| 1. |
Takaaki Yasuhara, Reona Kato, Motohiro Yamauchi, Yuki Uchihara, Lee Zou, Kiyoshi Miyagawa, Atsushi Shibata, RAP80 suppresses the vulnerability of R-loops during DNA double-strand break repair, Cell Reports, 10.1016/j.celrep.2022.110335, 38, 5, 110335-110335, 2022.02. |
| 2. |
Moe Moe Han, Miyako Hirakawa, Motohiro Yamauchi, Naoki Matsuda, Roles of the SUMO-related enzymes, PIAS1, PIAS4, and RNF4, in DNA double-strand break repair by homologous recombination, Biochemical and Biophysical Research Communications, 591, 95-101, 2021.12. |
| 3. |
Palina Kot, Takaaki Yasuhara, Atsushi Shibata, Miyako Hirakawa, Yu Abe, Motohiro Yamauchi, Naoki Matsuda, Mechanism of chromosome rearrangement arising from single-strand breaks, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2021.08.001, 572, 191-196, 2021.10. |
| 4. |
Motohiro Yamauchi, Mechanisms Underlying the Suppression of Chromosome Rearrangements by Ataxia-Telangiectasia Mutated, Genes, 10.3390/genes12081232, 12, 8, 1232-1232, 2021.08, Chromosome rearrangements are structural variations in chromosomes, such as inversions and translocations. Chromosome rearrangements have been implicated in a variety of human diseases. Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by a broad range of clinical and cellular phenotypes. At the cellular level, one of the most prominent features of A-T cells is chromosome rearrangement, especially that in T lymphocytes. The gene that is defective in A-T is ataxia-telangiectasia mutated (ATM). The ATM protein is a serine/threonine kinase and plays a central role in the cellular response to DNA damage, particularly DNA double-strand breaks. In this review, the mechanisms by which ATM suppresses chromosome rearrangements are discussed.. |
| 5. |
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, High linear energy transfer carbon-ion irradiation upregulates PD-L1 expression more significantly than X-rays in human osteosarcoma U2OS cells., Journal of radiation research, 10.1093/jrr/rrab050, 2021.07, 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.. |
| 6. |
Yoshihiko Hagiwara, Takahiro Oike, Atsuko Niimi, Motohiro Yamauchi, Hiro Sato, Siripan Limsirichaikul, Kathryn D Held, Takashi Nakano, Atsushi Shibata, Clustered DNA double-strand break formation and the repair pathway following heavy-ion irradiation., Journal of radiation research, 10.1093/jrr/rry096, 60, 1, 69-79, 2019.01, 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 ( |
| 7. |
Yasuhara T, Kato R, Hagiwara Y, Shiotani B, Yamauchi M, Nakada S, Shibata A, Miyagawa K, Human Rad52 Promotes XPG-Mediated R-loop Processing to Initiate Transcription-Associated Homologous Recombination Repair, Cell, 175, 2, 558-570, 2018.10. |
| 8. |
Yoshihiko Hagiwara, Hiro Sato, Tiara Bunga Mayang Permata, Atsuko Niimi, Motohiro Yamauchi, Takahiro Oike, Takashi Nakano, Atsushi Shibata, Analysis of programmed death-ligand 1 expression in primary normal human dermal fibroblasts after DNA damage., Human immunology, 10.1016/j.humimm.2018.05.008, 79, 8, 627-631, 2018.08, 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.. |
| 9. |
Hagiwara Y, Niimi A, Isono M, Yamauchi M, Yasuhara T, Limsirichaikul S, Oike T, Sato H, Held KD, Nakano T, Shibata A, 3D-structured illumination microscopy reveals clustered DNA double-strand break formation in widespread γH2AX foci after high LET heavy-ion particle radiation, Oncotarget, 10.18632/oncotarget.22679, 8, 65, 109370-109381, 2017.12. |
| 10. |
Motohiro Yamauchi, Atsushi Shibata, Keiji Suzuki, Masatoshi Suzuki, Atsuko Niimi, Hisayoshi Kondo, Miwa Miura, Miyako Hirakawa, Keiko Tsujita, Shunichi Yamashita, Naoki Matsuda, Regulation of pairing between broken DNA-containing chromatin regions by Ku80, DNA-PKcs, ATM, and 53BP1, SCIENTIFIC REPORTS, 10.1038/srep41812, 7, 41812, 2017.02, 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.. |
| 11. |
Atsuko Niimi, Motohiro Yamauchi, Siripan Limsirichaikul, Ryota Sekine, Takahiro Oike, Hiro Sato, Keiji Suzuki, Kathryn D. Held, Takashi Nakano, Atsushi Shibata, Identification of DNA Double Strand Breaks at Chromosome Boundaries Along the Track of Particle Irradiation, GENES CHROMOSOMES & CANCER, 10.1002/gcc.22367, 55, 8, 650-660, 2016.08, 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.. |
