Details of a Researcher - Shibata, Atsushi

Oyoshi, H; Du, JY; Sakai, SA; Yamashita, R; Okumura, M; Nakamura, M; Hirata, H; Sunakawa, H; Yano, T; Kojima, T; Kojima, M; Tsuchihara, K; Akimoto, T; Shibata, A; Suzuki, Y; Kageyama, S

CANCER SCIENCE 115 1972 - 1972 2024.03

ISSN 1347-9032

Relationship between HLA Class-1 expression and tumor microenvironment in inflammatory colorectal carcinogenesis

Okami, H; Ozawa, N; Yokobori, T; Shibasaki, Y; Komine, C; Okada, T; Shiraishi, T; Osone, K; Sakai, M; Sano, A; Ogawa, H; Sohda, M; Shirabe, K; Shibata, A; Saeki, H

CANCER SCIENCE 115 518 - 518 2024.03

ISSN 1347-9032

ATR signaling controls the bystander responses of human chondrosarcoma cells by promoting RAD51-dependent DNA repair

Nho Cong Luong, Hidemasa Kawamura, Hiroko Ikeda, Reiko T. Roppongi, Atsushi Shibata, Jiaxuan Hu, Jinmeng G. Jiang, David S. Yu, Kathryn D. Held

International Journal of Radiation Biology (Taylor & Francis group) 100 ( 5 ) 1 - 12 2024

Research paper (scientific journal), Joint Work, Accepted, ISSN 0955-3002

Purpose
Radiation-induced bystander effect (RIBE) frequently is seen as DNA damage in unirradiated bystander cells, but the repair processes initiated in response to that DNA damage are not well understood. RIBE-mediated formation of micronuclei (MN), a biomarker of persistent DNA damage, was previously observed in bystander normal fibroblast (AG01522) cells, but not in bystander human chondrosarcoma (HTB94) cells. The molecular mechanisms causing this disparity are not clear. Herein, we investigate the role of DNA repair in the bystander responses of the two cell lines.

Methods
Cells were irradiated with X-rays and immediately co-cultured with un-irradiated cells using a trans-well insert system in which they share the same medium. The activation of DNA damage response (DDR) proteins was detected by immunofluorescence staining or Western blotting. MN formation was examined by the cytokinesis-block MN assay, which is a robust method to detect persistent DNA damage.

Results
Immunofluorescent foci of γH2AX and 53BP1, biomarkers of DNA damage and repair, revealed a greater capacity for DNA repair in HTB94 cells than in AG01522 cells in both irradiated and bystander populations. Autophosphorylation of ATR at the threonine 1989 site was expressed at a greater level in HTB94 cells compared to AG01522 cells at the baseline and in response to hydroxyurea treatment or exposure to 1 Gy of X-rays. An inhibitor of ATR, but not of ATM, promoted MN formation in bystander HTB94 cells. In contrast, no effect of either inhibitor was observed in bystander AG01522 cells, indicating that ATR signaling might be a pivotal pathway to preventing the MN formation in bystander HTB94 cells. Supporting this idea, we found an ATR-dependent increase in the fractions of bystander HTB94 cells with pRPA2 S33 and RAD51 foci. A blocker of RAD51 facilitated MN formation in bystander HTB94 cells.

Conclusion
Our results indicate that HTB94 cells were likely more efficient in DNA repair than AG01522 cells, specifically via ATR signaling, which inhibited the bystander signal-induced MN formation. This study highlights the significance of DNA repair efficiency in bystander cell responses.

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Characterization of the signal transduction cascade for inflammatory gene expression in fibroblasts with ATM-ATR deficiencies after Ionizing radiation

Shunji Haruna, Ken Okuda, Akiko Shibata, Mayu Isono, Kohei Tateno, Hiro Sato, Takahiro Oike, Yuki Uchihara, Yu Kato, Atsushi Shibata

Radiotherapy and Oncology (Elsevier) 194 110198 - 110198 2024

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted, ISSN 0167-8140

Background and purpose
Ionizing radiation (IR) induces DNA double-strand breaks (DSBs), leading to micronuclei formation, which has emerged as a key mediator of inflammatory responses after IR. This study aimed to investigate the signaling cascade in inflammatory gene expression using fibroblasts harboring DNA damage response deficiency after exposure to IR.
Materials and methods
Micronuclei formation was examined in human dermal fibroblasts derived from patients with deficiencies in ATM, ATR, MRE11, XLF, Artemis, or BRCA2 after IR. RNA-sequencing analysis was performed to assess gene expression, pathway mapping, and the balance of transcriptional activity using the transcription factor–based downstream gene expression mapping (TDEM) method developed in this study.
Results
Deficiencies in ATM, ATR, or MRE11 led to increased micronuclei formation after IR compared to normal cells. RNA-seq analysis revealed significant upregulation of inflammatory expression in cells deficient in ATM, ATR, or MRE11 following IR. Pathway mapping analysis identified the upregulation of RIG-I, MDA-5, IRF7, IL6, and interferon stimulated gene expression after IR. These changes were pronounced in cells deficient in ATM, ATR, or MRE11. TDEM analysis suggested the differential activation of STAT1/3–pathway between ATM and ATR deficiency.
Conclusion
Enhanced micronuclei formation upon ATM, ATR, or MRE11 deficiency activated the cGAS/STING, RIG-I-MDA-5-IRF7-IL6 pathway, resulting in its downstream interferon stimulated gene expression following exposure to IR. Our study provides comprehensive information regarding the status of inflammation-related gene expression under DSB repair deficiency after IR. The generated dataset may be useful in developing functional biomarkers to accurately identify patients sensitive to radiotherapy.

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Inhibition of intracellular ATP synthesis impairs the recruitment of homologous recombination factors after ionizing radiation

Ryota Hayashi, Hikaru Okumura, Mayu Isono, Motohiro Yamauchi, Daiki Unami, Rahmartani Tania Lusi, Masamichi Yamamoto, Yu Kato, Yuki Uchihara, Atsushi Shibata

Journal of Radiation Research (Oxford University Press) 65 ( 3 ) 263 - 271 2024

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted, ISSN 0449-3060

Ionizing radiation (IR)-induced double-strand breaks (DSBs) are primarily repaired by non-homologous end joining or homologous recombination (HR) in human cells. DSB repair requires adenosine-5′-triphosphate (ATP) for protein kinase activities in the multiple steps of DSB repair, such as DNA ligation, chromatin remodeling, and DNA damage signaling via protein kinase and ATPase activities. To investigate whether low ATP culture conditions affect the recruitment of repair proteins at DSB sites, IR-induced foci were examined in the presence of ATP synthesis inhibitors. We found that p53 binding protein 1 foci formation was modestly reduced under low ATP conditions after IR, although phosphorylated histone H2AX and mediator of DNA damage checkpoint 1 foci formation were not impaired. Next, we examined the foci formation of breast cancer susceptibility gene I (BRCA1), replication protein A (RPA) and radiation 51 (RAD51), which are HR factors, in G2 phase cells following IR. Interestingly, BRCA1 and RPA foci in the G2 phase were significantly reduced under low ATP conditions compared to that under normal culture conditions. Notably, RAD51 foci were drastically impaired under low ATP conditions. These results suggest that HR does not effectively progress under low ATP conditions; in particular, ATP shortages impair downstream steps in HR, such as RAD51 loading. Taken together, these results suggest that the maintenance of cellular ATP levels is critical for DNA damage response and HR progression after IR.

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Involvement of the splicing factor SART1 in the BRCA1-dependent homologous recombination repair of DNA double-strand breaks

Kie Ozaki, Reona Kato, Takaaki Yasuhara, Yuki Uchihara, Miyako Hirakawa, Yu Abe, Hiroki Shibata, Reika Kawabata-Iwakawa, Aizhan Shakayeva, Palina Kot, Kiyoshi Miyagawa, Keiji Suzuki, Naoki Matsuda, Atsushi Shibata & Motohiro Yamauchi

Scientific Reports (Springer Science and Business Media LLC) 14(1):18455 ( 1 ) 18455 2024

Accepted, ISSN 2045-2322

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Comprehensive single cell analysis demonstrates radiotherapy-induced infiltration of macrophages expressing immunosuppressive genes into tumour in oesophageal squamous cell carcinoma

Hidekazu Oyoshi, Junyan Du, Shunsuke A Sakai, Riu Yamashita, Masayuki Okumura, Atsushi Motegi, Hidehiro Hojo, Masaki Nakamura, Hidenari Hirata, Hironori Sunakawa, Daisuke Kotani, Tomonori Yano, Takashi Kojima, Yuka Nakamura, Motohiro Kojima, Ayako Suzuki, Junko Zenkoh, Katsuya Tsuchihara, Tetsuo Akimoto, Atsushi Shibata, Yutaka Suzuki, Shun-Ichiro Kageyama

Science Advances (American Association for the Advancement of Science) 9 ( 50 ) eadh9069 2023.12

Research paper (scientific journal), Joint Work, Corresponding author, Accepted

Radiotherapy (RT) combined with immunotherapy is promising; however, the immune response signature in the clinical setting after RT remains unclear. Here, by integrative spatial and single-cell analyses using multiplex immunostaining (CODEX), spatial transcriptome (VISIUM), and single-cell RNA sequencing, we substantiated the infiltration of immune cells into tumors with dynamic changes in immunostimulatory and immunosuppressive gene expression after RT. In addition, our comprehensive analysis uncovered time- and cell type–dependent alterations in the gene expression profile after RT. Furthermore, myeloid cells showed prominent up-regulation of immune response–associated genes after RT. Notably, a subset of infiltrating tumor-associated myeloid cells showing PD-L1 positivity exhibited significant up-regulation of immunostimulatory (HMGB1 and ISG15), immunosuppressive (SIRPA and IDO1), and protumor genes (CXCL8, CCL3, IL-6, and IL-1AB), which can be targets of immunotherapy in combination with PD-L1. These datasets will provide information on the RT-induced gene signature to seek an appropriate target for personalized immunotherapy combined with RT and guide the timing of combination therapy.

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Regulation of DNA damage-induced HLA class I presentation

Yuki Uchihara, Atsushi Shibata

DNA Repair (Elsevier BV) 132 103590 - 103590 2023.12

ISSN 1568-7864

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時空間解析による放射線治療による食道がん患者組織での免疫環境変化の解析

大吉秀和, 山下理宇, 茂木厚, 北條秀博, 小谷大輔, 小島隆嗣, 中村優香, 小嶋基寛, 鈴木絢子, 善光純子, 土原一哉, 秋元哲夫, 柴田淳史, 鈴木穣, 影山俊一郎

日本癌治療学会学術集会抄録集 ((一社)日本癌治療学会) 61回 O45 - 2 2023.10

HLA Class I Expression Is Associated with DNA Damage and Immune Cell Infiltration into Dysplastic and Neoplastic Lesions in Ulcerative Colitis.

Okami H, Ozawa N, Sohda M, Yokobori T, Osone K, Erkhem-Ochir B, Dorjkhorloo G, Shiraishi T, Okada T, Sano A, Sakai M, Miyazaki T, Ogawa H, Yao T, Oike T, Sato H, Shirabe K, Shibata A, Saeki H

International journal of molecular sciences (MDPI) 24 ( 17 ) 13648 - 13648 2023.09

Research paper (scientific journal), Joint Work, Accepted, ISSN 1661-6596

Human leukocyte antigen class I (HLA-I) is considered a genetic pathogen for ulcerative colitis (UC). This study aimed to investigate the significance of DNA damage and HLA-I expression in infiltrating immune cells and immune checkpoint protein PD-L1 expression in dysplasia/colitic cancer (CC) and sporadic colorectal cancer (SCRC). We performed immunohistochemical staining for HLA-I, PD-L1, γH2AX (DNA damage marker), and immune cell markers such as CD8, FOXP3, CD68, and CD163 (in surgically resected specimens from 17 SCRC patients with 12 adjacent normal mucosa (NM) and 9 UC patients with 18 dysplasia/CC tumors. The ratio of membrane HLA-I-positive epithelial cells in UC and dysplasia/CC tissues was significantly higher than that in NM and SCRC. High HLA-I expression in dysplasia/CC was associated with high positivity of γH2AX and PD-L1 expression compared to SCRC. The infiltration of CD8-positive T cells and CD68-positive macrophages in HLA-I-high dysplasia/CC was significantly higher than in UC and SCRC. Dysplasia/CC specimens with DNA damage exhibited high levels of HLA-I-positive epithelial cells with high CD8- and CD68-positive immune cell infiltration compared to UC and SCRC specimens. Targeting DNA damage in UC may regulate immune cell infiltration, immune checkpoint proteins, and carcinogenesis by modulating DNA damage-induced HLA-I antigen presentation.

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Analysis of the relationship between LET, γH2AX foci volume and cell killing effect of carbon ions using high-resolution imaging technology

Takahiro Oike, Sangeeta Kakoti, Makoto Sakai, Akihiko Matsumura, Tatsuya Ohno, Atsushi Shibata

Journal of Radiation Research (Oxford University Press (OUP)) 64 ( 2 ) 335 - 344 2023.03

Research paper (scientific journal), Joint Work, Last author, Accepted, ISSN 0449-3060

Abstract

The strong cell killing effect of high linear energy transfer (LET) carbon ions is dependent on lethal DNA damage. Our recent studies suggest that induction of clusters of double-strand breaks (DSBs) in close proximity is one of the potential mechanisms. However, the relationship between LET, the degree of DSB clustering and the cell killing effect of carbon ions remains unclear. Here, we used high-resolution imaging technology to analyze the volume of γH2AX foci induced by monoenergetic carbon ions with a clinically-relevant range of LET (13–100 keV/μm). We obtained data from 3317 γH2AX foci and used a gaussian function to approximate the probability (p) that 1 Gy-carbon ions induce γH2AX foci of a given volume (vth) or greater per nucleus. Cell killing effects were assessed in clonogenic assays. The cell killing effect showed high concordance with p at vth = 0.7 μm3 across various LET values; the difference between the two was 4.7% ± 2.2%. This relationship was also true for clinical carbon ion beams harboring a mixed LET profile throughout a spread-out Bragg peak width (30–120 mm), with the difference at vth = 0.7 μm3 being 1.6% ± 1.2% when a Monte Carlo simulation-derived dose-averaged LET was used to calculate p. These data indicate that the cell killing effect of carbon ions is predictable by the ability of carbon ions to induce γH2AX foci containing clustered DSBs, which is linked to LET, providing the biological basis for LET modulation in the planning of carbon ion radiotherapy.

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Calreticulin Upregulation in Cervical Cancer Tissues From Patients After 10 Gy Radiation Therapy

Kohei Okada, Hiro Sato, Takuya Kumazawa, Yasumasa Mori, Tiara Bunga Mayang Permata, Yuki Uchihara, Shin-ei Noda, Keiji Suzuki, Hayato Ikota, Hideaki Yokoo, Soehartati Gondhowiardjo, Takashi Nakano, Tatsuya Ohno, Atsushi Shibata

Advances in Radiation Oncology (Elsevier BV) 8 ( 3 ) 101159 2022.12

Research paper (scientific journal), Joint Work, Last author, Accepted, ISSN 2452-1094

Purpose: Understanding the immune response during radiation therapy (RT) in a clinical setting is imperative for maximizing the efficacy of combined RT and immunotherapy. Calreticulin, a major damage-associated molecular pattern that is exposed on the cell surface after RT, is presumed to be associated with the tumor-specific immune response. Here, we examined changes in calreticulin expression in clinical specimens obtained before and during RT and analyzed its relationship with the density of CD8+ T cells in the same patient set. Methods and Materials: This retrospective analysis evaluated 67 patients with cervical squamous cell carcinoma who were treated with definitive RT. Tumor biopsy specimens were collected before RT and after 10 Gy irradiation. Calreticulin expression in tumor cells was evaluated via immunohistochemical staining. Subsequently, the patients were divided into 2 groups according to the level of calreticulin expression, and the clinical outcomes were compared. Finally, the correlation between calreticulin levels and density of stromal CD8+ T cells was evaluated. Results: The calreticulin expression significantly increased after 10 Gy (82% of patients showed an increase; P <.01). Patients with increased calreticulin levels tended to show better progression-free survival, but this was not statistically significant (P =.09). In patients with high expression of calreticulin, a positive trend was observed between calreticulin and CD8+ T cell density, but the association was not statistically significant (P =.06). Conclusions: Calreticulin expression increased after 10 Gy irradiation in tissue biopsies of patients with cervical cancer. Higher calreticulin expression levels are potentially associated with better progression-free survival and greater T cell positivity, but there was no statistically significant relationship between calreticulin upregulation and clinical outcomes or CD8+ T cell density. Further analysis will be required to clarify mechanisms underlying the immune response to RT and to optimize the RT and immunotherapy combination approach.

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DNA damage promotes HLA class I presentation by stimulating a pioneer round of translation-associated antigen production.

Yuki Uchihara, Tiara Bunga Mayang Permata, Hiro Sato, Reika Kawabata-Iwakawa, Sayako Katada, Wenchao Gu, Sangeeta Kakoti, Motohiro Yamauchi, Reona Kato, Soehartati Gondhowiardjo, Naoki Hosen, Takaaki Yasuhara, Atsushi Shibata

Molecular cell (Cell Press) 82 ( 14 ) 2557 - 2570 2022.07

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted, ISSN 10972765

Antigen presentation by the human leukocyte antigen (HLA) on the cell surface is critical for the transduction of the immune signal toward cytotoxic T lymphocytes. DNA damage upregulates HLA class I presentation; however, the mechanism is unclear. Here, we show that DNA-damage-induced HLA (di-HLA) presentation requires an immunoproteasome, PSMB8/9/10, and antigen-transporter, TAP1/2, demonstrating that antigen production is essential. Furthermore, we show that di-HLA presentation requires ATR, AKT, mTORC1, and p70-S6K signaling. Notably, the depletion of CBP20, a factor initiating the pioneer round of translation (PRT) that precedes nonsense-mediated mRNA decay (NMD), abolishes di-HLA presentation, suggesting that di-antigen production requires PRT. RNA-seq analysis demonstrates that DNA damage reduces NMD transcripts in an ATR-dependent manner, consistent with the requirement for ATR in the initiation of PRT/NMD. Finally, bioinformatics analysis identifies that PRT-derived 9-mer peptides bind to HLA and are potentially immunogenic. Therefore, DNA damage signaling produces immunogenic antigens by utilizing the machinery of PRT/NMD.

