Tsuyoshi Ikura

Kyoto University, Kioto, Kyōto, Japan

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Publications (40)261.36 Total impact

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    ABSTRACT: Histone modifications change upon the cellular response to ionizing radiation, and their cellular amounts could reflect the DNA damage response activity. We previously reported a sensitive and reliable method for the absolute quantification of γH2AX within cells, using liquid chromatography-tandem mass spectrometry (LC/MS/MS). The technique has broad adaptability to a variety of biological systems and can quantitate different modifications of histones. In this study, we applied it to quantitate the levels of γH2AX and K5-acetylated H2AX, and to compare the radiation responses between two cancer cell lines: HeLa and U-2 OS. The two cell lines have distinct properties in terms of their H2AX modifications. HeLa cells have relatively high γH2AX (3.1 %) against the total H2AX even in un-irradiated cells, while U-2 OS cells have an essentially undetectable level (nearly 0 %) of γH2AX. In contrast, the amounts of acetylated histones are lower in HeLa cells (9.3 %) and higher in U-2 OS cells (24.2 %) under un-irradiated conditions. Furthermore, after ionizing radiation exposure, the time-dependent increases and decreases in the amounts of histone modifications differed between the two cell lines, especially at the early time points. These results suggest that each biological system has distinct kinase/phosphatase and/or acetylase/deacetylase activities. In conclusion, for the first time, we have succeeded in simultaneously monitoring the absolute amounts of phosphorylated and acetylated cellular H2AX after ionizing radiation exposure. This multi-criteria assessment enables precise comparisons of the effects of radiation between any biological systems.
    Biophysik 06/2015; DOI:10.1007/s00411-015-0608-3 · 1.58 Impact Factor
  • Scientific Reports 06/2015; 5:10984. DOI:10.1038/srep10984 · 5.58 Impact Factor
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    ABSTRACT: Homologous recombinational repair (HR) is one of the major repair systems for DNA double-strand breaks. RAD51 is a key molecule in HR, and the RAD51 concentration in the cell nucleus increases after DNA damage induction. However, the mechanism that regulates the intracellular distribution of RAD51 is still unclear. Here, we show that hCAS/CSE1L associates with RAD51 in human cells. We found that hCAS/CSE1L negatively regulates the nuclear protein level of RAD51 under normal conditions. hCAS/CSE1L is also required to repress the DNA damage-induced focus formation of RAD51. Moreover, we show that hCAS/CSE1L plays roles in the regulation of the HR activity and in chromosome stability. These findings suggest that hCAS/CSE1L is responsible for controlling the HR activity by directly interacting with RAD51. © 2015 The Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.
    Genes to Cells 06/2015; DOI:10.1111/gtc.12262 · 2.86 Impact Factor
  • Shun Matsuda, Tsuyoshi Ikura, Tomonari Matsuda
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    ABSTRACT: Ser139-phosphorylated histone H2AX (γH2AX) is a useful biomarker of DNA double strand breaks. γH2AX has been conventionally detected by immunology-based methods using anti-γH2AX antibody, but quantitative analysis is difficult to perform with such methods. Here, we describe an absolute quantification method using liquid chromatography-triple quadrupole tandem mass spectrometry that is applicable to peptides derived from γH2AX (ATQA(pS)QEY) and unphosphorylated H2AX (ATQASQEY). Our method was successfully applied to histones extracted from human cervix adenocarcinoma HeLa S3 cells. The estimated number of molecules of γH2AX (ATQA(pS)QEY) per vehicle-treated HeLa S3 cell was 9.4 × 10(4) and increased to 6.2 × 10(5) molecules/cell after exposure to the DNA-damaging agent camptothecin (10 μM) for 1 h. The estimated total amount of H2AX (ATQA(pS)QEY + ATQASQEY) was 3.3-3.6 × 10(6) molecules/cell. Due to its broad adaptability and throughput performance, we believe that our method is a powerful tool for mechanistic studies of the DNA-damage response as well as for genotoxicity testing, cancer drug screening, clinical studies, and other fields.
