研究者詳細 - 柴田　淳史

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年03月

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年03月

ISSN 1347-9032

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

Luong NC, Kawamura H, Ikeda H, Roppongi RT, Shibata A, Hu J, Jiang JG, Yu DS, Held KD

International journal of radiation biology （Taylor & Francis group） 100 （ 5 ） 1 - 12 2024年

研究論文（学術雑誌）, 共著, 査読有り, 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.

Haruna S, Okuda K, Shibata A, Isono M, Tateno K, Sato H, Oike T, Uchihara Y, Kato Y, Shibata A

Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology （Elsevier） 194 110198 - 110198 2024年

研究論文（学術雑誌）, 共著, 最終著者, 責任著者, 査読有り, 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.

Hayashi R, Okumura H, Isono M, Yamauchi M, Unami D, Lusi RT, Yamamoto M, Kato Y, Uchihara Y, Shibata A

Journal of radiation research （Oxford University Press） 65 （ 3 ） 263 - 271 2024年

研究論文（学術雑誌）, 共著, 最終著者, 責任著者, 査読有り, 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.

Ozaki K, Kato R, Yasuhara T, Uchihara Y, Hirakawa M, Abe Y, Shibata H, Kawabata-Iwakawa R, Shakayeva A, Kot P, Miyagawa K, Suzuki K, Matsuda N, Shibata A, Yamauchi M

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

査読有り, ISSN 2045-2322

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Comprehensive single-cell analysis demonstrates radiotherapy-induced infiltration of macrophages expressing immunosuppressive genes into tumor in esophageal squamous cell carcinoma.

Oyoshi H, Du J, Sakai SA, Yamashita R, Okumura M, Motegi A, Hojo H, Nakamura M, Hirata H, Sunakawa H, Kotani D, Yano T, Kojima T, Nakamura Y, Kojima M, Suzuki A, Zenkoh J, Tsuchihara K, Akimoto T, Shibata A, Suzuki Y, Kageyama SI

Science advances （American Association for the Advancement of Science） 9 （ 50 ） eadh9069 2023年12月

研究論文（学術雑誌）, 共著, 責任著者, 査読有り

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.

Uchihara Y, Shibata A

DNA repair （Elsevier BV） 132 103590 - 103590 2023年12月

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年10月

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年09月

研究論文（学術雑誌）, 共著, 査読有り, 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年03月

研究論文（学術雑誌）, 共著, 最終著者, 査読有り, 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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