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Radiosensitization by the Selective Pan-FGFR Inhibitor LY2874455.

Narisa Dewi Maulany Darwis, Eisuke Horigome, Shan Li, Akiko Adachi, Takahiro Oike, Atsushi Shibata, Yuka Hirota, Tatsuya Ohno

Cells (MDPI) 11 ( 11 ) 2022.06

Research paper (scientific journal), Joint Work, Accepted

Ionizing radiation activates cytoprotective pathways in cancer cells. Fibroblast growth factor receptor (FGFR) is a key player in these pathways. Thus, FGFR signaling is a potential target to induce radiosensitization. LY2874455 is an orally administrable selective pan-FGFR inhibitor. However, the radiosensitizing effects of LY2874455 remain unclear. In this study, we addressed this issue by using radioresistant human cancer cell lines H1703 (FGFR1 mutant), A549 (FGFR1-4 wild-type), and H1299 (FGFR1-4 wild-type). At an X-ray dose corresponding to 50%-clonogenic survival as the endpoint, 100 nM LY2874455 increased the sensitivity of H1703, A549, and H1299 cells by 31%, 62%, and 53%, respectively. The combination of X-rays and LY2874455 led to a marked induction of mitotic catastrophe, a hallmark of radiation-induced cell death. Furthermore, combination treatment suppressed the growth of A549 xenografts to a significantly greater extent than either X-rays or the drug alone without noticeable toxicity. This is the first report to show the radiosensitizing effect of a selective pan-FGFR inhibitor. These data suggest the potential efficacy of LY2874455 as a radiosensitizer, warranting clinical validation.

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RAP80 suppresses the vulnerability of R-loops during DNA double-strand break repair

Takaaki Yasuhara, Reona Kato, Motohiro Yamauchi, Yuki Uchihara, Lee Zou, Kiyoshi Miyagawa, Atsushi Shibata

Cell Reports (Cell Press) 38 ( 5 ) 110335 - 110335 2022.02

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted, ISSN 2211-1247

Single-stranded DNA (ssDNA) arising as an intermediate of cellular processes on DNA is a potential vulnerability of the genome unless it is appropriately protected. Recent evidence suggests that R-loops, consisting of ssDNA and DNA-RNA hybrids, can form in the proximity of DNA double-strand breaks (DSBs) within transcriptionally active regions. However, how the vulnerability of ssDNA in R-loops is overcome during DSB repair remains unclear. Here, we identify RAP80 as a factor suppressing the vulnerability of ssDNA in R-loops, chromosome translocations, and deletions during DSB repair. Mechanistically, RAP80 prevents unscheduled nucleolytic processing of ssDNA in R-loops by CtIP. This mechanism promotes efficient DSB repair via transcription-associated end joining dependent on BRCA1, Polθ, and LIG1/3. Thus, RAP80 suppresses the vulnerability of R-loops during DSB repair, thereby precluding genomic abnormalities in a critical component of the genome caused by deleterious R-loop processing.

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Expression of non‑homologous end joining factor, Ku80, is negatively correlated with PD‑L1 expression in cancer cells after X‑ray irradiation

Takuya Kumazawa, Yasumasa Mori, Hiro Sato, Tiara Permata, Yuki Uchihara, Shin-Ei Noda, Kohei Okada, Sangeeta Kakoti, Keiji Suzuki, Hayato Ikota, Hideaki Yokoo, Soehartati Gondhowiardjo, Takashi Nakano, Tatsuya Ohno, Atsushi Shibata

Oncology Letters (Spandidos Publications) 23 ( 1 ) 29 - 29 2022.01

Research paper (scientific journal), Joint Work, Last author, Accepted, ISSN 17921074

The growing importance of antitumour immunity by cancer immunotherapy has prompted studies on radiotherapy-induced immune response. Previous studies have indicated that programmed cell death-1 ligand (PD-L1) expression is regulated by DNA damage signalling. However, PD-L1 up-regulation after radiotherapy has not been fully investigated at the clinical level, particularly in the context of expression of DNA repair factors. The present study examined the correlation of mRNA expression between PD-L1 and non-homologous end joining (NHEJ) factors using The Cancer Genome Atlas database analysis. Among NHEJ factors, Ku80 mRNA expression was negatively correlated with PD-L1 mRNA expression levels in several types of cancer (colon adenocarcinoma, breast invasive carcinoma, skin cutaneous melanoma, lung adenocarcinoma, head and neck squamous cell carcinoma, uterine corpus endometrial carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma). To verify the negative correlation in clinical samples, the present study analysed whether Ku80 expression levels affected PD-L1 up-regulation after radiotherapy using cervical squamous cell carcinoma samples. Quantitative evaluation using software analysis of immunohistochemically stained slides revealed that patients with low Ku80 positivity in biopsy specimens demonstrated increased PD-L1 expression levels after 10 Gy irradiation (Spearman's rank correlation coefficient=-0.274; P=0.017). Furthermore, PD-L1 induction levels in tumour cells after 10 Gy of irradiation were significantly inversely correlated with Ku80 expression levels (Spearman's rank correlation coefficient=-0.379; P<0.001). The present study also confirmed that short interfering RNA-mediated Ku80 depletion was associated with greater X-ray-induced PD-L1 up-regulation in HeLa cells. These results indicated that radiotherapy could enhance PD-L1 induction in tumour cells with low Ku80 expression in a clinical setting. Furthermore, these data highlighted Ku80 as a potential predictive biomarker for immune checkpoint therapy combined with radiotherapy.

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Carbon ion radiation and clustered DNA double-strand breaks

Atsushi Shibata

DNA Damage and Double Strand Breaks - Part A (Elsevier) 117 - 130 2022

ISSN 1874-6047

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64Cu-ATSM Predicts Efficacy of Carbon Ion Radiotherapy Associated with Cellular Antioxidant Capacity

Ankita Nachankar, Takahiro Oike, Hirofumi Hanaoka, Ayaka Kanai, Hiro Sato, Yukari Yoshida, Hideru Obinata, Makoto Sakai, Naoto Osu, Yuka Hirota, Akihisa Takahashi, Atsushi Shibata, Tatsuya Ohno

Cancers (MDPI AG) 13 ( 24 ) 6159 - 6159 2021.12

Research paper (scientific journal), Joint Work, Accepted

Carbon ion radiotherapy is an emerging cancer treatment modality that has a greater therapeutic window than conventional photon radiotherapy. To maximize the efficacy of this extremely scarce medical resource, it is important to identify predictive biomarkers of higher carbon ion relative biological effectiveness (RBE) over photons. We addressed this issue by focusing on cellular antioxidant capacity and investigated 64Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM), a potential radioligand that reflects an over-reduced intracellular environment. We found that the carbon ion RBE correlated with 64Cu-ATSM uptake both in vitro and in vivo. High RBE/64Cu-ATSM cells showed greater steady-state levels of antioxidant proteins and increased capacity to scavenge reactive oxygen species in response to X-rays than low RBE/64Cu-ATSM counterparts; this upregulation of antioxidant systems was associated with downregulation of TCA cycle intermediates. Furthermore, inhibition of nuclear factor erythroid 2-related factor 2 (Nrf2) sensitized high RBE/64Cu-ATSM cells to X-rays, thereby reducing RBE values to levels comparable to those in low RBE/64Cu-ATSM cells. These data suggest that the cellular activity of Nrf2-driven antioxidant systems is a possible determinant of carbon ion RBE predictable by 64Cu-ATSM uptake. These new findings highlight the potential clinical utility of 64Cu-ATSM imaging to identify high RBE tumors that will benefit from carbon ion radiotherapy.

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Mechanism of chromosome rearrangement arising from single-strand breaks

Palina Kot, Takaaki Yasuhara, Atsushi Shibata, Miyako Hirakawa, Yu Abe, Motohiro Yamauchi, Naoki Matsuda

Biochemical and Biophysical Research Communications (Elsevier BV) 572 191 - 196 2021.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006291X

Chromosome rearrangements, which are structural chromosomal abnormalities commonly found in human cancer, result from the misrejoining between two or more DNA double-strand breaks arising at different genomic regions. Consequently, chromosome rearrangements can generate fusion genes that promote tumorigenesis. The mechanisms of chromosome rearrangement have been studied using exogenous double-strand break inducers, such as radiation and nucleases. However, the mechanism underlying the occurrence of chromosome rearrangements in the absence of exogenous double-strand break-inducing stimuli is unclear. This study aimed to identify the major source of chromosome rearrangements and the DNA repair pathway that suppresses them. DNA repair factors that potentially suppress gene fusion were screened using The Cancer Genome Atlas dataset. In total, 22 repair factors whose expression levels were negatively correlated with the frequency of gene fusion were identified. More than 60% of these repair factors are involved in homologous recombination, a major double-strand break repair pathway. We hypothesized that DNA single-strand breaks are the source of double-strand breaks that lead to chromosome rearrangements. This study demonstrated that hydrogen peroxide (H2O2)-induced single-strand breaks gave rise to double-strand breaks in a replication-dependent manner. Additionally, H2O2 induced the formation of RPA and RAD51 foci, which indicated that double-strand breaks derived from single-strand breaks were repaired through homologous recombination. Moreover, treatment with H2O2 promoted the formation of radial chromosomes, a type of chromosome rearrangements, only upon the downregulation of homologous recombination factors, such as BRCA1 and CtIP. Thus, single-strand breaks are the major source of chromosome rearrangements when the expression of homologous recombination factors is downregulated.

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Genome Maintenance Mechanisms at the Chromatin Level

Hirotomo Takatsuka, Atsushi Shibata, Masaaki Umeda

International Journal of Molecular Sciences (MDPI AG) 22 ( 19 ) 10384 - 10384 2021.09

Genome integrity is constantly threatened by internal and external stressors, in both animals and plants. As plants are sessile, a variety of environment stressors can damage their DNA. In the nucleus, DNA twines around histone proteins to form the higher-order structure “chromatin”. Unraveling how chromatin transforms on sensing genotoxic stress is, thus, key to understanding plant strategies to cope with fluctuating environments. In recent years, accumulating evidence in plant research has suggested that chromatin plays a crucial role in protecting DNA from genotoxic stress in three ways: (1) changes in chromatin modifications around damaged sites enhance DNA repair by providing a scaffold and/or easy access to DNA repair machinery; (2) DNA damage triggers genome-wide alterations in chromatin modifications, globally modulating gene expression required for DNA damage response, such as stem cell death, cell-cycle arrest, and an early onset of endoreplication; and (3) condensed chromatin functions as a physical barrier against genotoxic stressors to protect DNA. In this review, we highlight the chromatin-level control of genome stability and compare the regulatory systems in plants and animals to find out unique mechanisms maintaining genome integrity under genotoxic stress.

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High linear energy transfer carbon-ion irradiation upregulates PD-L1 expression more significantly than X-rays in human osteosarcoma U2OS cells

Tiara Bunga Mayang Permata, Hiro Sato, Wenchao Gu, Sangeeta Kakoti, Yuki Uchihara, Yukihiko Yoshimatsu, Itaru Sato, Reona Kato, Motohiro Yamauchi, Keiji Suzuki, Takahiro Oike, Yoshito Tsushima, Soehartati Gondhowiardjo, Tatsuya Ohno, Takaaki Yasuhara, Atsushi Shibata

Journal of Radiation Research (Oxford University Press (OUP)) 62 ( 5 ) 773 - 781 2021.09

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted, ISSN 04493060

<title>Abstract</title>
Programmed death ligand 1 (PD-L1) expression on the surface of cancer cells affects the efficacy of anti-PD-1/PD-L1 immune checkpoint therapy. However, the mechanism underlying PD-L1 expression in cancer cells is not fully understood, particularly after ionizing radiation (IR). Here, we examined the impact of high linear energy transfer (LET) carbon-ion irradiation on the expression of PD-L1 in human osteosarcoma U2OS cells. We found that the upregulation of PD-L1 expression after high LET carbon-ion irradiation was greater than that induced by X-rays at the same physical and relative biological effectiveness (RBE) dose, and that the upregulation of PD-L1 induced by high LET carbon-ion irradiation was predominantly dependent on ataxia telangiectasia and Rad3-related (ATR) kinase activity. Moreover, we showed that the downstream signaling, e.g. STAT1 phosphorylation and IRF1 expression, was upregulated to a greater extent after high LET carbon-ion irradiation than X-rays, and that IRF1 upregulation was also ATR dependent. Finally, to visualize PD-L1 molecules on the cell surface in 3D, we applied immunofluorescence-based super-resolution imaging. The three-dimensional structured illumination microscopy (3D-SIM) analyses revealed substantial increases in the number of presented PD-L1 molecules on the cell surface after high LET carbon-ion irradiation compared with X-ray irradiation.

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ATM’s Role in the Repair of DNA Double-Strand Breaks

Atsushi Shibata, Penny A. Jeggo

Genes (MDPI AG) 12 ( 9 ) 1370 - 1370 2021.08

Ataxia telangiectasia mutated (ATM) is a central kinase that activates an extensive network of responses to cellular stress via a signaling role. ATM is activated by DNA double strand breaks (DSBs) and by oxidative stress, subsequently phosphorylating a plethora of target proteins. In the last several decades, newly developed molecular biological techniques have uncovered multiple roles of ATM in response to DNA damage—e.g., DSB repair, cell cycle checkpoint arrest, apoptosis, and transcription arrest. Combinational dysfunction of these stress responses impairs the accuracy of repair, consequently leading to dramatic sensitivity to ionizing radiation (IR) in ataxia telangiectasia (A-T) cells. In this review, we summarize the roles of ATM that focus on DSB repair.

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Quantitative volumetric analysis of the Golgi apparatus following X-ray irradiation by super-resolution 3D-SIM microscopy

Takahiro Oike, Yuki Uchihara, Tiara Bunga Mayang Permata, Soehartati Gondhowiardjo, Tatsuya Ohno, Atsushi Shibata

Medical Molecular Morphology (Springer Science and Business Media LLC) 54 ( 2 ) 166 - 172 2021.06

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted, ISSN 18601480

<title>Abstract</title>To obtain quantitative volumetric data for the Golgi apparatus after ionizing radiation (IR) using super-resolution three-dimensional structured illumination (3D-SIM) microscopy. Normal human retinal pigment epithelial (RPE) cells were irradiated with X-rays (10 Gy), followed by immunofluorescence staining of the Golgi marker RCAS1. 3D-SIM imaging was performed using DeltaVision OMX version 4 and SoftWoRx 6.1. Polygon rendering and spot signal identification were performed using Imaris 8.1.2. Differences between groups were assessed by Welch’s <italic>t</italic> test. RCAS1 signals in untreated cells were located adjacent to nuclei and showed a reticular morphology. Upon IR, the area of RCAS1 signals expanded while retaining the reticular morphology. Polygon rendering imaging revealed that the volume of RCAS1 at 48 h post-IR was greater than that for unirradiated cells (93.7 ± 19.0 μm3 vs. 33.0 ± 4.2 μm3, respectively; <italic>P</italic> < 0.001): a 2.8-fold increase. Spot signal imaging showed that the number of RCAS1 spot signals post-IR was greater than that for unirradiated cells [3.4 ± 0.8 (× 103) versus 1.3 ± 0.2 (× 103), respectively; <italic>P</italic> < 0.001]: a 2.7-fold increase. This is the first study to report quantitative volumetric data of the Golgi apparatus in response to IR using super-resolution 3D-SIM microscopy.

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Analysis of radiotherapy‑induced alteration of CD8+ T cells and PD‑L1 expression in patients with uterine cervical squamous cell carcinoma

Yasumasa Mori, Hiro Sato, Takuya Kumazawa, Tiara Permata, Yuya Yoshimoto, Kazutoshi Murata, Shin-Ei Noda, Takuya Kaminuma, Ken Ando, Takahiro Oike, Noriyuki Okonogi, Kohei Okada, Sangeeta Kakoti, Keiji Suzuki, Hayato Ikota, Hideaki Yokoo, Takashi Nakano, Tatsuya Ohno, Atsushi Shibata

Oncology Letters (Spandidos Publications) 21 ( 6 ) 446 - 446 2021.06

Research paper (scientific journal), Joint Work, Last author, Accepted, ISSN 17921074

Radiotherapy induces an immune response in the cancer microenvironment that may influence clinical outcome. The present study aimed to analyse the alteration of CD8+ T-cell infiltration and programmed death-ligand 1 (PD-L1) expression following radiotherapy in clinical samples from patients with uterine cervical squamous cell carcinoma. Additionally, the current study sought to analyse the association between these immune responses and clinical outcomes. A total of 75 patients who received either definitive chemoradiotherapy or radiotherapy were retrospectively analyzed. CD8+ T-cell infiltration and PD-L1 expression were determined by immunohistochemistry using biopsy specimens before radiotherapy (pre-RT) and after 10 Gy radiotherapy (post-10 Gy). The PD-L1+ rate was significantly increased from 5% (4/75) pre-RT to 52% (39/75) post-10 Gy (P<0.01). Despite this increase in the PD-L1+ rate post-10 Gy, there was no significant association between both pre-RT and post-10 Gy and overall survival (OS), locoregional control (LC) and progression-free survival (PFS). On the other hand, the CD8+ T-cell infiltration density was significantly decreased for all patients (median, 23.1% pre-RT vs. 16.9% post-10 Gy; P=0.038); however, this tended to increase in patients treated with radiotherapy alone (median, 17.7% pre-RT vs. 24.0% post-10 Gy; P=0.400). Notably, patients with high CD8+ T-cell infiltration either pre-RT or post-10 Gy exhibited positive associations with OS, LC and PFS. Thus, the present analysis suggested that CD8+ T-cell infiltration may be a prognostic biomarker for patients with cervical cancer receiving radiotherapy. Furthermore, immune checkpoint inhibitors may be effective in patients who have received radiotherapy, since radiotherapy upregulated PD-L1 expression in cervical cancer specimens.