    Analytical and Bioanalytical Chemistry 05/2015; DOI:10.1007/s00216-015-8725-z · 3.58 Impact Factor
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    ABSTRACT: DNA double-strand breaks (DSBs) are the major lethal lesion induced by ionizing radiation (IR). RAD51-dependent homologous recombination (HR) is one of the most important pathways in DSB repair and genome integrity maintenance. However, the mechanism of HR regulation by RAD51 remains unclear. To understand the mechanism of RAD51-dependent HR, we searched for interacting partners of RAD51 by a proteomics analysis and identified lamin B1 in human cells. Lamins are nuclear lamina proteins that play important roles in the structural organization of the nucleus and the regulation of chromosome functions. Immunoblotting analyses revealed that siRNA-mediated lamin B1 depletion repressed the DNA damage-dependent increase of RAD51 after IR. The repression was abolished by the proteasome inhibitor MG132, suggesting that lamin B1 stabilizes RAD51 by preventing proteasome-mediated degradation in cells with IR-induced DNA damage. We also showed that lamin B1 depletion repressed RAD51 focus formation and decreased the survival rates after IR. On the basis of these results, we propose that lamin B1 promotes DSB repair and cell survival by maintaining the RAD51 protein levels for HR upon DSB induction after IR.-Liu, N.-A., Sun, J., Kono, K., Horikoshi, Y., Ikura, T., Tong, X., Haraguchi, T., Tashiro, S. Regulation of homologous recombinational repair by lamin B1 in radiation-induced DNA damage. © FASEB.
    The FASEB Journal 03/2015; 29(6). DOI:10.1096/fj.14-265546 · 5.48 Impact Factor
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    ABSTRACT: The reorganization of damaged chromatin plays an important role in the regulation of the DNA damage response. A recent study revealed the presence of 2 vertebrate H2A.Z isoforms, H2A.Z-1 and H2A.Z-2. However, the roles of the vertebrate H2A.Z isoforms are still unclear. Thus, in this study we examined the roles of the vertebrate H2A.Z isoforms in chromatin reorganization after the induction of DNA double-strand breaks (DSBs).
    International journal of radiation oncology, biology, physics 07/2014; 89(4):736-44. DOI:10.1016/j.ijrobp.2014.03.031 · 4.18 Impact Factor
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    ABSTRACT: Homologous recombination plays essential roles in mitotic DNA double strand break (DSB) repair and meiotic genetic recombination. In eukaryotes, RAD51 promotes the central homologous-pairing step during homologous recombination, but is not sufficient to overcome the reaction barrier imposed by nucleosomes. RAD54, a member of the ATP-dependent nucleosome remodeling factor family, is required to promote the RAD51-mediated homologous pairing in nucleosomal DNA. In higher eukaryotes, most nucleosomes form higher-ordered chromatin containing the linker histone H1. However, the mechanism by which RAD51/RAD54-mediated homologous pairing occurs in higher-ordered chromatin has not been elucidated. In this study, we found that a histone chaperone, Nap1, accumulates on DSB sites in human cells, and DSB repair is substantially decreased in Nap1-knockdown cells. We determined that Nap1 binds to RAD54, enhances the RAD54-mediated nucleosome remodeling by evicting histone H1, and eventually stimulates the RAD51-mediated homologous pairing in higher-ordered chromatin containing histone H1.
    Scientific Reports 05/2014; 4:4863. DOI:10.1038/srep04863 · 5.58 Impact Factor
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    ABSTRACT: The Fanconi anemia (FA) pathway is critically involved in the maintenance of hematopoietic stem cells and the suppression of carcinogenesis. A key FA protein, FANCD2, is monoubiquitinated and accumulates in chromatin in response to DNA interstrand crosslinks (ICLs), where it coordinates DNA repair through mechanisms that are still poorly understood. Here, we report that CtIP protein directly interacts with FANCD2. A region spanning amino acids 166 to 273 of CtIP and monoubiquitination of FANCD2 are both essential for the FANCD2-CtIP interaction and mitomycin C (MMC)-induced CtIP foci. Remarkably, both FANCD2 and CtIP are critical for MMC-induced RPA2 hyperphosphorylation, an event that accompanies end resection of double-strand breaks. Collectively, our results reveal a role of monoubiquitinated FANCD2 in end resection that depends on its binding to CtIP during ICL repair.