| 12. |
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, Carbon-Ion Beam Irradiation Kills X-Ray-Resistant p53-Null Cancer Cells by Inducing Mitotic Catastrophe, PLoS One, 10.1371/journal.pone.0115121, 9, 12, e115121, 2014.12, 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.. |
| 13. |
Motohiro Yamauchi, Kensuke Otsuka, Hisayoshi Kondo, Nobuyuki Hamada, Masanori Tomita, Masayuki Takahashi, Satoshi Nakasono, Toshiyasu Iwasaki, Kazuo Yoshida, A novel in vitro survival assay of small intestinal stem cells after exposure to ionizing radiation, JOURNAL OF RADIATION RESEARCH, 10.1093/jrr/rrt123, 55, 2, 381-390, 2014.03, The microcolony assay developed by Withers and Elkind has been a gold standard to assess the surviving fraction of small intestinal stem cells after exposure to high (>= 8 Gy) doses of ionizing radiation (IR), but is not applicable in cases of exposure to lower doses. Here, we developed a novel in vitro assay that enables assessment of the surviving fraction of small intestinal stem cells after exposure to lower IR doses. The assay includes in vitro culture of small intestinal stem cells, which allows the stem cells to develop into epithelial organoids containing all four differentiated cell types of the small intestine. We used Lgr5-EGFP-IRES-CreERT2/ROSA26-tdTomato mice to identify Lgr5(+) stem cells and their progeny. Enzymatically dissociated single crypt cells from the duodenum and jejunum of mice were irradiated with 7.25, 29, 101, 304, 1000, 2000 and 4000 mGy of X-rays immediately after plating, and the number of organoids was counted on Day 12. Organoid-forming efficiency of irradiated cells relative to that of unirradiated controls was defined as the surviving fraction of stem cells. We observed a significant decrease in the surviving fraction of stem cells at >= 1000 mGy. Moreover, fluorescence-activated cell sorting analyses and passage of the organoids revealed that proliferation of stem cells surviving IR is significantly potentiated. Together, the present study demonstrates that the in vitro assay is useful for quantitatively assessing the surviving fraction of small intestinal stem cells after exposure to lower doses of IR as compared with previous examinations using the microcolony assay.. |
| 14. |
Masatoshi Suzuki, Motohiro Yamauchi, Yasuyoshi Oka, Keiji Suzuki, Shunichi Yamashita, Live-Cell Imaging Visualizes Frequent Mitotic Skipping During Senescence-Like Growth Arrest in Mammary Carcinoma Cells Exposed to Ionizing Radiation, INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS, 10.1016/j.ijrobp.2011.12.003, 83, 2, E241-E250, 2012.06, Purpose: Senescence-like growth arrest in human solid carcinomas is now recognized as the major outcome of radiotherapy. This study was designed to analyze cell cycle during the process of senescence-like growth arrest in mammary carcinoma cells exposed to X-rays.
Methods and Materials: Fluorescent ubiquitination-based cell cycle indicators were introduced into the human mammary carcinoma cell line MCF-7. Cell cycle was sequentially monitored by live-cell imaging for up to 5 days after exposure to 10 Gy of X-rays.
Results: Live-cell imaging revealed that cell cycle transition from G2 to G1 phase without mitosis, so-called mitotic skipping, was observed in 17.1% and 69.8% of G1- and G2-irradiated cells, respectively. Entry to G1 phase was confirmed by the nuclear accumulation of mKO(2)-hCdt1 as well as cyclin E, which was inversely correlated to the accumulation of G2-specific markers such as mAG-hGeminin and CENP-F. More than 90% of cells skipping mitosis were persistently arrested in G1 phase and showed positive staining for the senescent biochemical marker, which is senescence-associated beta-galactosidase, indicating induction of senescence-like growth arrest accompanied by mitotic skipping. While G2 irradiation with higher doses of X-rays induced mitotic skipping in approximately 80% of cells, transduction of short hairpin RNA (shRNA) for p53 significantly suppressed mitotic skipping, suggesting that ionizing radiation-induced mitotic skipping is associated with p53 function.