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Comparison of Clonogenic Survival Data Obtained by Pre- and Post-Irradiation Methods

Takahiro Oike, Yuka Hirota, Narisa Dewi Maulany Darwis, Atsushi Shibata, Tatsuya Ohno

Journal of Personalized Medicine (MDPI AG) 10 ( 4 ) 171 - 171 2020.10

Research paper (scientific journal), Joint Work, Accepted

Clonogenic assays are the gold standard to measure in vitro radiosensitivity, which use two cell plating methods, before or after irradiation (IR). However, the effect of the plating method on the experimental outcome remains unelucidated. By using common cancer cell lines, here we demonstrate that pre-IR and post-IR plating methods have a negligible effect on the clonogenic assay-derived photon sensitivity as assessed by SF2, SF4, SF6, SF8, D10, or D50 (N.B. SFx indicates the survival at X Gy; Dx indicates the dose providing X% survival). These data provide important biological insight that supports inter-study comparison and integrated analysis of published clonogenic assay data regardless of the plating method used.

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高LET線ならびに放射線類似薬により誘発されるDNA二本鎖切断の修復における修復経路の重要度の解析(Requirement of DNA double-strand break repair pathways for repairing high LET radiation- and radiomimetic agents-induced DNA damage)

黒沢綾, 高橋昭久, 武田茂樹, 柴田淳史, 足立典隆

日本放射線影響学会大会講演要旨集 ((一社)日本放射線影響学会) 63回 75 - 75 2020.10

ISSN 1347-8680

DNA Repair and Signaling in Immune-Related Cancer Therapy

Sangeeta Kakoti, Hiro Sato, Siddhartha Laskar, Takaaki Yasuhara, Atsushi Shibata

Frontiers in Molecular Biosciences (Frontiers Media SA) 7 205 - 205 2020.09

Research paper (scientific journal), Last author, Corresponding author, Accepted

Cancer therapy using immune checkpoint inhibitors (ICIs) is a promising clinical strategy for patients with multiple types of cancer. The expression of programmed cell death ligand-1 (PD-L1), an immune-suppressor ligand, in cancer cells is a factor that influences the efficacy of ICI therapy, particularly in the anti-programmed cell death protein-1 (PD-1)/PD-L1 antibody therapy. PD-L1 expression in cancer cells are associated with tumor mutation burden including microsatellite instability because the accumulation of mutations in the cancer genome can produce abnormal proteins via mutant mRNAs, resulting in neoantigen production and HLA-neoantigen complex presentation in cancer cells. HLA-neoantigen presentation promotes immune activity within tumor environment; therefore, known as hot tumor. Thus, as the fidelity of DNA repair affects the generation of genomic mutations, the status of DNA repair and signaling in cancer cells can be considered prior to ICI therapy. The Cancer Genome Atlas (TCGA) and The Cancer Immunome Atlas (TCIA) database analysis showed that tumor samples harboring mutations in any non-homologous end joining, homologous recombination, or DNA damage signaling genes exhibit high neoantigen levels. Alternatively, an urgent task is to understand how the DNA damage-associated cancer treatments change the status of immune activity in patients because multiple clinical trials on combination therapy are ongoing. Recent studies demonstrated that multiple pathways regulate PD-L1 expression in cancer cells. Here, we summarize the regulation of the immune response to ICI therapy, including PD-L1 expression, and also discuss the potential strategies to improve the efficacy of ICI therapy for poor responders from the viewpoint of DNA damage response before or after DNA damage-associated cancer treatment.

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Roles for the DNA-PK complex and 53BP1 in protecting ends from resection during DNA double-strand break repair

Atsushi Shibata, Penny A Jeggo

Journal of Radiation Research (Oxford University Press (OUP)) 61 ( 5 ) 718 - 726 2020.09

ISSN 0449-3060

<title>Abstract</title>
p53-binding protein 1 (53BP1) exerts distinct impacts in different situations involving DNA double-strand break (DSB) rejoining. Here we focus on how 53BP1 impacts upon the repair of ionising radiation-induced DSBs (IR-DSBs) and how it interfaces with Ku, the DNA end-binding component of canonical non-homologous end-joining (c-NHEJ), the major DSB repair pathway in mammalian cells. We delineate three forms of IR-DSB repair: resection-independent c-NHEJ, which rejoins most IR-DSBs with fast kinetics in G1 and G2, and Artemis and resection-dependent c-NHEJ and homologous recombination (HR), which repair IR-DSBs with slow kinetics in G1 and G2 phase, respectively. The fast component of DSB repair after X-ray exposure occurs via c-NHEJ with normal kinetics in the absence of 53BP1. Ku is highly abundant and has avid DNA end-binding capacity which restricts DNA end-resection and promotes resection-independent c-NHEJ at most IR-DSBs. Thus, 53BP1 is largely dispensable for resection-independent c-NHEJ. In contrast, 53BP1 is essential for the process of rejoining IR-DSBs with slow kinetics. This role requires 53BP1’s breast cancer susceptibility gene I (BRCA1) C-terminal (BRCT) 2 domain, persistent ataxia telangiectasia mutated (ATM) activation and potentially relaxation of compacted chromatin at heterochromatic-DSBs. In distinction, 53BP1 inhibits resection-dependent IR-DSB repair in G1 and G2, and this resection-inhibitory function can be counteracted by BRCA1. We discuss a model whereby most IR-DSBs are rapidly repaired by 53BP1-independent and resection-independent c-NHEJ due to the ability of Ku to inhibit resection, but, if delayed, then resection in the presence of Ku is triggered, the 53BP1 barrier comes into force and BRCA1 counteraction is required for resection.

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Induction of Micronuclei in Cervical Cancer Treated with Radiotherapy

Daijiro Kobayashi, Takahiro Oike, Kazutoshi Murata, Daisuke Irie, Yuka Hirota, Hiro Sato, Atsushi Shibata, Tatsuya Ohno

Journal of Personalized Medicine (MDPI AG) 10 ( 3 ) 110 - 110 2020.09

Research paper (scientific journal), Joint Work, Accepted

Micronuclei (MN) trigger antitumor immune responses via the cyclic GMP-AMP synthase-signaling effector stimulator of interferon genes (cGAS-STING) pathway. Radiotherapy induces MN in peripheral blood lymphocytes. However, data for solid tumors are lacking. Here, we analyzed MN post-radiotherapy in solid tumor samples. Tumor biopsy specimens were obtained from seven prospectively recruited patients with cervical cancer, before treatment and after receiving radiotherapy at a dose of 10 Gy (in five fractions). The samples were stained with 4′,6-diamidino-2-phenylindole dihydrochloride, and 200 nuclei per sample were randomly identified and assessed for the presence of MN or apoptosis, based on nuclear morphology. The median number of MN-harboring nuclei was significantly greater in samples from patients treated with radiotherapy than in pre-treatment samples (151 (range, 16–327) versus 28 (range, 0–61); p = 0.015). No significant differences in the number of apoptotic nuclei were observed between pre-treatment and 10 Gy samples (5 (range, 0–30) versus 12 (range, 2–30); p = 0.30). This is the first report to demonstrate MN induction by radiotherapy in solid tumors. The results provide clinical evidence of the activation of antitumor immune responses by radiotherapy.

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Roles for 53BP1 in the repair of radiation-induced DNA double strand breaks

Atsushi Shibata, Penny A. Jeggo

DNA Repair (Elsevier BV) 93 102915 - 102915 2020.09

ISSN 1568-7864

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Reporting of methodologies used for clonogenic assays to determine radiosensitivity

Takahiro Oike, Shuichiro Komatsu, Yuka Komatsu, Ankita Nachankar, Narisa Dewi Maulany Darwis, Atsushi Shibata, Tatsuya Ohno

Journal of Radiation Research (Oxford University Press (OUP)) 61 ( 6 ) 828 - 831 2020.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 0449-3060

<title>Abstract</title>
Radiotherapy treatment strategies should be personalized based on the radiosensitivity of individual tumors. Clonogenic assays are the gold standard method for in vitro assessment of radiosensitivity. Reproducibility is the critical factor for scientific rigor; however, this is reduced by insufficient reporting of methodologies. In reality, the reporting standards of methodologies pertaining to clonogenic assays remain unclear. To address this, we performed a literature search and qualitative analysis of the reporting of methodologies pertaining to clonogenic assays. A comprehensive literature review identified 1672 papers that report the radiosensitivity of human cancer cells based on clonogenic assays. From the identified papers, important experimental parameters (i.e. number of biological replicates, technical replicates, radiation source and dose rate) were recorded and analyzed. We found that, among the studies, (i) 30.5% did not report biological or technical replicates; (ii) 47.0% did not use biological or technical replicates; (iii) 3.8% did not report the radiation source; and (iv) 32.3% did not report the dose rate. These data suggest that reporting of methodologies pertaining to clonogenic assays in a considerable number of previously published studies is insufficient, thereby threatening reproducibility. This highlights the need to raise awareness of standardization of the methodologies used to conduct clonogenic assays.

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Relative Biological Effectiveness of Carbon Ions for Head-and-Neck Squamous Cell Carcinomas According to Human Papillomavirus Status

Naoto Osu, Daijiro Kobayashi, Katsuyuki Shirai, Atsushi Musha, Hiro Sato, Yuka Hirota, Atsushi Shibata, Takahiro Oike, Tatsuya Ohno

Journal of Personalized Medicine (MDPI AG) 10 ( 3 ) 71 - 71 2020.07

Research paper (scientific journal), Joint Work, Accepted

Carbon-ion radiotherapy (CIRT) has strong antitumor effects and excellent dose conformity. In head-and-neck squamous cell carcinoma (HNSCC), human papillomavirus (HPV) status is a prognostic factor for photon radiotherapy outcomes. However, the effect of HPV status on the sensitivity of HNSCCs to carbon ions remains unclear. Here, we showed that the relative biological effectiveness (RBE) of carbon ions over X-rays was higher in HPV-negative cells than in HSGc-C5 cells, which are used for CIRT dose establishment, whereas the RBE in HPV-positive cells was modest. These data indicate that CIRT is more advantageous in HPV-negative than in HPV-positive HNSCCs.

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RNF8 promotes high linear energy transfer carbon-ion-induced DNA double-stranded break repair in serum-starved human cells

Nakako Izumi Nakajima, Motohiro Yamauchi, Sangeeta Kakoti, Liu Cuihua, Reona Kato, Tiara Bunga Mayang Permata, Moito Iijima, Hirohiko Yajima, Takaaki Yasuhara, Shigeru Yamada, Sumitaka Hasegawa, Atsushi Shibata

DNA Repair (Elsevier BV) 91-92 102872 - 102872 2020.07

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted, ISSN 1568-7864

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DNA double-strand break end resection: a critical relay point for determining the pathway of repair and signaling

Yoko Katsuki, Penny A. Jeggo, Yuki Uchihara, Minoru Takata, Atsushi Shibata

Genome Instability & Disease (Springer Science and Business Media LLC) 1 ( 4 ) 155 - 171 2020.07

Accepted

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UBC13-Mediated Ubiquitin Signaling Promotes Removal of Blocking Adducts from DNA Double-Strand Breaks

Remi Akagawa, Hai Thanh Trinh, Liton Kumar Saha, Masataka Tsuda, Kouji Hirota, Shintaro Yamada, Atsushi Shibata, Masato T. Kanemaki, Shinichiro Nakada, Shunichi Takeda, Hiroyuki Sasanuma

iScience (Elsevier BV) 23 ( 4 ) 101027 - 101027 2020.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 2589-0042

Chemical modifications and adducts at DNA double-strand break (DSB) ends must be cleaned before re-joining by non-homologous end-joining (NHEJ). MRE11 nuclease is essential for efficient removal of Topoisomerase II (TOP2)-DNA adducts from TOP2 poison-induced DSBs. However, mechanisms in MRE11 recruitment to DSB sites in G1 phase remain poorly understood. Here, we report that TOP2-DNA adducts are expeditiously removed through UBC13-mediated polyubiquitination, which promotes DSB resection in G2 phase. We found that this ubiquitin signaling is required for efficient recruitment of MRE11 onto DSB sites in G1 by facilitating localization of RAP80 and BRCA1 to DSB sites and complex formation between BRCA1 and MRE11 at DSB sites. UBC13 and MRE11 are dispensable for restriction-enzyme-induced “clean” DSBs repair but responsible for over 50% and 70% of NHEJ-dependent repair of γ-ray-induced “dirty” DSBs, respectively. In conclusion, ubiquitin signaling promotes nucleolytic removal of DSB blocking adducts by MRE11 before NHEJ.

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Canonical DNA non-homologous end-joining; capacity versus fidelity.

Atsushi Shibata, Penny A Jeggo

The British journal of radiology 20190966 - 20190966 2020.01

Accepted

The significance of canonical DNA non-homologous end-joining (c-NHEJ) for DNA double strand break (DSB) repair has increased from lower organisms to higher eukaryotes, and plays the predominant role in human cells. Ku, the c-NHEJ end-binding component, binds DSBs with high efficiency enabling c-NHEJ to be the first choice DSB repair pathway, although alternative pathways can ensue after regulated steps to remove Ku. Indeed, radiation-induced DSBs are repaired rapidly in human cells. However, an important question is the fidelity with which radiation-induced DSBs are repaired, which is essential for assessing any harmful impacts caused by radiation exposure. Indeed, is compromised fidelity a price we pay for high capacity repair. Two subpathways of c-NHEJ have been revealed; a fast process that does not require nucleases or significant chromatin changes and a slower process that necessitates resection factors, and potentially more significant chromatin changes at the DSB. Recent studies have also shown that DSBs within transcriptionally active regions are repaired by specialised mechanisms, and the response at such DSBs encompasses a process of transcriptional arrest. Here, we consider the limitations of c-NHEJ that might result in DSB misrepair. We consider the common IR-induced misrepair events and discuss how they might arise via the distinct subpathways of c-NHEJ.

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p53 deficiency augments nucleolar instability after ionizing irradiation.

Sangeeta Kakoti, Motohiro Yamauchi, Wenchao Gu, Reona Kato, Takaaki Yasuhara, Yoshihiko Hagiwara, Siddhartha Laskar, Takahiro Oike, Hiro Sato, Kathryn D Held, Takashi Nakano, Atsushi Shibata

Oncology reports (Spandidos publications) 42 ( 6 ) 2293 - 2302 2019.12

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted

Ribosomes are important cellular components that maintain cellular homeostasis through overall protein synthesis. The nucleolus is a prominent subnuclear structure that contains ribosomal DNA (rDNA) encoding ribosomal RNA (rRNA), an essential component of ribosomes. Despite the significant role of the rDNA‑rRNA‑ribosome axis in cellular homeostasis, the stability of rDNA in the context of the DNA damage response has not been fully investigated. In the present study, the number and morphological changes of nucleolin, a marker of the nucleolus, were examined following ionizing radiation (IR) in order to investigate the impact of DNA damage on nucleolar stability. An increase in the number of nucleoli per cell was found in HCT116 and U2OS cells following IR. Interestingly, the IR‑dependent increase in nucleolar fragmentation was enhanced by p53 deficiency. In addition, the morphological analysis revealed several distinct types of nucleolar fragmentation following IR. The pattern of nucleolar morphology differed between HCT116 and U2OS cells, and the p53 deficiency altered the pattern of nucleolar morphology. Finally, a significant decrease in rRNA synthesis was observed in HCT116 p53‑/‑ cells following IR, suggesting that severe nucleolar fragmentation downregulates rRNA transcription. The findings of the present study suggest that p53 plays a key role in protecting the transcriptional activity of rDNA in response to DNA damage.

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Analysis of nucleolus stability after ionizing irradiation(和訳中)

Kakoti Sangeeta, 萩原慶彦, 佐藤浩央, 尾池貴洋, 中野隆史, 柴田淳史

日本放射線影響学会大会講演要旨集 ((一社)日本放射線影響学会) 62回 44 - 44 2019.11

ISSN 1347-8680

イオン線照射後の核安定性に関する分析(Analysis of nucleolus stability after ionizing irradiation)

Kakoti Sangeeta, 萩原慶彦, 佐藤浩央, 尾池貴洋, 中野隆史, 柴田淳史

日本放射線影響学会大会講演要旨集 ((一社)日本放射線影響学会) 62回 44 - 44 2019.11

ISSN 1347-8680

Combination of Anti-Cancer Drugs with Molecular Chaperone Inhibitors.

Maxim Shevtsov, Gabriele Multhoff, Elena Mikhaylova, Atsushi Shibata, Irina Guzhova, Boris Margulis

International journal of molecular sciences 20 ( 21 ) 2019.10

Accepted

Most molecular chaperones belonging to heat shock protein (HSP) families are known to protect cancer cells from pathologic, environmental and pharmacological stress factors and thereby can hamper anti-cancer therapies. In this review, we present data on inhibitors of the heat shock response (particularly mediated by the chaperones HSP90, HSP70, and HSP27) either as a single treatment or in combination with currently available anti-cancer therapeutic approaches. An overview of the current literature reveals that the co-administration of chaperone inhibitors and targeting drugs results in proteotoxic stress and violates the tumor cell physiology. An optimal drug combination should simultaneously target cytoprotective mechanisms and trigger the imbalance of the tumor cell physiology.

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Deep learning-assisted literature mining for in vitro radiosensitivity data.