    Cell Reports 04/2014; 7(4). DOI:10.1016/j.celrep.2014.04.005 · 7.21 Impact Factor
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    ABSTRACT: Histone H2AX phosphorylated at Ser139 (γ-H2AX) is a useful biomarker for DNA double-strand breaks. However, γ-H2AX detection has methodological disadvantages such as the requirement of expensive anti-γ-H2AX antibody and time-consuming handling for its staining. Mediator of DNA damage checkpoint 1 (MDC1) is a central adaptor protein which recruits various DNA damage response proteins to γ-H2AX and thus forms nuclear foci in the same location as γ-H2AX in response to DNA damage. Here, we describe an easy-to-use genotoxicity assay which combines enhanced green fluorescence protein (EGFP)-fused MDC1-expressing cells with a free R program for image-processing and quantification of foci area/nucleus. The workflow of this assay is simple: mutagen treatment, imaging, and R-processing. This assay does not need antibodies or staining handling and it detected the genotoxicity of a range of mutagens, including camptothecin (topoisomerase I inhibitior), cisplatin (crosslinker), and 4-nitroquinoline 1-oxide and benzo[a]pyrene (bulky DNA-adduct forming compounds), as increased fluorescence of EGFP-MDC1 foci. Furthermore, cotreatment with arabinofuranosyl cytosine/hydroxyurea and mutagens sensitized EGFP-MDC1 foci formation to bulky DNA adduct-type mutagens. Additionally, the established cells can be monitored in real-time using live cell imaging to obtain detailed dynamics of MDC1 in response to mutagens. The simple handling of this assay is expected to enable its full automation, thus making it useful for high-throughput genotoxicity screening of chemicals and monitoring of environmental mutagens.
    Genes and Environment 01/2014; 36(1):17-28. DOI:10.3123/jemsge.2014.001
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    ABSTRACT: The E3 ubiquitin ligase RNF20 regulates chromatin structure through ubiquitination of histone H2B, so that early homologous recombination repair (HRR) proteins can access the DNA in eukaryotes during repair. However, it remains unresolved how RNF20 itself approaches the DNA in the presence of chromatin structure. Here, we identified the histone chaperone FACT as a key protein in the early step of HRR. Depletion of SUPT16H, a component of FACT, caused pronounced defects in accumulations of repair proteins, consequently decreased HRR activity. This led to enhanced sensitivity to ionizing radiation (IR) and mitomycin-C in a fashion similar to RNF20-deficient cells, indicating that SUPT16H is essential for RNF20-mediated pathway. Indeed, SUPT16H directly bound to RNF20 in vivo, and mutation at the RNF20's RING-finger domain abolished its interaction and accumulation, as well as RAD51 and BRCA1 at sites of DSBs, whereas the localization of SUPT16H remained intact. Interestingly, PAF1, implicated in transcription as a mediator of FACT and RNF20 association, was dispensable for DNA damage-induced interaction of RNF20 with SUPT16H. Furthermore, depletion of SUPT16H caused pronounced defects in RNF20-mediated H2B ubiquitination and thereby, impaired accumulation of the chromatin remodeling factor SNF2h. Consistent with this observation, the defective phenotypes of SUPT16H were effectively counteracted by enforced nucleosome relaxation. Taken together, present results indicate a primary role of FACT in RNF20 recruitment and the resulting chromatin remodeling for initiation of HRR.
