Analysis of micronucleus/chromotrypsis formation process by inducible Dic.

• Project/Area Number: 18K11647
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 63020:Radiation influence-related
• Research Institution: Hiroshima University (2023); Fukushima Medical University (2018-2022)
• Principal Investigator: 津山 尚宏 広島大学, PSI GMP教育研究センター, 主幹特任学術研究員 (10335747)
• Co-Investigator(Kenkyū-buntansha): 阿部 悠 長崎大学, 原爆後障害医療研究所, 助教 (00722472) ; 柳 亜希 福島県立医科大学, 医学部, 助手 (60803525) ; 坂井 晃 福島県立医科大学, 医学部, 教授 (70284221)
• Project Period (FY): 2018-04-01 – 2024-03-31
• Project Status: Completed (Fiscal Year 2023)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2020: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2019: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2018: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
• Keywords: 二動原体染色体 / 微小核 / クロモトリプシス / 放射線発がん / ゲノム編集

Outline of Annual Research Achievements

電離放射線は細胞ゲノムDNAにDNA二本鎖切断(DSB)を引き起こす。修復の過程で元の切断端同士が再結合せず異なった切断端が再結合した場合、染色体転座や二動原体染色体などの染色体異常となる。セントロメアを持つ染色体断片同士、持たない断片同士が結合した二動原体染色体(Dic)および染色体断片は、分裂中期の染色体分配調節が正常に進行せず細胞分裂終了後に細胞核に取り込まれることなく微小核(MN)形成を形成することが多い。微小核は細胞質のDNAに対する自然免疫応答機構を介して炎症応答を惹起すると同時に核酸分解酵素の作用により断片化し、次の細胞周期で核内に取り込まれ修復されることにより複雑に融合した破砕染色体（クロモトリプシス）の原因となる。
この経路を効率よく解析するために誘導実験系の確立を試みた。HEK293細胞およびiPS細胞の14番染色体IgH座位および11番染色体CCND1座位5’領域のそれぞれに、loxPを相同組換により導入したクローンを作成した。loxPは転座あるいはDic が生成されるよう組み換えた。iCreあるいはERT2-Creを導入したが転座あるいはDicの誘導効率が低く、Dic解析に十分な細胞数を得ることができなかった。同様に、染色体転座については、転座前後のiPS細胞を用いて遺伝子発現の変動解析を行った。
CRISPR-Cas9を用いた染色体異常誘導系として、多発性骨髄腫で観察されるt(11;14), 線維形成性小細胞腫瘍のt(11;22), 非小細胞肺がんのt(5;6)を作成・誘導を確認した。それぞれの特異的転座と同時に同じ組み合わせの二動原体染色体が生じていることをゲノムDNAを用いたPCRにより確認した。shRNA導入によるノックダウン細胞を作成し、これらの染色体異常誘導系をそれぞれ導入し、染色体異常頻度の変動を検討した。

Research Products

• [Journal Article] Influence of matrix effects by inorganic ions in single-cell direct mass spectrometry (2023)
  • Author(s): Kato Y, Kudoh S, Sugiyama E, Sakane I, Tsuyama N, Toyo’oka T, Todoroki K, Hajime Mizuno
  • Journal Title: Medical Mass Spectrometry Volume: 7 Issue: 1 Pages: 43-52
  • DOI: 10.24508/mms.2023.06.003
  • ISSN: 2432-7441, 2432-745X
  • Year and Date: 2023-06-25
  • Peer Reviewed / Open Access
• [Journal Article] No increase in translocated chromosomal aberrations, an indicator of ionizing radiation exposure, in childhood thyroid cancer in Fukushima Prefecture. (2023)
  • Author(s): Sakai A, Tsuyama N, Ohira T, Sugai-Takahashi M, Ohba T, Azami Y, Matsumoto Y, Iwadate Manabu, Suzuki S, Sato M, Hosoya M, Ishikawa T, Suzuki S.
  • Journal Title: Scientific Reports Volume: 13 Issue: 1 Pages: 14254-14254
  • DOI: 10.1038/s41598-023-41501-x
  • Peer Reviewed / Open Access
• [Journal Article] RENEB Inter-Laboratory Comparison 2021: Inter-Assay Comparison of Eight Dosimetry Assays (2023)
  • Author(s): Port M, Barquinero JF, Endesfelder D, Moquet J, Oestreicher U, Terzoudi G, Trompier F, Vral A, Abe Y, Ainsbury L, Alkebsi L, Amundson SA, Badie C, Baeyens A, Balajee AS, Balazs K, Barnard S, Bassinet C, Beaton-Green LA, Beinke C, Bobyk L, Brochard P, Brzoska K, Bucher M, Ciesielski B, Cuceu C, Discher M et al.