Shuichiro Komatsu, Takahiro Oike, Yuka Komatsu, Yoshiki Kubota, Makoto Sakai, Toshiaki Matsui, Endang Nuryadi, Tiara Bunga Mayang Permata, Hiro Sato, Hidemasa Kawamura, Masahiko Okamoto, Takuya Kaminuma, Kazutoshi Murata, Naoko Okano, Yuka Hirota, Tatsuya Ohno, Jun-Ichi Saitoh, Atsushi Shibata, Takashi Nakano

Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology (Elsevier) 139 87 - 93 2019.10

Joint Work, Accepted

BACKGROUND AND PURPOSE: Integrated analysis of existing radiosensitivity data obtained by the gold-standard clonogenic assay has the potential to improve our understanding of cancer cell radioresistance. However, extraction of radiosensitivity data from the literature is highly labor-intensive. To aid in this task, using deep convolutional neural networks (CNNs) and other computer technologies, we developed an analysis pipeline that extracts radiosensitivity data derived from clonogenic assays from the literature. MATERIALS AND METHODS: Three classifiers (C1-3) were developed to identify publications containing radiosensitivity data derived from clonogenic assays. C1 uses Faster Regions CNN with Inception Resnet v2 (fRCNN-IRv2), VGG-16, and Optical Character Recognition (OCR) to identify publications that contain semi-logarithmic graphs showing radiosensitivity data derived from clonogenic assays. C2 uses fRCNN-IRv2 and OCR to identify publications that contain bar graphs showing radiosensitivity data derived from clonogenic assays. C3 is a program that identifies publications containing keywords related to radiosensitivity data derived from clonogenic assays. A program (iSF2) was developed using Mask RCNN and OCR to extract surviving fraction after 2-Gy irradiation (SF2) as assessed by clonogenic assays, presented in semi-logarithmic graphs. The efficacy of C1-3 and iSF2 was tested using seven datasets (1805 and 222 publications in total, respectively). RESULTS: C1-3 yielded sensitivity of 91.2% ± 3.4% and specificity of 90.7% ± 3.6%. iSF2 returned SF2 values that were within 2.9% ± 2.6% of the SF2 values determined by radiation oncologists. CONCLUSION: Our analysis pipeline is potentially useful to acquire radiosensitivity data derived from clonogenic assays from the literature.

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FGFR Signaling as a Candidate Therapeutic Target for Cancers Resistant to Carbon Ion Radiotherapy.

Narisa Dewi Maulany Darwis, Ankita Nachankar, Yasushi Sasaki, Toshiaki Matsui, Shin-Ei Noda, Kazutoshi Murata, Tomoaki Tamaki, Ken Ando, Noriyuki Okonogi, Shintaro Shiba, Daisuke Irie, Takuya Kaminuma, Takuya Kumazawa, Mai Anakura, Souichi Yamashita, Takashi Hirakawa, Sangeeta Kakoti, Yuka Hirota, Takashi Tokino, Akira Iwase, Tatsuya Ohno, Atsushi Shibata, Takahiro Oike, Takashi Nakano

International journal of molecular sciences (MDPI) 20 ( 18 ) 4563 2019.09

Research paper (scientific journal), Joint Work, Accepted

Radiotherapy is an essential component of cancer therapy. Carbon ion radiotherapy (CIRT) promises to improve outcomes compared with standard of care in many cancers. Nevertheless, clinicians often observe in-field recurrence after CIRT. This indicates the presence of a subset of cancers that harbor intrinsic resistance to CIRT. Thus, the development of methods to identify and sensitize CIRT-resistant cancers is needed. To address this issue, we analyzed a unique donor-matched pair of clinical specimens: a treatment-naïve tumor, and the tumor that recurred locally after CIRT in the same patient. Exon sequencing of 409 cancer-related genes identified enrichment of somatic mutations in FGFR3 and FGFR4 in the recurrent tumor compared with the treatment-naïve tumor, indicating a pivotal role for FGFR signaling in cancer cell survival through CIRT. Inhibition of FGFR using the clinically available pan-FGFR inhibitor LY2874455 sensitized multiple cancer cell lines to carbon ions at 3 Gy (RBE: relative biological effectiveness), the daily dose prescribed to the patient. The sensitizer enhancement ratio was 1.66 ± 0.17, 1.27 ± 0.09, and 1.20 ± 0.18 in A549, H1299, and H1703 cells, respectively. Our data indicate the potential usefulness of the analytical pipeline employed in this pilot study to identify targetable mutations associated with resistance to CIRT, and of LY21874455 as a sensitizer for CIRT-resistant cancers. The results warrant validation in larger cohorts.

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Robustness of Clonogenic Assays as a Biomarker for Cancer Cell Radiosensitivity.

Toshiaki Matsui, Endang Nuryadi, Shuichiro Komatsu, Yuka Hirota, Atsushi Shibata, Takahiro Oike, Takashi Nakano

International journal of molecular sciences (MDPI) 20 ( 17 ) 4148 2019.08

Research paper (scientific journal), Joint Work, Accepted

Photon radiation therapy is a major curative treatment for cancer. However, the lack of robust predictive biomarkers for radiosensitivity precludes personalized radiation therapy. Clonogenic assays are the gold standard method for measuring the radiosensitivity of cancer cells. Although a large number of publications describe the use of clonogenic assays to measure cancer cell radiosensitivity, the robustness of results from different studies is unclear. To address this, we conducted a comprehensive detailed literature search of 256 common cancer cell lines and identified the eight cell lines most-frequently examined for photon sensitivity using clonogenic assays. Survival endpoints and experimental parameters from all 620 relevant experiments were compiled and analyzed. We found that the coefficients of variation for SF2 (surviving fraction after 2 Gy irradiation) and for D10 (dose that yields a surviving fraction of 10%) were below 30% for all cell lines, indicating that SF2 and D10 have acceptable inter-assay precision. These data support further analysis of published data on clonogenic assays using SF2 and D10 as survival endpoints, which facilitates robust identification of biological profiles representative of cancer cell sensitivity to photons.

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Radiosensitivity Differences between EGFR Mutant and Wild-Type Lung Cancer Cells are Larger at Lower Doses

Mai Anakura, Ankita Nachankar, Daijiro Kobayashi, Napapat Amornwichet, Yuka Hirota, Atsushi Shibata, Takahiro Oike, Takashi Nakano

International Journal of Molecular Sciences (MDPI) 20 ( 15 ) 3635 2019.07

Research paper (scientific journal), Joint Work, Accepted

In the era of precision medicine, radiotherapy strategies should be determined based on genetic profiles that predict tumor radiosensitivity. Accordingly, pre-clinical research aimed at discovering clinically applicable genetic profiles is needed. However, how a given genetic profile affects cancer cell radiosensitivity is unclear. To address this issue, we performed a pilot in vitro study by utilizing EGFR mutational status as a model for genetic profile. Clonogenic assays of EGFR mutant (n = 6) and wild-type (n = 9) non-small cell lung carcinoma (NSCLC) cell lines were performed independently by two oncologists. Clonogenic survival parameters SF2, SF4, SF6, SF8, mean inactivation dose (MID), D10, D50, α, and β were obtained using the linear quadratic model. The differences in the clonogenic survival parameters between the EGFR mutant and wild-type cell lines were assessed using the Mann-Whitney U test. As a result, for both datasets, the p values for SF2, SF4, D50, α, and α/β were below 0.05, and those for SF2 were lowest. These data indicate that a genetic profile of NSCLC cell lines might be predictive for their radiation response; i.e., EGFR mutant cell lines might be more sensitive to low dose- and low fraction sized-irradiation.

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Base excision repair regulates PD-L1 expression in cancer cells.

Tiara Bunga Mayang Permata, Yoshihiko Hagiwara, Hiro Sato, Takaaki Yasuhara, Takahiro Oike, Soehartati Gondhowiardjo, Kathryn D Held, Takashi Nakano, Atsushi Shibata

Oncogene (Springer nature) 38 ( 23 ) 4452 - 4466 2019.06

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted

Programmed death-ligand 1 (PD-L1) is a key factor influencing cancer immunotherapy; however, the regulation of PD-L1 expression in cancer cells remains unclear, particularly regarding DNA damage, repair and its signalling. Herein, we demonstrate that oxidative DNA damage induced by exogenously applied hydrogen peroxide (H2O2) upregulates PD-L1 expression in cancer cells. Further, depletion of the base excision repair (BER) enzyme DNA glycosylase augments PD-L1 upregulation in response to H2O2. PD-L1 upregulation in BER-depleted cells requires ATR/Chk1 kinase activities, demonstrating that PD-L1 upregulation is mediated by DNA damage signalling. Further analysis of The Cancer Genome Atlas revealed that the expression of PD-L1 is negatively correlated with that of the BER/single-strand break repair (SSBR) and tumours with low BER/SSBR gene expression show high microsatellite instability and neoantigen production. Hence, these results suggest that PD-L1 expression is regulated in cancer cells via the DNA damage signalling and neoantigen-interferon-γ pathway under oxidative stress.

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Elucidation of the Mechanism Regulating Transcription Associated DNA Double Strand Break Repair and Its Pathway.

Atsushi Shibata, Takaaki Yasuhara

Impact (Science Impact Ltd) 2019 ( 3 ) 50 - 51 2019.03

Research paper (scientific journal), Joint Work, Lead author, Corresponding author, Accepted

The ability to repair breaks and mutations in DNA is essential for a cell's – and therefore organism's – survival. Breaks and other mutations arise naturally over time and are dealt with by the cell using several different pathways. Incorrect or non-existent DNA repair can lead directly to either cell death or the development of a cancerous cell. A break in double strand in the DNA is particularly serious. Dr Atsushi Shibata, a Principle Investigator and Senior Lecturer at Gunma University, Japan explains that the cell has two key ways to deal with this sort of break. 'These are homologous recombination repair (HRR) and non-homologous end-joining (NHEJ),' he says. 'HRR is the more accurate pathway, while NHEJ provides a quicker fix, but it has a risk of error under some circumstance.' This makes HRR perfectly suited to repairing important parts of the genome whilst NHEJ is better suited to less key regions.
The molecular mechanisms behind these repair pathways have been extensively studied. However, the question of how a particular pathway is selected over another in different situations is less well characterised, particularly in human normal cells as well as cancer cells. Uncovering the signalling pathways behind this choice the cell makes is both very important to our fundamental understanding of DNA repair and to its potential use in tackling cancer. Investigating these pathways Shibata is collaborating with Dr Takaaki Yasuhara from the University of Tokyo. They have revealed some of the inner workings behind the choice of repair pathway.
Previously, Shibata has shown that the NHEJ was the default choice for repairing DSBs, but that HRR is then selected if the re-joining does not occur properly. He has also shown a fundamental role of MRE11 nuclease in determining the type of repair that occurs at a DSB. Additionally, Shibata has shown the crucial role BRCA1 has in directing the repair of DSB towards the HRR pathway. In short, his work in the field is internationally recognised. 'These studies have contributed to the establishment of research basis for the recent discovery in the study of transcription-associated HRR,' observes Shibata.

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Clustered DNA double-strand break formation and the repair pathway following heavy-ion irradiation.

Yoshihiko Hagiwara, Takahiro Oike, Atsuko Niimi, Motohiro Yamauchi, Hiro Sato, Siripan Limsirichaikul, Kathryn D Held, Takashi Nakano, Atsushi Shibata

Journal of radiation research (Oxford University Press) 60 ( 1 ) 69 - 79 2019.01

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted

Photons, such as X- or γ-rays, induce DNA damage (distributed throughout the nucleus) as a result of low-density energy deposition. In contrast, particle irradiation with high linear energy transfer (LET) deposits high-density energy along the particle track. High-LET heavy-ion irradiation generates a greater number and more complex critical chromosomal aberrations, such as dicentrics and translocations, compared with X-ray or γ irradiation. In addition, the formation of >1000 bp deletions, which is rarely observed after X-ray irradiation, has been identified following high-LET heavy-ion irradiation. Previously, these chromosomal aberrations have been thought to be the result of misrepair of complex DNA lesions, defined as DNA damage through DNA double-strand breaks (DSBs) and single-strand breaks as well as base damage within 1-2 helical turns (<3-4 nm). However, because the scale of complex DNA lesions is less than a few nanometers, the large-scale chromosomal aberrations at a micrometer level cannot be simply explained by complex DNA lesions. Recently, we have demonstrated the existence of clustered DSBs along the particle track through the use of super-resolution microscopy. Furthermore, we have visualized high-level and frequent formation of DSBs at the chromosomal boundary following high-LET heavy-ion irradiation. In this review, we summarize the latest findings regarding the hallmarks of DNA damage structure and the repair pathway following heavy-ion irradiation. Furthermore, we discuss the mechanism through which high-LET heavy-ion irradiation may induce dicentrics, translocations and large deletions.

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A historical reflection on our understanding of radiation-induced DNA double strand break repair in somatic mammalian cells; interfacing the past with the present

Atsushi Shibata, Penny Jeggo

International Journal of Radiation Biology 2019

Accepted

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Regulation of PD-L1 expression in response to DNA damage in cancer cells: Implications for precision medicine

Hiro Sato, Penny A. Jeggo, Atsushi Shibata, Sangeeta Kakoti, Motohiro Yamauchi, Wenchao Gu, Reona Kato, Takaaki Yasuhara, Yoshihiko Hagiwara, Siddhartha Laskar, Takahiro Oike, Hiro Sato, Kathryn D. Held, Takashi Nakano, Atsushi Shibata, Corresponding author

Cancer Science 2019

Accepted

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Novel Approaches to Improve the Efficacy of Immuno-Radiotherapy.

Maxim Shevtsov, Hiro Sato, Gabriele Multhoff, Atsushi Shibata

Frontiers in oncology 9 156 - 156 2019

Accepted

Radiotherapy (RT) has been applied for decades as a treatment modality in the management of various types of cancer. Ionizing radiation induces tumor cell death, which in turn can either elicit protective anti-tumor immune responses or immunosuppression in the tumor micromilieu that contributes to local tumor recurrence. Immunosuppression is frequently accompanied by the attraction of immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs), M2 tumor-associated macrophages (TAMs), T regulatory cells (Tregs), N2 neutrophils, and by the release of immunosuppressive cytokines (TGF-β, IL-10) and chemokines. Immune checkpoint pathways, particularly of the PD-1/PD-L1 axis, have been determined as key regulators of cancer immune escape. While IFN-dependent upregulation of PD-L1 has been extensively investigated, up-to-date studies indicated the importance of DNA damage signaling in the regulation of PD-L1 expression following RT. DNA damage dependent PD-L1 expression is upregulated by ATM/ATR/Chk1 kinase activities and cGAS/STING-dependent pathway, proving the role of DNA damage signaling in PD-L1 induced expression. Checkpoint blockade immunotherapies (i.e., application of anti-PD-1 and anti-PD-L1 antibodies) combined with RT were shown to significantly improve the objective response rates in therapy of various primary and metastatic malignancies. Further improvements in the therapeutic potential of RT are based on combinations of RT with other immunotherapeutic approaches including vaccines, cytokines and cytokine inducers, and an adoptive immune cell transfer (DCs, NK cells, T cells). In the current review we provide immunological rationale for a combination of RT with various immunotherapies as well as analysis of the emerging preclinical evidences for these therapies.

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相同組換え修復への経路切り替えを促進する時空間的53BP1動態制御機構

磯野真由, 萩原慶彦, 佐藤浩央, 尾池貴洋, 西良太郎, 中田慎一郎, 中野隆史, 鈴木啓司, 柴田淳史

日本放射線影響学会大会講演要旨集 ((一社)日本放射線影響学会) 61回 51 - 51 2018.11

ISSN 1347-8680

重粒子線誘発DSBクラスター領域でのDNA end resection促進機構の解明

萩原慶彦, 安原崇哲, 山内基弘, 尾池貴洋, 佐藤浩央, 中野隆史, 柴田淳史

日本放射線影響学会大会講演要旨集 ((一社)日本放射線影響学会) 61回 46 - 46 2018.11

ISSN 1347-8680

Human Rad52 Promotes XPG-Mediated R-loop Processing to Initiate Transcription-Associated Homologous Recombination Repair.

Takaaki Yasuhara, Reona Kato, Yoshihiko Hagiwara, Bunsyo Shiotani, Motohiro Yamauchi, Shinichiro Nakada, Atsushi Shibata, Kiyoshi Miyagawa

Cell (Cell Press) 175 ( 2 ) 558 - 570 2018.10

Research paper (scientific journal), Joint Work, Corresponding author, Accepted, ISSN 0092-8674

Given that genomic DNA exerts its function by being transcribed, it is critical for the maintenance of homeostasis that DNA damage, such as double-strand breaks (DSBs), within transcriptionally active regions undergoes accurate repair. However, it remains unclear how this is achieved. Here, we describe a mechanism for transcription-associated homologous recombination repair (TA-HRR) in human cells. The process is initiated by R-loops formed upon DSB induction. We identify Rad52, which is recruited to the DSB site in a DNA-RNA-hybrid-dependent manner, as playing pivotal roles in promoting XPG-mediated R-loop processing and initiating subsequent repair by HRR. Importantly, dysfunction of TA-HRR promotes DSB repair via non-homologous end joining, leading to a striking increase in genomic aberrations. Thus, our data suggest that the presence of R-loops around DSBs within transcriptionally active regions promotes accurate repair of DSBs via processing by Rad52 and XPG to protect genomic information in these critical regions from gene alterations.

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Mutational analysis of uterine cervical cancer that survived multiple rounds of radiotherapy.