    Journal of Cell Science 12/2013; 127(4). DOI:10.1242/jcs.135855 · 5.33 Impact Factor
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    ABSTRACT: Human histone H2A.B (formerly H2A.Bbd), a non-allelic H2A variant, exchanges rapidly as compared to canonical H2A, and preferentially associates with actively transcribed genes. We found that H2A.B transiently accumulated at DNA replication and repair foci in living cells. To explore the biochemical function of H2A.B, we performed nucleosome reconstitution analyses using various lengths of DNA. Two types of H2A.B nucleosomes, octasome and hexasome, were formed with 116, 124, or 130 base pairs (bp) of DNA, and only the octasome was formed with 136 or 146 bp DNA. In contrast, only hexasome formation was observed by canonical H2A with 116 or 124 bp DNA. A small-angle X-ray scattering analysis revealed that the H2A.B octasome is more extended, due to the flexible detachment of the DNA regions at the entry/exit sites from the histone surface. These results suggested that H2A.B rapidly and transiently forms nucleosomes with short DNA segments during chromatin reorganization.
    Scientific Reports 12/2013; 3:3510. DOI:10.1038/srep03510 · 5.58 Impact Factor
  • International Journal of Radiation OncologyBiologyPhysics 10/2013; 87(2):S22. DOI:10.1016/j.ijrobp.2013.06.062 · 4.18 Impact Factor
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    ABSTRACT: Genetic information encoded in chromosomal DNA is challenged by intrinsic and exogenous sources of DNA damage. DNA double-strand breaks (DSBs) are extremely dangerous DNA lesions. RAD51 plays a central role in homologous recombinational DSB repair, by facilitating the recombination of damaged DNA with intact DNA in eukaryotes. RAD51 accumulates at sites containing DNA damage to form nuclear foci. However, the mechanism of RAD51 accumulation at sites of DNA damage is still unclear. Posttranslational modifications of proteins, such as phosphorylation, acetylation and ubiquitination, play a role in the regulation of protein localization and dynamics. Recently, the covalent binding of small ubiquitin-like modifier (SUMO) proteins to target proteins, termed SUMOylation, at sites containing DNA damage has been determined to play a role in the regulation of the DNA damage response. Here we show that the SUMOylation E2, UBC9, and E3, PIAS1 and PIAS4 enzymes are required for RAD51 accretion at sites containing DNA damage in human cells. Moreover, we identified a SUMO-interacting motif (SIM) in RAD51, which is necessary for RAD51 accumulation at sites of DNA damage. These findings suggest that the SUMO-SIM system plays an important role in DNA repair, through the regulation of RAD51 dynamics.
    Journal of Cell Science 09/2013; 126(22). DOI:10.1242/jcs.133744 · 5.33 Impact Factor
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    ABSTRACT: Poly(ADP-ribose) polymerase (PARP) plays a critical role in responding to DNA damage, by activating DNA repair pathways responsible for cellular survival. PARP inhibition is used to treat certain solid cancers, such as breast and ovarian cancers. However, its effectiveness with other solid cancers, such as esophageal squamous cell carcinoma (ESCC), has not been clarified. We evaluated the effects of PARP inhibition on the survival of human esophageal cancer cells, with a special focus on the induction and repair of DNA double-strand breaks. The effects were monitored by colony formation assays and DNA damage responses, with immunofluorescence staining of γH2AX and RAD51. We found that PARP inhibition synergized with cisplatin, and the cells were highly sensitive, in a similar manner to the combination of cisplatin and 5-fluorouracil (5-FU). Comparable increases in RAD51 foci formation were observed after each combinational treatment with cisplatin and either 3-aminobenzamide (3-AB) or 5-FU in three human esophageal cancer cell lines, TE11, TE14 and TE15. In addition, decreasing the amount of RAD51 by RNA interference rendered the TE11 cells even more hypersensitive to these combinational treatments. Our findings suggested that the homologous recombinational repair pathway may be involved in the synergism between cisplatin and either 3-AB or 5-FU, and that 3-AB and 5-FU may similarly modify the cisplatin-induced DNA damage to types requiring the recruitment of RAD51 proteins for their repair. Understanding these mechanisms could be useful for improving the clinical outcome of ESCC patients, who suffer from aggressive diseases that presently lack effective treatment options. This article is protected by copyright. All rights reserved.