  • Journal Title: Radiation Research Volume: 199 Issue: 6 Pages: 535-555
  • DOI: 10.1667/rade-22-00207.1
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] RENEB Inter-Laboratory Comparison 2021: The Dicentric Chromosome Assay. (2023)
  • Author(s): Endesfelder D, Oestreicher U, Bucher M, Beinke C, Siebenwirth C, Ainsbury E, Moquet J, Gruel G, Gregoire E, Martinez JS, Vral A, Baeyens A, Valente M, Montoro A, Terzoudi G, Triantopoulou S, Pantelias A, Monteiro Gil O, Prieto MJ, Domene MM, Zafiropoulos D, Barquinero JF, Pujol-Canadell M, Lumniczky K et al.
  • Journal Title: Radiation Research Volume: 199 Issue: 6 Pages: 556-570
  • DOI: 10.1667/rade-22-00202.1
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] RENEB Inter-Laboratory Comparison 2021: The FISH-Based Translocation Assay (2023)
  • Author(s): Barquinero JF, Abe Y, Aneva N, Endesfelder D, Georgieva D, Goh V, Gregoire E, Hristova R, Lee Y, Martinez JS, Meher PK, Miura T, Port M, Pujol-Canadell M, Prieto-Rodriguez MJ, Seong KM, Suto Y, Takebayashi K, Tsuyama N, Wojcik A, Yoon HJ, Abend M.
  • Journal Title: Radiation Research Volume: 199 Issue: 6 Pages: 583-590
  • DOI: 10.1667/rade-22-00203.1
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Chromosomal translocation t(11;14) and p53 deletion induced by the CRISPR/Cas9 system in normal B cell-derived iPS cells (2021)
  • Author(s): Yusuke Azami, Naohiro Tsuyama, Yu Abe, Misaki Sugai-Takahashi, Ken-Ichi Kudo, Akinobu Ota, Karnan Sivasundaram , Moe Muramatsu, Tomonari Shigemura, Megumi Sasatani, Yuko Hashimoto, Shigehira Saji, Kenji Kamiya, Ichiro Hanamura, Takayuki Ikezoe, Masafumi Onodera, Akira Sakai.
  • Journal Title: Sci Rep Volume: 11 Issue: 1 Pages: 5216-5216
  • DOI: 10.1038/s41598-021-84628-5
  • Peer Reviewed / Open Access
• [Journal Article] Induction of t(11;14) IgH enhancer/promoter-Cyclin D1 gene translocation using CRISPR/Cas9. (2019)
  • Author(s): Tsuyama N, Abe Y, Yanagi A, Yanai Y, Sugai M, Katafuchi A, Kawamura F, Kamiya K, Sakai A.
  • Journal Title: Oncology Letters. 18 : 275-282, 2019. Volume: 18 Pages: 275-282
  • DOI: 10.3892/ol.2019.10303
  • Peer Reviewed / Open Access
• [Journal Article] Dose-response curves for analyzing of dicentric chromosomes and chromosome translocation following doses of 1,000 mGy or less based on irradiated peripheral blood samples from 5 healthy individuals. (2018)
  • Author(s): Abe Y, Yoshida MA, Fujioka K, Kurosu Y, Ujiie R, Yanagi A, Tsuyama N, Miura T, Inaba T, Kamiya K, Sakai A
  • Journal Title: Journal of Radiation Research Volume: 59 Issue: 1 Pages: 35-42
  • DOI: 10.1093/jrr/rrx052
  • Peer Reviewed / Open Access
• [Presentation] Analysis of differential gene expression in t(11;14) carrying iPS cells induced by genome editing and Cre-loxP. (2023)
  • Author(s): Naohiro Tsuyama, Yu Abe, Misaki Sugai Takahashi, Kenji Kamiya, Akira Sakai
  • Organizer: The 7th International Symposium of the Network-type Joint Usage/Research Center for Radiation Disaster Medical Science
  • Int'l Joint Research
• [Presentation] Analysis of differential gene expression in t(11;14) carrying iPS cells induced by genome editing (2022)
  • Author(s): Naohiro Tsuyama, Kenichi Kudo, Misaki Sugai Takahashi, Yusuke Azami, Kenji Kamiya, Akira Sakai.