Endang Nuryadi, Yasushi Sasaki, Yoshihiko Hagiwara, Tiara Bunga Mayang Permata, Hiro Sato, Shuichiro Komatsu, Yuya Yoshimoto, Kazutoshi Murata, Ken Ando, Nobuteru Kubo, Noriyuki Okonogi, Yosuke Takakusagi, Akiko Adachi, Mototaro Iwanaga, Keisuke Tsuchida, Tomoaki Tamaki, Shin-Ei Noda, Yuka Hirota, Atsushi Shibata, Tatsuya Ohno, Takashi Tokino, Takahiro Oike, Takashi Nakano

Oncotarget 9 32642 - 32652 2018.08

Research paper (scientific journal), Joint Work, Accepted

Radiotherapy is an essential component of cancer therapy. Despite advances in cancer genomics, the mutation signatures of radioresistant tumors have not yet been fully elucidated. To address this issue, we analyzed a unique set of clinical specimens from a uterine cervical cancer that repeatedly locally recurred after multiple rounds of radiotherapy. Exon sequencing of 409 cancer-related genes in the treatment-naïve tumor and the tumors that recurred after initial and secondary radiotherapy identified (i) activating mutations in PIK3CA and KRAS, and putative inactivating mutations in SMAD4, as trunk mutation signatures that persisted over the clinical course; and (ii) mutations in KMT2A, TET1, and NLRP1 as acquired mutation signatures observed only in recurrent tumors after radiotherapy. Comprehensive mining of published in vitro genomics data pertaining to radiosensitivity revealed that simultaneous mutations in KRAS and SMAD4, which have not been described previously in uterine cervical cancer, are associated with cancer cell radioresistance. The association between this mutation signature and radioresistance was validated by isogenic cell-based experiments. These results provide proof-of-principle for the analytical pipeline employed in this study, which explores clinically relevant mutation signatures for radioresistance, and demonstrate that this approach is worth pursuing with larger cohorts in the future.

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The pendulum of the Ku-Ku clock.

Shibata A, Jeggo P, Löbrich M

DNA repair 2018.08

Accepted, ISSN 1568-7864

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Analysis of programmed death-ligand 1 expression in primary normal human dermal fibroblasts after DNA damage.

Yoshihiko Hagiwara, Hiro Sato, Tiara Bunga Mayang Permata, Atsuko Niimi, Motohiro Yamauchi, Takahiro Oike, Takashi Nakano, Atsushi Shibata

Human immunology (Elsevier) 79 ( 8 ) 627 - 631 2018.05

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted, ISSN 0198-8859

Programmed cell death-1 (PD-1) and its ligand (programmed death-ligand 1, PD-L1) are key factors that regulate a cytotoxic immune reaction. Anti-PD-1 therapy provides significant clinical benefits for patients with cancer, even those with advanced-stage cancer. We have recently demonstrated that DNA damage signaling from DNA double-strand breaks (DSBs) promotes PD-L1 upregulation in cancer cells. In the present study, we aimed to investigate PD-L1 expression in primary normal human dermal fibroblasts (NHDFs) in response to DSBs. We demonstrated that PD-L1 expression in NHDFs is not upregulated after ionizing radiation (IR). In addition, interferon (IFN) regulatory factor 1 (IRF1) and signal transducer and activator of transcription 1 (STAT1) phosphorylation do not respond in NHDFs after IR. In contrast, IFNγ treatment upregulates PD-L1 and IRF1 expressions and STAT1 phosphorylation. The nonresponsiveness was also observed after treatment with other DNA-damaging agents, such as camptothecin and etoposide. Treatment with a histone deacetylase inhibitor (HDACi), which causes chromatin relaxation and restores gene silencing, upregulates PD-L1 without exogenous DNA damage; however, IR-dependent upregulation is not observed in NHDFs treated with HDACi. Taken together, our data suggest that DNA-damage signaling is insufficient for upregulating PD-L1 in NHDFs.

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ゲノム恒常性維持機構の破綻と疾患発症の分子メカニズム DNA二本鎖切断修復経路による免疫チェックポイント標的分子PD-L1の発現制御機構

柴田淳史, 佐藤浩央, 新美敦子, 安原崇哲, 萩原慶彦, Permata Tiara, 磯野真由, Nuryadi Endang, 尾池貴洋, 吉本由哉, 鈴木義行, 河野浩二, 宮川清, 中野隆史

生命科学系学会合同年次大会 (生命科学系学会合同年次大会運営事務局) 2017年度 [3PW02 - 6] 2017.12

重粒子線に特徴的なDNA損傷の可視化と細胞運命決定に与える影響の解析

新美敦子, 萩原慶彦, リムシリチャイクル・シリパン, 尾池貴洋, 佐藤浩央, 中野隆史, 柴田淳史

生命科学系学会合同年次大会 (生命科学系学会合同年次大会運営事務局) 2017年度 [4P2T14 - 0597)] 2017.12

3D-structured illumination microscopy reveals clustered DNA double-strand break formation in widespread γH2AX foci after high LET heavy-ion particle radiation.

Yoshihiko Hagiwara, Atsuko Niimi, Mayu Isono, Motohiro Yamauchi, Takaaki Yasuhara, Siripan Limsirichaikul, Takahiro Oike, Hiro Sato, Kathryn D Held, Takashi Nakano, Atsushi Shibata

Oncotarget 8 109370 - 109381 2017.11

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted

DNA double-strand breaks (DSBs) induced by ionising radiation are considered the major cause of genotoxic mutations and cell death. While DSBs are dispersed throughout chromatin after X-rays or γ-irradiation, multiple types of DNA damage including DSBs, single-strand breaks and base damage can be generated within 1-2 helical DNA turns, defined as a complex DNA lesion, after high Linear Energy Transfer (LET) particle irradiation. In addition to the formation of complex DNA lesions, recent evidence suggests that multiple DSBs can be closely generated along the tracks of high LET particle irradiation. Herein, by using three dimensional (3D)-structured illumination microscopy, we identified the formation of 3D widespread γH2AX foci after high LET carbon-ion irradiation. The large γH2AX foci in G2-phase cells encompassed multiple foci of replication protein A (RPA), a marker of DSBs undergoing resection during homologous recombination. Furthermore, we demonstrated by 3D analysis that the distance between two individual RPA foci within γH2AX foci was approximately 700 nm. Together, our findings suggest that high LET heavy-ion particles induce clustered DSB formation on a scale of approximately 1 μm3. These closely localised DSBs are considered to be a risk for the formation of chromosomal rearrangement after heavy-ion irradiation.

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DNA double-strand break repair pathway regulates PD-L1 expression in cancer cells

Hiro Sato, Atsuko Niimi, Takaaki Yasuhara, Tiara Bunga Mayang Permata, Yoshihiko Hagiwara, Mayu Isono, Endang Nuryadi, Ryota Sekine, Takahiro Oike, Sangeeta Kakoti, Yuya Yoshimoto, Kathryn D. Held, Yoshiyuki Suzuki, Koji Kono, Kiyoshi Miyagawa, Takashi Nakano, Atsushi Shibata

NATURE COMMUNICATIONS (NATURE PUBLISHING GROUP) 8 ( 1 ) 1751 2017.11

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted, ISSN 2041-1723

Accumulating evidence suggests that exogenous cellular stress induces PD-L1 upregulation in cancer. A DNA double-strand break (DSB) is the most critical type of genotoxic stress, but the involvement of DSB repair in PD-L1 expression has not been investigated. Here we show that PD-L1 expression in cancer cells is upregulated in response to DSBs. This upregulation requires ATM/ATR/Chk1 kinases. Using an siRNA library targeting DSB repair genes, we discover that BRCA2 depletion enhances Chk1-dependent PD-L1 upregulation after X-rays or PARP inhibition. In addition, we show that Ku70/80 depletion substantially enhances PD-L1 upregulation after X-rays. The upregulation by Ku80 depletion requires Chk1 activation following DNA end-resection by Exonuclease 1. DSBs activate STAT1 and STAT3 signalling, and IRF1 is required for DSB-dependent PD-L1 upregulation. Thus, our findings reveal the involvement of DSB repair in PD-L1 expression and provide mechanistic insight into how PD-L1 expression is regulated after DSBs.

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Aquarius is required for proper CtIP expression and homologous recombination repair

Ryo Sakasai, Mayu Isono, Mitsuo Wakasugi, Mitsumasa Hashimoto, Yumi Sunatani, Tadashi Matsui, Atsushi Shibata, Tsukasa Matsunaga, Kuniyoshi Iwabuchi

SCIENTIFIC REPORTS (NATURE PUBLISHING GROUP) 7 ( 1 ) 13808 2017.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 2045-2322

Accumulating evidence indicates that transcription is closely related to DNA damage formation and that the loss of RNA biogenesis factors causes genome instability. However, whether such factors are involved in DNA damage responses remains unclear. We focus here on the RNA helicase Aquarius (AQR), a known R-loop processing factor, and show that its depletion in human cells results in the accumulation of DNA damage during S phase, mediated by R-loop formation. We investigated the involvement of Aquarius in DNA damage responses and found that AQR knockdown decreased DNA damage-induced foci formation of Rad51 and replication protein A, suggesting that Aquarius contributes to homologous recombination (HR)-mediated repair of DNA double-strand breaks (DSBs). Interestingly, the protein level of CtIP, a DSB processing factor, was decreased in AQR-knockdown cells. Exogenous expression of Aquarius partially restored CtIP protein level; however, CtIP overproduction did not rescue defective HR in AQR-knockdown cells. In accordance with these data, Aquarius depletion sensitized cells to genotoxic agents. We propose that Aquarius contributes to the maintenance of genomic stability via regulation of HR by CtIP-dependent and -independent pathways.

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One-step Protocol for Evaluation of the Mode of Radiation-induced Clonogenic Cell Death by Fluorescence Microscopy

Daijiro Kobayashi, Atsushi Shibata, Takahiro Oike, Takashi Nakano

JOVE-JOURNAL OF VISUALIZED EXPERIMENTS (JOURNAL OF VISUALIZED EXPERIMENTS) ( 128 ) e56338 2017.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 1940-087X

Research on ionizing radiation (IR)-induced clonogenic cell death is important for understanding the effect of IR on malignant tumors and normal tissues. Here, we describe a quick and cost-effective one-step assay for simultaneously assessing the major modes of clonogenic cell death induced by IR, i.e., apoptosis, mitotic catastrophe, and cellular senescence. In this method, cells grown on a cover slip are irradiated with Xrays and stained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI). Using fluorescence microscopy, apoptosis, mitotic catastrophe, and cellular senescence are identified based on the characteristic morphologies of the DAPI-stained nuclei. Apoptosis is determined by the presence of apoptotic bodies (i.e., condensed and fragmented nuclei). Mitotic catastrophe is determined by the presence of nuclei that exhibit two or more distinct lobes and micronuclei. Cellular senescence is determined by the presence of senescence-associated heterochromatic foci (i.e., nuclear DNA containing 30-50 bright, dense foci). This approach allows the experimenter to easily screen for clonogenic cell death modes using various cell lines, treatment settings, and/or time points, with the goal of elucidating the mechanisms of cell death in the target cells and conditions of interest.

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Regulation of repair pathway choice at two-ended DNA double-strand breaks

Atsushi Shibata

MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS 803 51 - 55 2017.10

Accepted, ISSN 0027-5107

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超解像度顕微鏡による重粒子線誘発クラスターDNA二本鎖切断の空間分布解析

新美敦子, 萩原慶彦, リムシリチャイクル・シリパン, 尾池貴洋, 佐藤浩央, 中野隆史, 柴田淳史

日本放射線影響学会大会講演要旨集 ((一社)日本放射線影響学会) 60回 156 - 156 2017.10

ISSN 1347-8680

放射線照射により誘導される免疫チェックポイント標的分子PD-L1の発現制御機構

柴田淳史, 佐藤浩央, 新美敦子, 安原崇哲, 萩原慶彦, パーマタ・ティアラ, 磯野真由, ナーヤディ・エンダング, 尾池貴洋, 吉本由哉, 鈴木義行, 河野浩二, 宮川清, 中野隆史

日本放射線影響学会大会講演要旨集 ((一社)日本放射線影響学会) 60回 116 - 116 2017.10

ISSN 1347-8680

Inhibition of the HDAC/Suv39/G9a pathway restores the expression of DNA damage-dependent major histocompatibility complex class I-related chain A and B in cancer cells

Nakako Izumi Nakajima, Atsuko Niimi, Mayu Isono, Takahiro Oike, Hiro Sato, Takashi Nakano, Atsushi Shibata

Oncology Reports (Spandidos publications) 38 ( 2 ) 693 - 702 2017.06

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted, ISSN 1021-335X

Immunotherapy is expected to be promising as a next generation cancer therapy. Immunoreceptors are often activated constitutively in cancer cells, however, such levels of ligand expression are not effectively recognized by the native immune system due to tumor microenvironmental adaptation. Studies have demonstrated that natural-killer group 2, member D (NKG2D), a major activating immunoreceptor, responds to DNA damage. The upregulation of major histocompatibility complex class I-related chain A and B (MICA/B) (members of NKG2D ligands) expression after DNA damage is associated with NK cell-mediated killing of cancer cells. However, the regulation of DNA damage-induced MICA/B expression has not been fully elucidated in the context of the types of cancer cell lines. In the present study, we found that MICA/B expression varied between cancer cell lines after DNA damage. Screening in terms of chromatin remodeling identified that inhibitors related to chromatin relaxation via post-translational modification on histone H3K9, i.e. HDAC, Suv39 or G9a inhibition, restored DNA damage-dependent MICA/B expression in insensitive cells. In addition, we revealed that the restored MICA/B expression was dependent on ATR as well as E2F1, a transcription factor. We further revealed that low-dose treatment of an HDAC inhibitor was sufficient to restore MICA/B expression in insensitive cells. Finally, we demonstrated that HDAC inhibition restored DNA damage-dependent cytotoxic NK activity against insensitive cells. Thus, the present study revealed that DNA damage-dependent MICA/B expression in insensitive cancer cells can be restored by chromatin relaxation via the HDAC/Suv39/G9a pathway. Collectively, manipulation of chromatin status by therapeutic cancer drugs may potentiate the antitumor effect by enhancing immune activation following radiotherapy and DNA damage-associated chemotherapy.

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DNA Double-Strand Break Resection Occurs during Non-homologous End Joining in G1 but Is Distinct from Resection during Homologous Recombination

Ronja Biehs, Monika Steinlage, Olivia Barton, Szilvia Juhasz, Julia Kuenzel, Julian Spies, Atsushi Shibata, Penny A. Jeggo, Markus Loebrich

MOLECULAR CELL (CELL PRESS) 65 ( 4 ) 671 - + 2017.02

Research paper (scientific journal), Joint Work, Corresponding author, Accepted, ISSN 1097-2765

Canonical non-homologous end joining (c-NHEJ) repairs DNA double-strand breaks (DSBs) in G1 cells with biphasic kinetics. We show that DSBs repaired with slow kinetics, including those localizing to heterochromatic regions or harboring additional lesions at the DSB site, undergo resection prior to repair by c-NHEJ and not alt-NHEJ. Resection-dependent c-NHEJ represents an inducible process during which Plk3 phosphorylates CtIP, mediating its interaction with Brca1 and promoting the initiation of resection. Mre11 exonuclease, EXD2, and Exo1 execute resection, and Artemis endonuclease functions to complete the process. If resection does not commence, then repair can ensue by c-NHEJ, but when executed, Artemis is essential to complete resection-dependent c-NHEJ. Additionally, Mre11 endonuclease activity is dispensable for resection in G1. Thus, resection in G1 differs from the process in G2 that leads to homologous recombination. Resection-dependent c-NHEJ significantly contributes to the formation of deletions and translocations in G1, which represent important initiating events in carcinogenesis.

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Regulation of pairing between broken DNA-containing chromatin regions by Ku80, DNA-PKcs, ATM, and 53BP1

Motohiro Yamauchi, Atsushi Shibata, Keiji Suzuki, Masatoshi Suzuki, Atsuko Niimi, Hisayoshi Kondo, Miwa Miura, Miyako Hirakawa, Keiko Tsujita, Shunichi Yamashita, Naoki Matsuda

SCIENTIFIC REPORTS (NATURE PUBLISHING GROUP) 7 41812 2017.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 2045-2322

Chromosome rearrangement is clinically and physiologically important because it can produce oncogenic fusion genes. Chromosome rearrangement requires DNA double-strand breaks (DSBs) at two genomic locations and misrejoining between the DSBs. Before DSB misrejoining, two DSB-containing chromatin regions move and pair with each other; however, the molecular mechanism underlying this process is largely unknown. We performed a spatiotemporal analysis of ionizing radiation-induced foci of p53-binding protein 1 (53BP1), a marker for DSB-containing chromatin. We found that some 53BP1 foci were paired, indicating that the two damaged chromatin regions neighboured one another. We searched for factors regulating the foci pairing and found that the number of paired foci increased when Ku80, DNA-PKcs, or ATM was absent. In contrast, 53BP1 depletion reduced the number of paired foci and dicentric chromosomes-an interchromosomal rearrangement. Foci were paired more frequently in heterochromatin than in euchromatin in control cells. Additionally, the reduced foci pairing in 53BP1-depleted cells was rescued by concomitant depletion of a heterochromatin building factor such as kRPP ASSOCIATED box-associated protein 1 or chromodomain helicase DNA-binding protein 3. These findings indicate that pairing between DSB-containing chromatin regions was suppressed by Ku80, DNA-PKcs, and ATM, and this pairing was promoted by 53BP1 through chromatin relaxation.