    Cancer Science 09/2013; DOI:10.1111/cas.12281 · 3.53 Impact Factor
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    ABSTRACT: When DNA replication is stalled at sites of DNA damage, a cascade of responses is activated in the cell to halt cell cycle progression and promote DNA repair. A pathway initiated by the kinase Ataxia teleangiectasia and Rad3 related (ATR) and its partner ATR interacting protein (ATRIP) plays an important role in this response. The Fanconi anemia (FA) pathway is also activated following genomic stress, and defects in this pathway cause a cancer-prone hematologic disorder in humans. Little is known about how these two pathways are coordinated. We report here that following cellular exposure to DNA cross-linking damage, the FA core complex enhances binding and localization of ATRIP within damaged chromatin. In cells lacking the core complex, ATR-mediated phosphorylation of two functional response targets, ATRIP and FANCI, is defective. We also provide evidence that the canonical ATR activation pathway involving RAD17 and TOPBP1 is largely dispensable for the FA pathway activation. Indeed DT40 mutant cells lacking both RAD17 and FANCD2 were synergistically more sensitive to cisplatin compared with either single mutant. Collectively, these data reveal new aspects of the interplay between regulation of ATR-ATRIP kinase and activation of the FA pathway.
    Nucleic Acids Research 05/2013; 41(14). DOI:10.1093/nar/gkt467 · 9.11 Impact Factor
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    ABSTRACT: 5-Fluorouracil (5-FU) is one of the most well established chemotherapeutic agents in the treatment of esophageal cancer. Ribonucleotide reductase M1 (RRM-1) is the rate‑limiting enzyme in de novo DNA synthesis, and has been considered to play an important role in the 5-FU metabolic pathway. However, the means by which RRM-1 participates in the anticancer effects of 5-FU and cisplatin (CDDP) have not been well studied. Here, we show that RRM-1 significantly contributes to the induction of DNA damage by 5-FU in esophageal cancer cell lines. An assay of γ-H2AX focus formation, a marker of DNA damage, after 5-FU treatment revealed good correlation with the levels of RRM-1 protein expression. Moreover, the increased sensitivity and RAD51 focus formation induced by the combination treatment of 5-FU and CDDP were significantly repressed by RRM-1 depletion. These results suggest that RRM-1 is involved not only in the induction of DNA damage by 5-FU but also in the synergistic cytotoxic effect in the combination therapy of 5-FU and CDDP.
    International Journal of Oncology 04/2013; 42(6). DOI:10.3892/ijo.2013.1899 · 3.03 Impact Factor
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    ABSTRACT: Although nuclear actin and Arps (actin-related proteins) are often identified as components of multi-protein chromatin-modifying enzyme complexes, such as chromatin remodeling and histone acetyltransferase (HAT) complexes, their molecular functions still remain largely elusive. Here, we investigated the role of human Arp4 (BAF53, also known as actin-like protein 6A) in Brg1-containing chromatin remodeling complexes. Depletion of Arp4 by RNA interference impaired the integrity of these complexes and accelerated the degradation of Brg1, indicating a crucial role in their maintenance, at least in certain human cell lines. We further found that Arp4 can form a heterocomplex with β-actin. Based on structural similarities between conventional actin and Arp4, and the assumption that actin-Arp4 binding might mimic actin-actin binding, we introduced a series of mutations in Arp4 that might be expected to impair its interaction with β-actin. Some of them indeed caused reduced binding to β-actin. Interestingly, such mutant Arp4 proteins also showed reduced incorporation into Brg1 complexes, and their interaction with Myc-associated complexes as well as Tip60 HAT complexes were also impaired. Based on these findings, we propose that β-actin-Arp4 complex formation might be a crucial feature in some chromatin-modifying enzyme complexes, such as the Brg1 complex.