  • Organizer: 第65回日本放射線影響学会
• [Presentation] Optimization of chromosome specimen preparation for biodosimetry using chromosome aberration frequency. (2021)
  • Author(s): Naohiro Tsuyama, Yu Abe, Misaki Sugai-Takahashi, Kenichi Kudo, Yusuke Azami, Miwa Fukami, Tomisato Miura, Kenji Kamiya, Akira Sakai
  • Organizer: 第64回日本放射線影響学会
• [Presentation] Study for exploring myeloma-initiating cell using normal B cell-derived iPS cells. (2021)
  • Author(s): Yusuke Azami, Naohiro Tsuyama, Yu Abe, Misaki Sugai-Takahashi, Ken-ichi Kudo, Miwa Fukami, Akinobu Ota, Karnan Sivasundaram, Moe Muramatsu, Tomonari Shigemura, Megumi Sasatani, Yuko Hashimoto, Shigehira Saji, Kenji Kamiya, Ichiro Hanamura, Takayuki Ikezoe, Masafumi Onodera, Akira Sakai
  • Organizer: 第83回日本血液学会学術集会
• [Presentation] Chromosomal translocation t(11;14) induced by Cre-loxP system in normal B cell-derived iPS cells. (2020)
  • Author(s): Misaki Sugai, Naohiro Tsuyama, Yu Abe*, Yusuke Azami*, Akinobu Ota, Karnan Sivasundaram, Moe Muramatsu, Tomonari Shigemura, Megumi Sasatani, Yuko Hashimoto, Kenji Kamiya, Ichiro Hanamura, Takayuki Ikezoe, Masafumi Onodera, Akira Sakai.
  • Organizer: 第82回日本血液学会学術集会
• [Presentation] Induction of sequence-specific chromosomal aberration using CRISPR/Cas9 and Cre-loxP. (2020)
  • Author(s): Naohiro Tsuyama, Yu Abe, Misaki Sugai, Yusuke Azami, Ken-ichi Kudo, Kenji Kamiya, Akira Sakai.
  • Organizer: 第63回日本放射線影響学会
• [Presentation] Investigation of naturally occurring chromosome aberrations and individual radiosensitivity in Japanese. (2020)
  • Author(s): Yu Abe, Misaki Sugai, Naohiro Tsuyama, Ken-ichi Kudo, Yusuke Azami, Akira Sakai
  • Organizer: 第63回日本放射線影響学会
• [Presentation] Chromosomal Translocation t(11;14) Induced by the Cre-loxP System in Normal B cell-derived iPS Cells for the Study of Myeloma-initiating Cells. (2020)
  • Author(s): Misaki Sugai, Naohiro Tsuyama, Yu Abe, Yusuke Azami, Kenichi Kudo, Akinobu Ota, Karnan Sivasundaram, Moe Muramatsu, Tomonari Shigemura, Megumi Sasatani, Yuko Hashimoto, Kenji Kamiya, Ichiro Hanamura, Takayuki Ikezoe, Masafumi Onodera, Akira Sakai.
  • Organizer: 62th ASH Annual Meeting.
