 124 and 125: 3-52 Imaging and Biodistribution of
Page 126 and 127: 3-54 Improvement of Spatial Resolut
Page 128 and 129: 3-56 The Optimum Conditions in the
Page 130 and 131: 3-58 Evaluation of Cisplatin Concen
Page 132 and 133: 3-60 JAEA-Review 2010-065 Analysis
Page 134 and 135: 3-62 JAEA-Review 2010-065 Sensitivi
Page 136 and 137: JAEA-Review 2010-065 4-14 The Effec
Page 138 and 139: JAEA-Review 2010-065 4-39 Relations
Page 141 and 142: 4-01 Hydrogen Gasochromism of WO3 F
Page 143 and 144: 4-03 Synthesis of Single-Crystallin
Page 145 and 146: 4-05 Synergy Effects in Electron/Io
Page 147 and 148: Fabrication of Diluted Magnetic Sem
Page 149 and 150: 4-09 Gas Permeation Characteristics
Page 151 and 152: 4-11 Control of Radial Size of Poly
Page 153 and 154: 4-13 Behavior of N Atoms in Nitridi
Page 155 and 156: 4-15 JAEA-Review 2010-065 Annealing
Page 157 and 158: Low Temperature Ion Channeling of F
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4-19 Radiation-Induced Electrical D
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4-21 Study on Cu Precipitation in E
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4-23 Evaluation of Fluorescence Mat
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4-25 Evaluation of the ZrC Layer fo
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4-27 Structure Analysis of K/Si(111
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4-29 LET Effect on the Radiation In
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4-31 Stabilization of Measurement S
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Systematic Measurement of Neutron a
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4-35 Measurement of Neutron Fluence
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4-37 Evaluation of the Response Cha
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4-39 Relationship between Internucl
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4-41 Analysis of Radiation Damage a
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4-43 Positive Secondary Ion Emissio
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4-45 JAEA-Review 2010-065 Ion Induc
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4-47 Fabrication of Dielectrophoret
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4-49 Fast Single-Ion Hit System for
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4-51 Development of Beam Generation
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JAEA-Review 2010-065 5. Status of I
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Operation of the AVF Cyclotron T. N
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Operation of Electron Accelerator a
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5-06 FACILITY USE PROGRAM in Takasa
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5-08 Radioactive Waste Management i
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Appendix 2 List of Related Patents
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Appendix 4 Type of Research Collabo
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表1.SI 基本単位基本量 SI
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