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Mitotic catastrophe is a putative mechanism underlying the weak correlation between sensitivity to carbon ions and cisplatin

Daijiro Kobayashi, Takahiro Oike, Atsushi Shibata, Atsuko Niimi, Yoshiki Kubota, Makoto Sakai, Napapat Amornwhichet, Yuya Yoshimoto, Yoshihiko Hagiwara, Yuka Kimura, Yuka Hirota, Hiro Sato, Mayu Isono, Yukari Yoshida, Takashi Kohno, Tatsuya Ohno, Takashi Nakano

SCIENTIFIC REPORTS (NATURE PUBLISHING GROUP) 7 40588 2017.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 2045-2322

In cancer therapy today, carbon ion radiotherapy is used mainly as monotherapy, whereas cisplatin is used concomitantly with X-ray radiotherapy. The effectiveness of concomitant carbon ions and cisplatin is unclear. To obtain the information on the mechanisms potentially shared between carbon ions or X-rays and cisplatin, we assessed the correlation of sensitivity to the single treatments. In 20 human cancer cell lines, sensitivity to X-rays strongly correlated with sensitivity to cisplatin, indicating the presence of potentially shared target mechanisms. Interestingly, the correlation of sensitivity to carbon ions and cisplatin was much weaker than that of sensitivity to X-rays and cisplatin, indicating the presence of potentially different target mechanisms between carbon ions and cisplatin. Assessment of clonogenic cell death by 4',6-diamidino-2-phenylindole dihydrochloride staining showed that mitotic catastrophe was more efficiently induced by carbon ions than by the same physical dose of X-rays, while apoptosis and senescence were not. These data indicate that the correlation of sensitivity to carbon ions and cisplatin is weaker than that of sensitivity to X-rays and cisplatin, which are helpful as biological basis to understand the potentially shared mechanism among these treatments. Further investigation is mandatory to elucidate the clinical efficacy of carbon ions and cisplatin combination.

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BRCA1 Directs the Repair Pathway to Homologous Recombination by Promoting 53BP1 Dephosphorylation

Mayu Isono, Atsuko Niimi, Takahiro Oike, Yoshihiko Hagiwara, Hiro Sato, Ryota Sekine, Yukari Yoshida, Shin-Ya Isobe, Chikashi Obuse, Ryotaro Nishi, Elena Petricci, Shinichiro Nakada, Takashi Nakano, Atsushi Shibata

CELL REPORTS (CELL PRESS) 18 ( 2 ) 520 - 532 2017.01

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted, ISSN 2211-1247

BRCA1 promoteshomologous recombination (HR) by activating DNA-end resection. By contrast, 53BP1 forms a barrier that inhibits DNA-end resection. Here, we show that BRCA1 promotes DNA-end resection by relieving the 53BP1-dependent barrier. We show that 53BP1 is phosphorylated by ATM in S/G(2) phase, promoting RIF1 recruitment, which inhibits resection. 53BP1 is promptly dephosphorylated and RIF1 released, despite remaining unrepaired DNA double-strand breaks (DSBs). When resection is impaired by CtIP/MRE11 endonuclease inhibition, 53BP1 phosphorylation and RIF1 are sustained due to ongoing ATM signaling. BRCA1 depletion also sustains 53BP1 phosphorylation and RIF1 recruitment. We identify the phosphatase PP4C as having a major role in 53BP1 dephosphorylation and RIF1 release. BRCA1 or PP4C depletion impairs 53BP1 repositioning, EXO1 recruitment, and HR progression. 53BP1 or RIF1 depletion restores resection, RAD51 loading, and HR in PP4C-depleted cells. Our findings suggest that BRCA1 promotes PP4C-dependent 53BP1 dephosphorylation and RIF1 release, directing repair toward HR.

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DNA二本鎖切断修復経路選択制に対する線エネルギー付与依存性の解析

萩原慶彦, 磯野真由, 新美敦子, 尾池貴洋, 佐藤浩央, 中野隆史, 柴田淳史

日本放射線影響学会大会講演要旨集 ((一社)日本放射線影響学会) 59回 3 - 3 2016.10

ISSN 1347-8680

非小細胞肺癌細胞においてEGFR変異状況は炭素イオン線照射療法の相対的生物学的有効性に影響を及ぼす(EGFR mutation status affects the relative biological effectiveness of carbon-ion irradiation in non-small cell lung carcinoma cells)

佐藤浩央, Amornwichet Napapat, 尾池貴洋, 柴田淳史, 磯野真由, 吉田由香里, 大野達也, 中野隆史

日本癌治療学会学術集会抄録集 ((一社)日本癌治療学会) 54回 P15 - 1 2016.10

Identification of DNA Double Strand Breaks at Chromosome Boundaries Along the Track of Particle Irradiation

Atsuko Niimi, Motohiro Yamauchi, Siripan Limsirichaikul, Ryota Sekine, Takahiro Oike, Hiro Sato, Keiji Suzuki, Kathryn D. Held, Takashi Nakano, Atsushi Shibata

GENES CHROMOSOMES & CANCER (Wiley Periodicals, Inc.) 55 ( 8 ) 650 - 660 2016.04

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted, ISSN 1045-2257

Chromosomal translocations arise from misrejoining of DNA double strand breaks (DSBs) between loci located on two chromosomes. One current model suggests that spatial proximity of potential chromosomal translocation partners influences translocation probability. Ionizing radiation (IR) is a potent inducer of translocations. Accumulating evidence demonstrates that particle irradiation more frequently causes translocations compared with X-ray irradiation. This observation has led to the hypothesis that the high frequency of translocations after particle irradiation may be due to the formation of DSBs at chromosome boundaries along the particle track, because such DSBs can be misrejoined between distinct chromosomes. In this study, we simultaneously visualized the site of IR-induced DSBs and chromosome position by combining Immunofluorescence and fluorescence in situ hybridization. Importantly, the frequency of gamma H2AX foci at the chromosome boundary of chromosome 1 after carbon-ion irradiation was >4-fold higher than that after X-ray irradiation. This observation is consistent with the idea that particle irradiation generates DSBs at the boundaries of two chromosomes along the track. Further, we showed that resolution of gamma H2AX foci at chromosome boundaries is prevented by inhibition of DNA-PKcs activity, indicating that the DSB repair is NHEJ-dependent. Finally, we found that gamma H2AX foci at chromosome boundaries after carbon-ion irradiation contain DSBs undergoing DNA-end resection, which promotes repair utilizing microhomology mediated end-joining during translocation. Taken together, our study suggests that the frequency of DSB formation at chromosome boundaries is associated with the incidence of chromosomal translocations, supporting the notion that the spatial proximity between breaks is an important factor in translocation formation. (C) 2016 Wiley Periodicals, Inc.

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Visualization of complex DNA double-strand breaks in a tumor treated with carbon ion radiotherapy

Takahiro Oike, Atsuko Niimi, Noriyuki Okonogi, Kazutoshi Murata, Akihiko Matsumura, Shin-Ei Noda, Daijiro Kobayashi, Mototaro Iwanaga, Keisuke Tsuchida, Tatsuaki Kanai, Tatsuya Ohno, Atsushi Shibata, Takashi Nakano

SCIENTIFIC REPORTS (NATURE PUBLISHING GROUP) 6 22275 2016.03

Research paper (scientific journal), Joint Work, Corresponding author, Accepted, ISSN 2045-2322

Carbon ion radiotherapy shows great potential as a cure for X-ray-resistant tumors. Basic research suggests that the strong cell-killing effect induced by carbon ions is based on their ability to cause complex DNA double-strand breaks (DSBs). However, evidence supporting the formation of complex DSBs in actual patients is lacking. Here, we used advanced high-resolution microscopy with deconvolution to show that complex DSBs are formed in a human tumor clinically treated with carbon ion radiotherapy, but not in a tumor treated with X-ray radiotherapy. Furthermore, analysis using a physics model suggested that the complexity of radiotherapy-induced DSBs is related to linear energy transfer, which is much higher for carbon ion beams than for X-rays. Visualization of complex DSBs in clinical specimens will help us to understand the anti-tumor effects of carbon ion radiotherapy.

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RAD51 and BRCA2 Enhance Oncolytic Adenovirus Type 5 Activity in Ovarian Cancer

Laura A. Tookman, Ashley K. Browne, Claire M. Connell, Gemma Bridge, Carin K. Ingemarsdotter, Suzanne Dowson, Atsushi Shibata, Michelle Lockley, Sarah A. Martin, Iain A. McNeish

MOLECULAR CANCER RESEARCH (AMER ASSOC CANCER RESEARCH) 14 ( 1 ) 44 - 55 2016.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 1541-7786

Homologous recombination (HR) function is critically important in high-grade serous ovarian cancer (HGSOC). HGSOC with intact HR has a worse prognosis and is less likely to respond to platinum chemotherapy and PARP inhibitors. Oncolytic adenovirus, a novel therapy for human malignancies, stimulates a potent DNA damage response that influences overall antitumor activity. Here, the importance of HR was investigated by determining the efficacy of adenovirus type 5 (Ad5) vectors in ovarian cancer. Using matched BRCA2-mutant and wild-type HGSOC cells, it was demonstrated that intact HR function promotes viral DNA replication and augments overall efficacy, without influencing viral DNA processing. These data were confirmed in a wider panel of HR competent and defective ovarian cancer lines. Mechanistically, both BRCA2 and RAD51 localize to viral replication centers within the infected cell nucleus and that RAD51 localization occurs independently of BRCA2. In addition, a direct interaction was identified between RAD51 and adenovirus E2 DNA binding protein. Finally, using functional assays of HR competence, despite inducing degradation of MRE11, Ad5 infection does not alter cellular ability to repair DNA double-strand break damage via HR. These data reveal that Ad5 redistributes critical HR components to viral replication centers and enhances cytotoxicity. (C)2015 AACR.

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多様なDNA損傷応答の統合制御機構2015 ゲノム不安定性の病態解明研究 BRCA1によるDSB end resectionの時空間的制御

磯野真由, 新美敦子, 尾池貴洋, 萩原慶彦, 佐藤浩央, 関根亮太, 吉田由香里, Petricci Elena, 中田慎一郎, 中野隆史, 柴田淳史

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 ((公社)日本生化学会) 88回・38回 [3W2 - 1] 2015.12

高LET重粒子線照射により誘発される2つの異なる染色体間にまたがるクラスターDNA損傷の同定

新美敦子, 山内基弘, リムシリチャイクル・シリパン, 関根崚太, 磯野真由, 尾池貴洋, 佐藤浩央, 鈴木啓司, 中野隆史, 柴田淳史

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 ((公社)日本生化学会) 88回・38回 [1P0630] - [1P0630] 2015.12

炭素線特異的なDNA end resection機序の解析

萩原慶彦, 磯野真由, 新美敦子, 尾池貴洋, 佐藤浩央, 関根崚太, 中野隆史, 柴田淳史

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 ((公社)日本生化学会) 88回・38回 [1P0651] - [1P0651] 2015.12

炭素イオン線特異的なDNA二本鎖切断修復開始機構の解析

萩原慶彦, 磯野真由, 新美敦子, 尾池貴洋, 佐藤浩央, 関根崚太, 中野隆史, 柴田淳史

放射線ワークショップ講演論文集 ((一社)日本放射線影響学会) 1回 6 - 7 2015.10

SETDB1, HP1 and SUV39 promote repositioning of 53BP1 to extend resection during homologous recombination in G2 cells

Meryem Alagoz, Yoko Katsuki, Hideaki Ogiwara, Tomoo Ogi, Atsushi Shibata, Andreas Kakarougkas, Penny Jeggo

NUCLEIC ACIDS RESEARCH (OXFORD UNIV PRESS) 43 ( 16 ) 7931 - 7944 2015.09

Research paper (scientific journal), Joint Work, Accepted, ISSN 0305-1048

Recent studies have shown that homologous recombination ( HR) requires chromatin repression as well as relaxation at DNA double strand breaks ( DSBs). HP1 and SUV39H1/ 2 are repressive factors essential for HR. Here, we identify SETDB1 as an additional compacting factor promoting HR. Depletion of HP1, SUV39, SETDB1 or BRCA1 confer identical phenotypes. The repressive factors, like BRCA1, are dispensable for the initiation of resection but promote the extension step causing diminished RPA or RAD51 foci and HR in irradiated G2 cells. Depletion of the compacting factors does not inhibit BRCA1 recruitment but at 8 h post IR, BRCA1 foci are smaller and aberrantly positioned compared to control cells. BRCA1 promotes 53BP1 repositioning to the periphery of enlarged foci and formation of a devoid core with BRCA1 becoming enlarged and localized internally to 53BP1. Depletion of the compacting factors precludes these changes at irradiation- induced foci. Thus, the repressive factors are required for BRCA1 function in promoting the repositioning of 53BP1 during HR. Additionally, depletion of these repressive factors in undamaged cells causes diminished sister chromatid association at centromeric sequences. We propose a model for how these findings may be functionally linked.

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Carbon-ion beams effectively induce growth inhibition and apoptosis in human neural stem cells compared with glioblastoma A172 cells

Mayu Isono, Yukari Yoshida, Akihisa Takahashi, Takahiro Oike, Atsushi Shibata, Yoshiki Kubota, Tatsuaki Kanai, Tatsuya Ohno, Takashi Nakano

JOURNAL OF RADIATION RESEARCH (OXFORD UNIV PRESS) 56 ( 5 ) 856 - 861 2015.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 0449-3060

Carbon-ion radiotherapy (CIRT) holds promise in the treatment of glioblastoma, an aggressive X-ray-resistant brain tumor. However, since glioblastoma cells show a highly invasive nature, carbon-ion (C-ion) irradiation of normal tissues surrounding the tumor is inevitable. Recent studies have revealed the existence of neural stem cells in the adult brain. Therefore, the damaging effect of C-ion beams on the neural stem cells has to be carefully considered in the treatment planning of CIRT. Here, we investigated the growth and death mode of human neural stem cells (hNSCs) and glioblastoma A172 cells after X-ray or C-ion beam irradiation. The X-ray dose resulting in a 50% growth rate (D-50) was 0.8 Gy in hNSCs and 3.0 Gy in A172 cells, while the D-50 for C-ion beams was 0.4 Gy in hNSCs and 1.6 Gy in A172 cells; the relative biological effectiveness value of C-ion beams was 2.0 in hNSCs and 1.9 in A172 cells. Importantly, both X-rays and C-ion beams preferentially induced apoptosis, not necrosis, in hNSCs; however, radiation-induced apoptosis was less evident in A172 cells. The apoptosis-susceptible nature of the irradiated hNSCs was associated with prolonged upregulation of phosphorylated p53, whereas the apoptosis-resistant nature of A172 cells was associated with a high basal level of nuclear factor kappa B expression. Taken together, these data indicate that apoptosis is the major cell death pathway in hNSCs after irradiation. The high sensitivity of hNSCs to C-ion beams underscores the importance of careful target volume delineation in the treatment planning of CIRT for glioblastoma.

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The EGFR mutation status affects the relative biological effectiveness of carbon-ion beams in non-small cell lung carcinoma cells

Napapat Amornwichet, Takahiro Oike, Atsushi Shibata, Chaitanya S. Nirodi, Hideaki Ogiwara, Haruhiko Makino, Yuka Kimura, Yuka Hirota, Mayu Isono, Yukari Yoshida, Tatsuya Ohno, Takashi Kohno, Takashi Nakano

SCIENTIFIC REPORTS (NATURE PUBLISHING GROUP) 5 11305 2015.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 2045-2322

Carbon-ion radiotherapy (CIRT) holds promise to treat inoperable locally-advanced non-small cell lung carcinoma (NSCLC), a disease poorly controlled by standard chemoradiotherapy using X-rays. Since CIRT is an extremely limited medical resource, selection of NSCLC patients likely to benefit from it is important; however, biological predictors of response to CIRT are ill-defined. The present study investigated the association between the mutational status of EGFR and KRAS, driver genes frequently mutated in NSCLC, and the relative biological effectiveness (RBE) of carbon-ion beams over X-rays. The assessment of 15 NSCLC lines of different EGFR/KRAS mutational status and that of isogenic NSCLC lines expressing wild-type or mutant EGFR revealed that EGFR-mutant NSCLC cells, but not KRAS-mutant cells, show low RBE. This was attributable to (i) the high X-ray sensitivity of EGFR-mutant cells, since EGFR mutation is associated with a defect in non-homologous end joining, a major pathway for DNA double-strand break (DSB) repair, and (ii) the strong cell-killing effect of carbon-ion beams due to poor repair of carbon-ion beam-induced DSBs regardless of EGFR mutation status. These data highlight the potential of EGFR mutation status as a predictor of response to CIRT, i. e., CIRT may show a high therapeutic index in EGFR mutation-negative NSCLC.

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Pre-Exposure to Ionizing Radiation Stimulates DNA Double Strand Break End Resection, Promoting the Use of Homologous Recombination Repair

Nakako Izumi Nakajima, Yoshihiko Hagiwara, Takahiro Oike, Ryuichi Okayasu, Takeshi Murakami, Takashi Nakano, Atsushi Shibata

PLOS ONE (PUBLIC LIBRARY SCIENCE) 10 ( 3 ) e0122582 2015.03

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted, ISSN 1932-6203

The choice of DNA double strand break (DSB) repair pathway is determined at the stage of DSB end resection. Resection was proposed to control the balance between the two major DSB repair pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). Here, we examined the regulation of DSB repair pathway choice at two-ended DSBs following ionizing radiation (IR) in G2 phase of the cell cycle. We found that cells pre-exposed to low-dose IR preferred to undergo HR following challenge IR in G2, whereas NHEJ repair kinetics in G1 were not affected by pre-IR treatment. Consistent with the increase in HR usage, the challenge IR induced Replication protein A (RPA) foci formation and RPA phosphorylation, a marker of resection, were enhanced by pre-IR. However, neither major DNA damage signals nor the status of core NHEJ proteins, which influence the choice of repair pathway, was significantly altered in pre-IR treated cells. Moreover, the increase in usage of HR due to pre-IR exposure was prevented by treatment with ATM inhibitor during the incubation period between pre-IR and challenge IR. Taken together, the results of our study suggest that the ATM-dependent damage response after pre-IR changes the cellular environment, possibly by regulating gene expression or post-transcriptional modifications in a manner that promotes resection.

