Details of a Researcher - Shibata, Atsushi

Papers - Shibata, Atsushi

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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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