    Journal of Cell Science 05/2012; 125(Pt 16):3870-82. DOI:10.1242/jcs.104349 · 5.33 Impact Factor
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    ABSTRACT: The analysis of dicentric chromosomes in human peripheral blood lymphocytes (PBLs) by Giemsa staining is the most established method for biological dosimetry. However, this method requires a well-trained person because of the difficulty in detecting aberrations rapidly and accurately. Here, we applied a fluorescence in situ hybridization (FISH) technique, using telomere and centromere peptide nucleic acid (PNA) probes, to solve the problem of biological dosimetry in radiation emergency medicine. A comparison by a well-trained observer found that FISH analysis of PBLs for the dose estimation was more accurate than the conventional Giemsa analysis, especially in samples irradiated at high doses. These results show that FISH analysis with centromeric/telomeric PNA probes could become the standard method for biological dosimetry in radiation emergency medicine.
    Radiation Research 04/2012; 177(5):533-8. DOI:10.2307/41545107 · 2.45 Impact Factor
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    ABSTRACT: Cellular senescence prevents the aberrant proliferation of damaged cells. The transcription factor Bach1 binds to p53 to repress cellular senescence, but it is still unclear how the Bach1-p53 interaction is regulated. We found that the Bach1-p53 interaction was inhibited by oncogenic Ras, bleomycin, and hydrogen peroxide. Proteomics analysis of Bach1 complex revealed its interaction with p19(ARF), a tumor suppressor that competitively inhibited the Bach1-p53 interaction when overexpressed within cells. Reduction of MDM2 expression in wild-type murine embryonic fibroblasts (MEFs) did not result in slower proliferation, showing that Bach1 plays a role in keeping the proliferation of MEFs independent of MDM2. Consistent with this interpretation, expression of p21 was highly induced in MEFs when both Bach1 and MDM2 were abrogated. The level of Bach1 protein was reduced on knockdown of p53. These results suggest that p53 activation involves its dissociation from Bach1, achieved in part by the competitive binding of p19(ARF) to Bach1. The p19(ARF)-Bach1 interaction constitutes a regulatory pathway of p53 in parallel with the p19(ARF)-MDM2 pathway.
    Cancer Science 02/2012; 103(5):897-903. DOI:10.1111/j.1349-7006.2012.02244.x · 3.53 Impact Factor
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    ABSTRACT: A deficiency in the SMN gene product causes the motor neuron degenerative disease spinal muscular atrophy. GEMIN2 was identified as an SMN-interacting protein, and the SMN-GEMIN2 complex constitutes part of the large SMN complex, which promotes the assembly of the spliceosomal small nuclear ribonucleoprotein (snRNP). In addition to its splicing function, we previously found that GEMIN2 alone stimulates RAD51-mediated recombination in vitro, and functions in DNA double-strand-break (DSB) repair through homologous recombination in vivo. However, the function of SMN in homologous recombination has not been reported. In the present study, we successfully purified the SMN-GEMIN2 complex as a fusion protein. The SMN-GEMIN2 fusion protein complemented the growth-defective phenotype of GEMIN2-knockout cells. The purified SMN-GEMIN2 fusion protein enhanced the RAD51-mediated homologous pairing much more efficiently than GEMIN2 alone. SMN-GEMIN2 possessed DNA-binding activity, which was not observed with the GEMIN2 protein, and significantly stimulated the secondary duplex DNA capture by the RAD51-single-stranded DNA complex during homologous pairing. These results provide the first evidence that the SMN-GEMIN2 complex plays a role in homologous recombination, in addition to spliceosomal snRNP assembly.
    Biochemistry 08/2011; 50(32):6797-805. DOI:10.1021/bi200828g · 3.19 Impact Factor

Publication Stats

2k Citations
261.36 Total Impact Points


  • 2010–2014
    • Kyoto University
      • Radiation Biology Center
      Kioto, Kyōto, Japan
  • 2012
    • Tohoku University
      • Department of Medical Biochemistry
  • 2003–2012
    • Hiroshima University
      • • Department of Cellular Biology
      • • Department of Experimental Oncology
      Hiroshima-shi, Hiroshima-ken, Japan
  • 2005
    • Dana-Farber Cancer Institute
      Boston, Massachusetts, United States
  • 2000
    • Harvard Medical School
      Boston, Massachusetts, United States