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Carbon-Ion Beam Irradiation Kills X-Ray-Resistant p53-Null Cancer Cells by Inducing Mitotic Catastrophe

Napapat Amornwichet, Takahiro Oike, Atsushi Shibata, Hideaki Ogiwara, Naoto Tsuchiya, Motohiro Yamauchi, Yuka Saitoh, Ryota Sekine, Mayu Isono, Yukari Yoshida, Tatsuya Ohno, Takashi Kohno, Takashi Nakano

PLoS One (PUBLIC LIBRARY SCIENCE) 9 ( 12 ) e115121 2014.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 1932-6203

Background and Purpose: To understand the mechanisms involved in the strong killing effect of carbon-ion beam irradiation on cancer cells with TP53 tumor suppressor gene deficiencies.
Materials and Methods: DNA damage responses after carbon-ion beam or X-ray irradiation in isogenic HCT116 colorectal cancer cell lines with and without TP53 (p53(+/+) and p53(-/-), respectively) were analyzed as follows: cell survival by clonogenic assay, cell death modes by morphologic observation of DAPI-stained nuclei, DNA double-strand breaks (DSBs) by immunostaining of phosphorylated H2AX (gamma H2AX), and cell cycle by flow cytometry and immunostaining of Ser10-phosphorylated histone H3.
Results: The p53(-/-) cells were more resistant than the p53(+/+) cells to X-ray irradiation, while the sensitivities of the p53(+/+) and p53(-/-) cells to carbon-ion beam irradiation were comparable. X-ray and carbon-ion beam irradiations predominantly induced apoptosis of the p53(+/+) cells but not the p53(-/-) cells. In the p53(-/-) cells, carbon-ion beam irradiation, but not X-ray irradiation, markedly induced mitotic catastrophe that was associated with premature mitotic entry with harboring long-retained DSBs at 24 h post-irradiation.
Conclusions: Efficient induction of mitotic catastrophe in apoptosis-resistant p53-deficient cells implies a strong cancer cell-killing effect of carbon-ion beam irradiation that is independent of the p53 status, suggesting its biological advantage over X-ray treatment.

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DNA2重鎖切断修復の分子機構研究の最先端 DNA二本鎖切断修復経路を決定する分子メカニズム

柴田淳史, 尾池貴洋, テイナー・ジョン, ペトリシ・エレーナ, ジェゴ・ペニー, 中野隆史

日本放射線影響学会大会講演要旨集 ((一社)日本放射線影響学会) 57回 65 - 65 2014.09

ISSN 1347-8680

神経幹細胞および神経膠芽腫細胞における増殖率を指標としたX線および炭素線の感受性

磯野真由, 吉田由香里, 八高知子, 高橋昭久, 尾池貴洋, 柴田淳史, 金井達明, 大野達也, 中野隆史

日本放射線影響学会大会講演要旨集 ((一社)日本放射線影響学会) 57回 130 - 130 2014.09

ISSN 1347-8680

DNA Double-strand Break Repair in a Cellular Context

A. Shibata, P. A. Jeggo

CLINICAL ONCOLOGY 26 ( 5 ) 243 - 249 2014.05

ISSN 0936-6555

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DNA Double-Strand Break Repair Pathway Choice Is Directed by Distinct MRE11 Nuclease Activities

Atsushi Shibata, Davide Moiani, Andrew S. Arvai, Jefferson Perry, Shane M. Harding, Marie-Michelle Genois, Ranjan Maity, Sari van Rossum-Fikkert, Aryandi Kertokalio, Filippo Romoli, Amani Ismail, Ermal Ismalaj, Elena Petricci, Matthew J. Neale, Robert G. Bristow, Jean-Yves Masson, Claire Wyman, Penny A. Jeggo, John A. Tainer

MOLECULAR CELL (CELL PRESS) 53 ( 1 ) 7 - 18 2014.01

Research paper (scientific journal), Joint Work, Lead author, Accepted, ISSN 1097-2765

MRE11 within the MRE11-RAD50-NBS1 (MRN) complex acts in DNA double-strand break repair (DSBR), detection, and signaling; yet, how its endo- and exonuclease activities regulate DSBR by non-homologous end-joining (NHEJ) versus homologous recombination (HR) remains enigmatic. Here, we employed structure-based design with a focused chemical library to discover specific MRE11 endo- or exonuclease inhibitors. With these inhibitors, we examined repair pathway choice at DSBs generated in G2 following radiation exposure. While nuclease inhibition impairs radiation-induced replication protein A (RPA) chromatin binding, suggesting diminished resection, the inhibitors surprisingly direct different repair outcomes. Endonuclease inhibition promotes NHEJ in lieu of HR, while exonuclease inhibition confers a repair defect. Collectively, the results describe nuclease-specific MRE11 inhibitors, define distinct nuclease roles in DSB repair, and support a mechanism whereby MRE11 endonuclease initiates resection, thereby licensing HR followed by MRE11 exonuclease and EXO1/BLM bidirectional resection toward and away from the DNA end, which commits to HR.

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Co-operation of BRCA1 and POH1 relieves the barriers posed by 53BP1 and RAP80 to resection

Andreas Kakarougkas, Amani Ismail, Yoko Katsuki, Raimundo Freire, Atsushi Shibata, Penny A. Jeggo

NUCLEIC ACIDS RESEARCH (OXFORD UNIV PRESS) 41 ( 22 ) 10298 - 10311 2013.09

Research paper (scientific journal), Joint Work, Accepted, ISSN 0305-1048

In G2 phase cells, DNA double-strand break repair switches from DNA non-homologous end-joining to homologous recombination. This switch demands the promotion of resection. We examine the changes in 53BP1 and RAP80 ionizing radiation induced foci (IRIF) in G2 phase, as these are factors that restrict resection. We observed a 2-fold increase in the volume of 53BP1 foci by 8 h, which is not seen in G1 cells. Additionally, an IRIF core devoid of 53BP1 arises where RPA foci form, with BRCA1 IRIF forming between 53BP1 and replication protein A (RPA). Ubiquitin chains assessed using alpha-FK2 antibodies are similarly repositioned. Repositioning of all these components requires BRCA1's BRCT but not the ring finger domain. 53BP1, RAP80 and ubiquitin chains are enlarged following POH1 depletion by small interfering RNA, but a devoid core does not form and RPA foci formation is impaired. Co-depletion of POH1 and RAP80, BRCC36 or ABRAXAS allows establishment of the 53BP1 and ubiquitin chain-devoid core. Thus, the barriers posed by 53BP1 and RAP80 are relieved by BRCA1 and POH1, respectively. Analysis of combined depletions shows that these represent distinct but interfacing barriers to promote loss of ubiquitin chains in the IRIF core, which is required for subsequent resection. We propose a model whereby BRCA1 impacts on 53BP1 to allow access of POH1 to RAP80. POH1-dependent removal of RAP80 within the IRIF core enables degradation of ubiquitin chains, which promotes loss of 53BP1. Thus, POH1 represents a novel component regulating the switch from non-homologous end-joining to homologous recombination.

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Opposing roles for 53BP1 during homologous recombination

Andreas Kakarougkas, Amani Ismail, Karolin Klement, Aaron A. Goodarzi, Sandro Conrad, Raimundo Freire, Atsushi Shibata, Markus Lobrich, Penny A. Jeggo

NUCLEIC ACIDS RESEARCH (OXFORD UNIV PRESS) 41 ( 21 ) 9719 - 9731 2013.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 0305-1048

Although DNA non-homologous end-joining repairs most DNA double-strand breaks (DSBs) in G2 phase, late repairing DSBs undergo resection and repair by homologous recombination (HR). Based on parallels to the situation in G1 cells, previous work has suggested that DSBs that undergo repair by HR predominantly localize to regions of heterochromatin (HC). By using H3K9me3 and H4K20me3 to identify HC regions, we substantiate and extend previous evidence, suggesting that HC-DSBs undergo repair by HR. Next, we examine roles for 53BP1 and BRCA1 in this process. Previous studies have shown that 53BP1 is pro-non-homologous end-joining and anti-HR. Surprisingly, we demonstrate that in G2 phase, 53BP1 is required for HR at HC-DSBs with its role being to promote phosphorylated KAP-1 foci formation. BRCA1, in contrast, is dispensable for pKAP-1 foci formation but relieves the barrier caused by 53BP1. As 53BP1 is retained at irradiation-induced foci during HR, we propose that BRCA1 promotes displacement but retention of 53BP1 to allow resection and any necessary HC modifications to complete HR. In contrast to this role for 53BP1 in HR in G2 phase, we show that it is dispensable for HR in S phase, where HC regions are likely relaxed during replication.

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Visualisation of gamma H2AX Foci Caused by Heavy Ion Particle Traversal; Distinction between Core Track versus Non-Track Damage

Nakako Izumi Nakajima, Holly Brunton, Ritsuko Watanabe, Amruta Shrikhande, Ryoichi Hirayama, Naruhiro Matsufuji, Akira Fujimori, Takeshi Murakami, Ryuichi Okayasu, Penny Jeggo, Atsushi Shibata

PLOS ONE (PUBLIC LIBRARY SCIENCE) 8 ( 14 ) e70107 2013.08

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted, ISSN 1932-6203

Heavy particle irradiation produces complex DNA double strand breaks (DSBs) which can arise from primary ionisation events within the particle trajectory. Additionally, secondary electrons, termed delta-electrons, which have a range of distributions can create low linear energy transfer (LET) damage within but also distant from the track. DNA damage by delta-electrons distant from the track has not previously been carefully characterised. Using imaging with deconvolution, we show that at 8 hours after exposure to Fe (similar to 200 keV/mu m) ions, gamma H2AX foci forming at DSBs within the particle track are large and encompass multiple smaller and closely localised foci, which we designate as clustered gamma H2AX foci. These foci are repaired with slow kinetics by DNA non-homologous end-joining (NHEJ) in G1 phase with the magnitude of complexity diminishing with time. These clustered foci (containing 10 or more individual foci) represent a signature of DSBs caused by high LET heavy particle radiation. We also identified simple gamma H2AX foci distant from the track, which resemble those arising after X-ray exposure, which we attribute to low LET delta-electron induced DSBs. They are rapidly repaired by NHEJ. Clustered gamma H2AX foci induced by heavy particle radiation cause prolonged checkpoint arrest compared to simple gamma H2AX foci following X-irradiation. However, mitotic entry was observed when similar to 10 clustered foci remain. Thus, cells can progress into mitosis with multiple clusters of DSBs following the traversal of a heavy particle.

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Endogenously induced DNA double strand breaks arise in heterochromatic DNA regions and require ataxia telangiectasia mutated and Artemis for their repair

Lisa Woodbine, H. Brunton, A. A. Goodarzi, A. Shibata, P. A. Jeggo

NUCLEIC ACIDS RESEARCH (OXFORD UNIV PRESS) 39 ( 16 ) 6986 - 6997 2011.09

Research paper (scientific journal), Joint Work, Accepted, ISSN 0305-1048

Ataxia telangiectasia (ATM) mutated and Artemis, the proteins defective in ataxia telangiectasia and a class of Radiosensitive-Severe Combined Immunodeficiency (RS-SCID), respectively, function in the repair of DNA double strand breaks (DSBs), which arise in heterochromatic DNA (HC-DSBs) following exposure to ionizing radiation (IR). Here, we examine whether they have protective roles against oxidative damage induced and/or endogenously induced DSBs. We show that DSBs generated following acute exposure of G0/G1 cells to the oxidative damaging agent, tert-butyl hydroperoxide (TBH), are repaired with fast and slow components of similar magnitude to IR-induced DSBs and have a similar requirement for ATM and Artemis. Strikingly, DSBs accumulate in ATM(-/-) mouse embryo fibroblasts (MEFs) and in ATM or Artemis-defective human primary fibroblasts maintained for prolonged periods under confluence arrest. The accumulated DSBs localize to HC-DNA regions. Collectively, the results provide strong evidence that oxidatively induced DSBs arise in HC as well as euchromatic DNA and that Artemis and ATM function in their repair. Additionally, we show that Artemis functions downstream of ATM and is dispensable for HC-relaxation and for pKAP-1 foci formation. These findings are important for evaluating the impact of endogenously arising DNA DSBs in ATM and Artemis-deficient patients.

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Analysis of Human Syndromes with Disordered Chromatin Reveals the Impact of Heterochromatin on the Efficacy of ATM-Dependent G(2)/M Checkpoint Arrest

Holly Brunton, Aaron A. Goodarzi, Angela T. Noon, Amruta Shrikhande, R. Scott Hansen, Penny A. Jeggo, Atsushi Shibata

MOLECULAR AND CELLULAR BIOLOGY (Taylor and Francis Group) 31 ( 19 ) 4022 - 4035 2011.07

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted, ISSN 0270-7306

Heterochromatin (HC) poses a barrier to gamma H2AX focus expansion and DNA double-strand break (DSB) repair, the latter being relieved by ATM-dependent KAP-1 phosphorylation. Using high-resolution imaging, we show here that the HC superstructure markedly restricts ATM signaling to cell cycle checkpoint proteins. The impact of HC is greater than anticipated from the percentage of HC-DNA and, in distinction to DSB repair, ATM only partly overcomes the constraints posed by HC. Importantly, we examine ATM signaling in human syndromes with disordered HC. After depletion of MeCP2 and DNMT3B, proteins defective in the Rett and immunodeficiency with centromere instability and facial anomalies (ICF) syndromes, respectively, we demonstrate enhanced gamma H2AX signal expansion at HC-chromocenters in mouse NIH 3T3 cells, which have visible HC-chromocenters. Previous studies have shown that the G(2)/M checkpoint is inefficient requiring multiple DSBs to initiate arrest. MeCP2 and DNMT3B depletion leads to hypersensitive radiation-induced G(2)/M checkpoint arrest despite normal DSB repair. Cell lines from Rett, ICF, and Hutchinson-Guildford progeria syndrome patients similarly showed hyperactivated ATM signaling and hypersensitive and prolonged G(2)/M checkpoint arrest. Collectively, these findings reveal that heterochromatin contributes to the previously described inefficient G(2)/M checkpoint arrest and demonstrate how the signaling response can be uncoupled from DSB repair.

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Factors determining DNA double-strand break repair pathway choice in G2 phase

Atsushi Shibata, Sandro Conrad, Julie Birraux, Verena Geuting, Olivia Barton, Amani Ismail, Andreas Kakarougkas, Katheryn Meek, Gisela Taucher-Scholz, Markus Lobrich, Penny A. Jeggo

EMBO JOURNAL (EMBO press) 30 ( 6 ) 1079 - 1092 2011.05

Research paper (scientific journal), Joint Work, Lead author, Accepted, ISSN 0261-4189

DNA non-homologous end joining (NHEJ) and homologous recombination (HR) function to repair DNA double-strand breaks (DSBs) in G2 phase with HR preferentially repairing heterochromatin-associated DSBs (HC-DSBs). Here, we examine the regulation of repair pathway usage at two-ended DSBs in G2. We identify the speed of DSB repair as a major component influencing repair pathway usage showing that DNA damage and chromatin complexity are factors influencing DSB repair rate and pathway choice. Loss of NHEJ proteins also slows DSB repair allowing increased resection. However, expression of an autophosphorylation-defective DNA-PKcs mutant, which binds DSBs but precludes the completion of NHEJ, dramatically reduces DSB end resection at all DSBs. In contrast, loss of HR does not impair repair by NHEJ although CtIP-dependent end resection precludes NHEJ usage. We propose that NHEJ initially attempts to repair DSBs and, if rapid rejoining does not ensue, then resection occurs promoting repair by HR. Finally, we identify novel roles for ATM in regulating DSB end resection; an indirect role in promoting KAP-1-dependent chromatin relaxation and a direct role in phosphorylating and activating CtIP. The EMBO Journal (2011) 30, 1079-1092. doi:10.1038/emboj.2011.27; Published online 11 February 2011

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Genomic DNA damage and ATR-Chk1 signaling determine oncolytic adenoviral efficacy in human ovarian cancer cells

Claire M. Connell, Atsushi Shibata, Laura A. Tookman, Kyra M. Archibald, Magdalena B. Flak, Katrina J. Pirlo, Michelle Lockley, Sally P. Wheatley, Iain A. McNeish

JOURNAL OF CLINICAL INVESTIGATION (American Society for Clinical Investigation) 121 ( 4 ) 1283 - 1297 2011.04

Research paper (scientific journal), Joint Work, Lead author, Accepted, ISSN 0021-9738

Oncolytic adenoviruses replicate selectively within and lyse malignant cells. As such, they are being developed as anticancer therapeutics. However, the sensitivity of ovarian cancers to adenovirus cytotoxicity varies greatly, even in cells of similar infectivity. Using both the adenovirus E1A-CR2 deletion mutant dl922-947 and WT adenovirus serotype 5 in a panel of human ovarian cancer cell lines that cover a 3-log range of sensitivity, we observed profound overreplication of genomic DNA only in highly sensitive cell lines. This was associated with the presence of extensive genomic DNA damage. Inhibition of ataxia telangiectasia and Rad3-related checkpoint kinase 1 (ATR-Chk1), but not ataxia telangiectasia mutated (ATM), promoted genomic DNA damage and overreplication in resistant and partially sensitive cells. This was accompanied by increased adenovirus cytotoxicity both in vitro and in vivo in tumor-bearing mice. We also demonstrated that Cdc25A was upregulated in highly sensitive ovarian cancer cell lines after adenovirus infection and was stabilized after loss of Chk1 activity. Knockdown of Cdc25A inhibited virus-induced DNA damage in highly sensitive cells and blocked the effects of Chk1 inhibition in resistant cells. Finally, inhibition of Chk1 decreased homologous recombination repair of virus-induced genomic DNA double-strand breaks. Thus, virus-induced host cell DNA damage signaling and repair are key determinants of oncolytic adenoviral activity, and promoting unscheduled DNA synthesis and/or impeding homologous recombination repair could potentiate the effects of oncolytic adenoviruses in the treatment of ovarian cancer.