  • Int'l Joint Research
• [Presentation] CRISPR/Cas9を用いた染色体転座t(11;14) 誘導系の作製と染色体異常解析への応用 (2019)
  • Author(s): 津山尚宏、阿部悠、柳亜紀、菅井美咲、神谷研二、坂井晃
  • Organizer: 第4回放射線災害・医科学研究拠点カンファランス
• [Presentation] Chromosomal translocation t(11;14) and p53 deletion in B cell-derived iPS cells by CRISPR/Cas9. (2019)
  • Author(s): Aki Yanagi, Naohiro Tsuyama, Misaki Sugai, Yu Abe, Yukari Yanai, Akinobu Ota, Karnan Sivasundaram, Lobna Alkebsi, Moe Muramatsu, Tomonari Shigemura, Megumi Sasatani, Yuko Hashimoto, Kenji Kamiya, Ichiro Hanamura, Takayuki Ikezoe, Masafumi Onodera, Akira Sakai
  • Organizer: 第81回日本血液学会学術集会
• [Presentation] CRISPR/Cas9を用いた配列特異的DNA二本鎖切断と染色体異常の解析 (2019)
  • Author(s): Naohiro Tsuyama, Yu Abe, Aki Yanagi, Misaki Sugai, Kenji Kamiya, Akira Sakai
  • Organizer: 第61回日本放射線影響学会
• [Presentation] Introduction of Chromosomal Translocation t(11; 14) and a p53 Deletion into Normal B Cell-derived iPSCs to Elucidate the Cellular Origin of Myeloma Cells. (2019)
  • Author(s): Yanagi A, Tsuyama N, Sugai M, Abe Y, Azami Y, Yanai Y, Ota A, Sivasundaram K, Muramatsu M, Shigemura T, Sasatani M, Hashimot Y, Kamiya K, Hanamura I, Ikezoe T, Onodera M, Sakai A
  • Organizer: 61th ASH Annual Meeting
  • Int'l Joint Research
• [Presentation] Analysis of the number of chromosome aberrations induced by three consecutive CT examinations. (2019)
  • Author(s): Yu Abe, Hideyoshi Noji, Misaki Sugai, Yumiko Kurosu, Naohiro Tsuyama, Aki Yanagi, Yukari Yanai, Takashi Ohba, Tetsuo Ishikawa, Tomisato Miura, Kenji Kamiya, Mitsuaki A. Yoshida, Akia Sakai
  • Organizer: 放射線災害・医科学研究拠点 第３回国際シンポジウム
  • Int'l Joint Research
• [Presentation] Induction of t(11;14) IgH enhancer/promoter-cyclin D1 gene translocation using CRISPR/Cas9. (2019)
  • Author(s): Naohiro Tsuyama, Yu Abe, Aki Yanagi, Misaki Sugai, Kenji Kamiya, Akira Sakai
  • Organizer: 放射線災害・医科学研究拠点 第３回国際シンポジウム
  • Int'l Joint Research
• [Presentation] CRISPR/Cas9を用いたt(11; 14)染色体転座誘発 (2018)
  • Author(s): 津山尚宏、阿部悠、柳亜紀、菅井美咲、神谷研二、坂井晃
  • Organizer: 第61回日本放射線影響学会
• [Presentation] Investigation of the cumulative number of chromosome aberrations induced by three consecutive CT scans. (2018)
  • Author(s): Yu Abe, Hideyoshi Noji, Misaki Sugai, Yumiko Kurosu, Takashi Ohba, Naohiro Tsuyama, Aki Yanagi, Yukari Yanai, Tetsuo Ishikawa, Tomisato Miura, Kenji Kamiya, Mitsuaki A Yoshida, Akira Sakai
  • Organizer: American Association for Cancer Research Annual Meeting 2018.
  • Int'l Joint Research
• [Presentation] Construction of dose response curves for cytogenetic biodosimetry in the low dose range based on five persons. (2018)
  • Author(s): Yu Abe, Mitsuaki A Yoshida, Kurumi Fujioka, Yumiko Kurosu, Risa Ujiie, Aki Yanagi, Naohiro Tsuyama, Tomisato Miura, Toshiya Inaba, Kenji Kamiya, Akira Sakai
  • Organizer: EPRBioDose 2018
  • Int'l Joint Research
• [Presentation] Analysis of the number of chromosome aberrations induced by three consecutive CT examinations. (2018)
  • Author(s): Abe Y, Noji H, Sugai M, Kurosu Y, Tsuyama N, Yanagi A, Yanai Y, Ohba T, Ishikawa T, Miura T, Kamiya K, Yoshida MA, Sakai A
  • Organizer: 第61回日本放射線影響学会
• [Presentation] ttempt to make abnormal B lymphocytes as myeloma-initiating cells from iPSs. (2018)
  • Author(s): 柳 亜希、津山尚宏、柳井祐佳里、阿部 悠、菅井美咲、太田明伸、シバスンダラン カルナン、笹谷めぐみ、神谷研二、花村一朗、池添隆之、小野寺雅史、坂井 晃
  • Organizer: 第80回日本血液学会学術集会
• [Presentation] Attempt to prove the existence of abnormal B lymphocyte as myeloma-initiating cells from B cell-derived induced pluripotent stem cells. (2018)
  • Author(s): Yanagi A, Tsuyama N, Yanai Y, Abe Y, Sugai M, Ota A, Sivasundaram K, Shigemura T, Sasatani M, Kamiya K, Hanamura I, Ikezoe T, Onodera M, Sakai A
  • Organizer: 60th ASH Annual Meeting.
  • Int'l Joint Research