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Role of ATM and the Damage Response Mediator Proteins 53BP1 and MDC1 in the Maintenance of G(2)/M Checkpoint Arrest

Atsushi Shibata, Olivia Barton, Angela T. Noon, Kirsten Dahm, Dorothee Deckbar, Aaron A. Goodarzi, Markus Loebrich, Penny A. Jeggo

MOLECULAR AND CELLULAR BIOLOGY (the American Society for Microbiology) 30 ( 13 ) 3371 - 3383 2010.07

Research paper (scientific journal), Joint Work, Lead author, Accepted, ISSN 0270-7306

ATM-dependent initiation of the radiation-induced G(2)/M checkpoint arrest is well established. Recent results have shown that the majority of DNA double-strand breaks (DSBs) in G(2) phase are repaired by DNA nonhomologous end joining (NHEJ), while similar to 15% of DSBs are slowly repaired by homologous recombination. Here, we evaluate how the G(2)/M checkpoint is maintained in irradiated G(2) cells, in light of our current understanding of G(2) phase DSB repair. We show that ATM-dependent resection at a subset of DSBs leads to ATR-dependent Chk1 activation. ATR-Seckel syndrome cells, which fail to efficiently activate Chk1, and small interfering RNA (siRNA) Chk1-treated cells show premature mitotic entry. Thus, Chk1 significantly contributes to maintaining checkpoint arrest. Second, sustained ATM signaling to Chk2 contributes, particularly when NHEJ is impaired by XLF deficiency. We also show that cells lacking the mediator proteins 53BP1 and MDC1 initially arrest following radiation doses greater than 3 Gy but are subsequently released prematurely. Thus, 53BP1(-/-) and MDC1(-/-) cells manifest a checkpoint defect at high doses. This failure to maintain arrest is due to diminished Chk1 activation and a decreased ability to sustain ATM-Chk2 signaling. The combined repair and checkpoint defects conferred by 53BP1 and MDC1 deficiency act synergistically to enhance chromosome breakage.

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Sensitization to Radiation and Alkylating Agents by Inhibitors of Poly(ADP-ribose) Polymerase Is Enhanced in Cells Deficient in DNA Double-Strand Break Repair

Dana A. Loeser, Atsushi Shibata, Akiko K. Shibata, Lisa J. Woodbine, Penny A. Jeggo, Anthony J. Chalmers

MOLECULAR CANCER THERAPEUTICS (American ) 9 ( 6 ) 1775 - 1787 2010.06

Joint Work, Lead author, Accepted, ISSN 1535-7163

As single agents, chemical inhibitors of poly(ADP-ribose) polymerase (PARP) are nontoxic and have clinical efficacy against BRCA1- and BRCA2-deficient tumors. PARP inhibitors also enhance the cytotoxicity of ionizing radiation and alkylating agents but will only improve clinical outcomes if tumor sensitization exceeds effects on normal tissues. It is unclear how tumor DNA repair proficiency affects the degree of sensitization. We have previously shown that the radiosensitizing effect of PARP inhibition requires DNA replication and will therefore affect rapidly proliferating tumors more than normal tissues. Because many tumors exhibit defective DNA repair, we investigated the impact of double-strand break (DSB) repair integrity on the sensitizing effects of the PARP inhibitor olaparib. Sensitization to ionizing radiation and the alkylating agent methylmethane sulfonate was enhanced in DSB repair-deficient cells. In Artemis(-/-) and ATM(-/-) mouse embryo fibroblasts, sensitization was replication dependent and associated with defective repair of replication-associated damage. Radiosensitization of Ligase IV(-/-) mouse embryo fibroblasts was independent of DNA replication and is explained by inhibition of "alternative" end joining. After methylmethane sulfonate treatment, PARP inhibition promoted replication-independent accumulation of DSB, repair of which required Ligase IV. Our findings predict that the sensitizing effects of PARP inhibitors will be more pronounced in rapidly dividing and/or DNA repair defective tumors than normal tissues and show their potential to enhance the therapeutic ratio achieved by conventional DNA-damaging agents. Mol Cancer Ther; 9(6); 1775-87. (C)2010 AACR.

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53BP1-dependent robust localized KAP-1 phosphorylation is essential for heterochromatic DNA double-strand break repair

Angela T. Noon, Atsushi Shibata, Nicole Rief, Markus Loebrich, Grant S. Stewart, Penelope A. Jeggo, Aaron A. Goodarzi

NATURE CELL BIOLOGY (NATURE PUBLISHING GROUP) 12 ( 2 ) 177 - 184 2010.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 1465-7392

DNA double-strand breaks (DSBs) trigger ATM (ataxia telangiectasia mutated) signalling and elicit genomic rearrangements and chromosomal fragmentation if misrepaired or unrepaired. Although most DSB repair is ATM-independent, similar to 15% of ionizing radiation (IR)-induced breaks persist in the absence of ATM-signalling(1). 53BP1 (p53-binding protein 1) facilitates ATM-dependent DSB repair but is largely dispensable for ATM activation or checkpoint arrest. ATM promotes DSB repair within heterochromatin by phosphorylating KAP-1 (KRAB-associated protein 1, also known as TIF1 beta, TRIM28 or KRIP-1; ref. 2). Here, we show that the ATM signalling mediator proteins MDC1, RNF8, RNF168 and 53BP1 are also required for heterochromatic DSB repair. Although KAP-1 phosphorylation is critical for 53BP1-mediated repair, overall phosphorylated KAP-1 (pKAP-1) levels are only modestly affected by 53BP1 loss. pKAP-1 is transiently pan-nuclear but also forms foci overlapping with gamma H2AX in heterochromatin. Cells that do not form 53BP1 foci, including human RIDDLE (radiosensitivity, immunodeficiency, dysmorphic features and learning difficulties) syndrome cells, fail to form pKAP-1 foci. 53BP1 amplifies Mre11-NBS1 accumulation at late-repairing DSBs, concentrating active ATM and leading to robust, localized pKAP-1. We propose that ionizing-radiation induced foci (IRIF) spatially concentrate ATM activity to promote localized alterations in regions of chromatin otherwise inhibitory to repair.

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gamma H2AX foci analysis for monitoring DNA double-strand break repair Strengths, limitations and optimization

Markus Loebrich, Atsushi Shibata, Andrea Beucher, Anna Fisher, Michael Ensminger, Aaron A. Goodarzi, Olivia Barton, Penny A. Jeggo

CELL CYCLE 9 ( 4 ) 662 - 669 2010.02

Accepted, ISSN 1538-4101

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ATM and Artemis promote homologous recombination of radiation-induced DNA double-strand breaks in G2

Andrea Beucher, Julie Birraux, Leopoldine Tchouandong, Olivia Barton, Atsushi Shibata, Sandro Conrad, Aaron A. Goodarzi, Andrea Krempler, Penny A. Jeggo, Markus Loebrich

EMBO JOURNAL (EMBO Press) 28 ( 21 ) 3413 - 3427 2009.11

Joint Work, Accepted, ISSN 0261-4189

Homologous recombination (HR) and non-homologous end joining (NHEJ) represent distinct pathways for repairing DNA double-strand breaks (DSBs). Previous work implicated Artemis and ATM in an NHEJ-dependent process, which repairs a defined subset of radiation-induced DSBs in G1-phase. Here, we show that in G2, as in G1, NHEJ represents the major DSB-repair pathway whereas HR is only essential for repair of similar to 15% of X-or gamma-ray-induced DSBs. In addition to requiring the known HR proteins, Brca2, Rad51 and Rad54, repair of radiation-induced DSBs by HR in G2 also involves Artemis and ATM suggesting that they promote NHEJ during G1 but HR during G2. The dependency for ATM for repair is relieved by depleting KAP-1, providing evidence that HR in G2 repairs heterochromatin-associated DSBs. Although not core HR proteins, ATM and Artemis are required for efficient formation of single-stranded DNA and Rad51 foci at radiation-induced DSBs in G2 with Artemis function requiring its endonuclease activity. We suggest that Artemis endonuclease removes lesions or secondary structures, which inhibit end resection and preclude the completion of HR or NHEJ. The EMBO Journal (2009) 28, 3413-3427. doi: 10.1038/emboj.2009.276; Published online 24 September 2009

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Role of Parp-1 in suppressing spontaneous deletion mutation in the liver and brain of mice at adolescence and advanced age

Atsushi Shibata, Daisuke Maeda, Hideki Ogino, Masahiro Tsutsumi, Takehiko Nohmi, Hitoshi Nakagama, Takashi Sugimura, Hirobumi Teraoka, Mitsuko Masutani

MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS (ELSEVIER SCIENCE BV) 664 ( 1-2 ) 20 - 27 2009.05

Research paper (scientific journal), Joint Work, Lead author, Accepted, ISSN 0027-5107

Poly(ADP-ribose) polymerase-1 knockout (Parp-1(-/-)) mice show increased frequency of spontaneous liver tumors compared to wild-type mice after aging. To understand the impact of Parp-1 deficiency on mutations during aging, in this study, we analyzed spontaneous mutations in Parp-1(-/-) aged mice. Parp-1(-/-) mice showed tendencies of higher mutation frequencies of the red/gam genes at 18 months of age, compared to Parp-1(+/+) mice, in the liver and brain. Complex-type deletions, accompanying small insertion were observed only in Parp-1(-/-) mice in the liver and brain. Further analysis in the liver showed that the frequency of single base deletion mutations at non-repeat or short repeat sequences was 5.8-fold higher in Parp-1(-/-) than in Parp-1(+/+) mice (p < 0.05). A 3.2-fold higher tendency of the deletion frequency of two bases or more was observed in Parp-1(-/-) mice compared to Parp-1(+/+) mice (p = 0.084). These results support the model that Parp-1 is involved in suppressing imprecise repair of endogenous DNA damage leading to deletion mutation during aging. The mutation frequencies of the gpt gene in the brain were found to be 3-fold lower in Parp-1(-/-) than in Parp-1(+/+) mice at 4 months of age (p < 0.01), implying that Parp-1 may be positively involved in imprecise DNA repair in the brain. On the other hand, the frequencies of gpt mutation showed an increase at 18 months of age in the Parp-1(-/-) (p < 0.05) but not in Parp-1 brains, suggesting that Parp-1 deficiency causes an increase of point mutations in the brain by aging. (c) 2009 Elsevier B.V. All rights reserved.

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The Maintenance of ATM Dependent G2/M Checkpoint Arrest Following Exposure to Ionizing Radiation

Atsushi Shibata, Olivia Barton, Angela T. Noon, Kirsten Dahm, Dorothee Deckbar, Aaron A. Goodarzi, Markus Lobrich, Penny A. Jeggo

Acta Medica Nagasakiensia (長崎大学医学部) ( 53 ) 19 - 21 2009.03

Research paper (bulletin of university, research institution), Joint Work, Lead author, Accepted, ISSN 00016055

The G2/M checkpoint is important in preventing cells with unrepaired DNA double strand breaks (DSBs) entering mitosis, an event which is
likely to result in genomic instability. We recently reported that heckpoint arrest is maintained until close to completion of DSB repair and that the duration of checkpoint arrest depends on the dose and DSB repair capacity rather than lasting for a fixed period of time. ATM leads to phosphorylation of Chk1/2 inG2 phase following exposure to ionizing radiation. These transducer kinases can phosphorylate and inhibit Cdc25
activity, which is the phosphatase regulatingmitotic entry. In this study we dissect three processes that contribute to the maintenance of checkpoint arrest in irradiated G2 phase cells. First, the ATR-Chk1 pathway contributes to maintaining checkpoint arrest, although it is dispensable for the initial activation of checkpoint arrest. Second, ongoing ATM to Chk2 signalling from unrepaired DSBs contributes to checkpoint arrest. This process plays a greater role in a repair defective background. Finally, slow decay of the initially activated Chk2 also contributes to the maintenance of checkpoint arrest. 53BP1 and MDC1 defective cells show an initial checkpoint defect after low doses but are proficient in initial activation of arrest after high doses. After higher radiation doses, however, 53BP1– /– and MDC1–/– MEFs fail to maintain checkpoint arrest. Furthermore 53BP1– /– andMDC1–/ –MEFs display elevated mitotic breakage even after high doses.We show that the defect in the maintenance of checkpoint arrest conferred by 53BP1 and MDC1 deficiency substantially enhances chromosome breakage.

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Parp-1欠損による短塩基反復配列の不安定性への影響(Impact of Parp-1 deficiency on instability of short nucleotide repeat sequences)

前田大介, 柴田淳史, 荻野秀樹, 中釜斉, 益谷美都子

日本癌学会総会記事 (日本癌学会) 66回 49 - 49 2007.08

Accepted, ISSN 0546-0476

Differential involvement of phosphatidylinositol 3-kinase-related protein kinases in hyperphosphorylation of replication protein A2 in response to replication-mediated DNA double-strand breaks.

Ryo Sakasai, Keitaro Shinohe, Yosuke Ichijima, Naoyuki Okita, Atsushi Shibata, Kinji Asahina, Hirobumi Teraoka

Genes to cells : devoted to molecular & cellular mechanisms (Wiley-Blackwell Publishing Ltd.) 11 ( 3 ) 237 - 246 2006.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 1356-9597

Replication protein A2 (RPA2), a component of the RPA heterotrimer, is hyperphosphorylated and forms nuclear foci in response to camptothecin (CPT) that directly induces replication-mediated DNA double-strand breaks (DSBs). Ataxia-telangiectasia mutated and Rad3-related kinase (ATR) and DNA-dependent protein kinase (DNA-PK) are activated by CPT, and RPA2 is hyperphosphorylated in a DNA-PK-dependent manner. To distinguish the roles of phosphatidylinositol 3-kinase-related protein kinases including DNA-PK, ataxia-telangiectasia mutated (ATM), and ATR, in the response to replication-mediated DSBs, we analyzed RPA2 focus formation and hyperphosphorylation during exposure to CPT. ATR knock-down with siRNA suppressed CPT-induced RPA2 hyperphosphorylation and focus formation. CPT-induced RPA2 focus formation was normally observed in DNA-PK- or ATM-deficient cells. Comparison between CPT and hydroxyurea (HU) indirectly inducing DSBs showed that RPA2 hyperphosphorylation is DNA-PK-dependent in CPT-treated cells and DNA-PK-independent in HU-treated cells. Although RPA2 foci rapidly formed in response to HU and CPT, the RPA2 hyperphosphorylation in HU-treated cells occurred later than in the CPT-treated cells, indicating that the DNA-PK dependency of RPA2 hyperphosphorylation is likely to be related to the mode of DSB induction. These results suggest that DNA-PK is responsible for the RPA2 hyperphosphorylation following ATR-dependent RPA2 focus formation in response to replication-mediated DSBs directly induced by CPT.

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Parp-1 deficiency causes an increase of deletion mutations and insertions/rearrangements in vivo after treatment with an alkylating agent

A Shibata, N Kamada, K Masumura, T Nohmi, S Kobayashi, H Teraoka, H Nakagama, T Sugimura, H Suzuki, M Masutani

ONCOGENE (NATURE PUBLISHING GROUP) 24 ( 8 ) 1328 - 1337 2005.02

Research paper (scientific journal), Joint Work, Lead author, Accepted, ISSN 0950-9232

Accumulated evidence suggests that Parp-1 is involved in DNA repair processes, including base excision repair, single-strand and double-strand break repairs. To understand the precise role of Parp-1 in genomic stability in vivo, we carried out mutation analysis using Parp-1 knockout (Parp-1(-/-)) mice harboring two marker genes, gpt and red/gam genes. Spontaneous mutant frequencies of both genes in the bone marrows and livers did not differ significantly between Parp-1(-/-) and Parp-1(+/+) mice (P > 0.05). After treatment with an alkylating agent, N-nitrosobis(2-hydroxypropyl)amine (BHP), the mutant frequency of the red/gam genes in the liver in Parp-1(-/-) mice was 1.6-fold higher than that in Parp-1(+/+) mice (P<0.05). Categorization of the mutations revealed that deletions larger than 1 kb or those accompanying 1-5 lip insertions at the deletion junctions, as well as rearrangements, were more frequently observed in Parp-1(-/-) than in Parp-1(+/+) mice (P<0.05, respectively). In contrast, mutant frequencies of the gpt gene in the livers of Parp-1(-/-) and Parp-1(+/+) mice after BHP treatment were both elevated and there was no significant difference between the genotypes. These results indicate that Parp-I is implicated in suppressing deletion mutations in vivo, especially those accompanying small insertions or rearrangements. Published online 20 December 2004.

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Efficient method for mapping and characterizing structures of deletion mutations in gpt delta mice using Southern blot analysis with oligo DNA probes

A Shibata, M Masutani, N Kamada, K Masumura, H Nakagama, S Kobayashi, H Teraoka, H Suzuki, T Nohmi

ENVIRONMENTAL AND MOLECULAR MUTAGENESIS (WILEY-LISS) 43 ( 3 ) 204 - 207 2004.01

Research paper (scientific journal), Joint Work, Lead author, Accepted, ISSN 0893-6692

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Improvement of the Spi(-) assay for mutations in gpt delta mice by including magnesium ions during plaque formation

A Shibata, M Masutani, T Nozaki, N Kamada, H Fujihara, K Masumura, H Nakagama, T Sugimura, S Kobayashi, H Suzuki, T Nohmi

ENVIRONMENTAL AND MOLECULAR MUTAGENESIS (WILEY-LISS) 41 ( 5 ) 370 - 372 2003.06

Research paper (scientific journal), Joint Work, Lead author, Accepted, ISSN 0893-6692

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Inhibition of poly(ADP-ribose) glycohydrolase activity by cyclic peptide antibiotics containing piperazic acid residues

Mitsuko Masutani, Takashi Shimokawa, Masayuki Igarashi, Masa Hamada, Atsushi Shibata, Sachiho Oami, Tadashige Nozaki, Hitoshi Nakagama, Takashi Sugimura, Tomio Takeuchi, Makoto Hori

Proceedings of the Japan Academy Series B: Physical and Biological Sciences (日本学士院) 78 ( 1 ) 15 - 17 2002.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 0386-2208

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