Conclusions: The present study found the pathway of senescence-like growth arrest in G1 phase without mitotic entry following G2-irradiation. (C) 2012 Elsevier Inc.. |
| 15. |
Motohiro Yamauchi, Keiji Suzuki, Yasuyoshi Oka, Masatoshi Suzuki, Hisayoshi Kondo, Shunichi Yamashita, Mode of ATM-dependent suppression of chromosome translocation, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2011.11.006, 416, 1-2, 111-118, 2011.12, It is well documented that deficiency in ataxia telangiectasia mutated (ATM) protein leads to elevated frequency of chromosome translocation, however, it remains poorly understood how ATM suppresses translocation frequency. In the present study, we addressed the mechanism of ATM-dependent suppression of translocation frequency. To know frequency of translocation events in a whole genome at once, we performed centromere/telomere FISH and scored dicentric chromosomes, because dicentric and translocation occur with equal frequency and by identical mechanism. By centromere/telomere FISH analysis, we confirmed that chemical inhibition or RNAi-mediated knockdown of ATM causes 2 to 2.5-fold increase in dicentric frequency at first mitosis after 2 Gy of gamma-irradiation in G0/G1. The FISH analysis revealed that ATM/p53-dependent G1 checkpoint suppresses dicentric frequency, since RNAi-mediated knockdown of p53 elevated dicentric frequency by 1.5-fold. We found ATM also suppresses dicentric occurrence independently of its checkpoint role, as ATM inhibitor showed additional effect on dicentric frequency in the context of p53 depletion and Chk1/2 inactivation. Epistasis analysis using chemical inhibitors revealed that ATM kinase functions in the same pathway that requires kinase activity of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to suppress dicentric frequency. From the results in the present study, we conclude that ATM minimizes translocation frequency through its commitment to G1 checkpoint and DNA double-strand break repair pathway that requires kinase activity of DNA-PKcs. (C) 2011 Elsevier Inc. All rights reserved.. |
| 16. |
Yasuyoshi Oka, Motohiro Yamauchi, Masatoshi Suzuki, Shunichi Yamashita, Keiji Suzuki, Persistence and Dynamics of DNA Damage Signal Amplification Determined by Microcolony Formation and Live-cell Imaging, JOURNAL OF RADIATION RESEARCH, 10.1269/jrr.10164, 52, 6, 766-774, 2011.11, Cell cycle checkpoints are essential cellular process protecting the integrity of the genome from DNA damaging agents. In the present study, we developed a microcolony assay, in which normal human diploid fibroblast-like cells exposed to ionizing radiation, were plated onto coverslips at very low density (3 cells/cm(2)). Cells were grown for up to 3 days, and phosphorylated ATM at Ser1981 and 53BP1 foci were analyzed as the markers for an amplified DNA damage signal. We observed a dose-dependent increase in the fraction of non-dividing cells, whose increase was compromised by knocking down p53 expression. While large persistent foci were predominantly formed in non-dividing cells, we observed some growing colonies that contained cells with large foci. As each microcolony was derived from a single cell, it appeared that some cells could proliferate with large foci. A live-imaging analysis using hTERT-immortalized normal human diploid cells transfected with the EGFP-tagged 53BP1 gene revealed that the formation of persistent large foci was highly dynamic. Delayed appearance and disappearance of large foci were frequently observed in exposed cells visualized 12-72 hours after X-irradiation. Thus, our results indicate that amplified DNA damage signal could be ignored, which may be explained in part by the dynamic nature of the amplification process.. |
| 17. |
Oka Y, Suzuki K, Yamauchi M, Mitsutake N, Yamashita S, Recruitment of the cohesin loading factor NIPBL to DNA double-strand breaks depends on MDC1, RNF168 and HP1γ in human cells, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2011.07.021, 411, 4, 762-767, 2011.08. |
| 18. |
K. Suzuki, M. Yamauchi, S. Yamashita, ATM-DEPENDENT CELLULAR RESPONSE TO DNA DOUBLE STRAND BREAKS PLAYS A PIVOTAL ROLE IN THE MAINTENANCE OF THE INTEGRITY OF THE GENOME, RADIATION PROTECTION DOSIMETRY, 10.1093/rpd/ncq533, 143, 2-4, 279-283, 2011.02, ATM-dependent cellular response to DNA double strand breaks plays a pivotal role in the maintenance of the integrity of the genome. Upon irradiation, activated ataxia-telangiectasia mutated (ATM) proteins phosphorylate various downstream mediators and effectors, such as histone H2AX, MDC1, 53BP1 and NBS1. These proteins create discrete foci within the nuclei, which are detectable under fluorescence microscopes. Interestingly, the size of the foci is also increasing as increasing the time after irradiation. Particularly, the residual foci form large foci, the sizes of which reach approximately 2 mm in diameter. We confirmed that such 'foci growth' is a mechanism, by which DNA damage signal is amplified. Especially, a proper DNA damage response of cells to lower doses of ionising radiation required amplification of the ATM-dependent damage signal by recruiting the DNA damage checkpoint factors to the site of chromatin.. |
| 19. |
Keiji Suzuki, Motohiro Yamauchi, Yasuyoshi Oka, Masatoshi Suzuki, Shunichi Yamashita, Creating localized DNA double-strand breaks with microirradiation, NATURE PROTOCOLS, 10.1038/nprot.2010.183, 6, 2, 134-139, 2011.02, We describe a protocol for creating localized DNANA double-strand breaks (DSBs) with minimal requirements that can be applied in cell biology and molecular biology. This protocol is based on the combination of 5-bromo-2'-deoxyuridine (BrdU) labeling and ultraviolet C (UVC) irradiation through porous membranes. Cells are labeled with 10 mu M BrdU for 48-72 h, washed with Ca(2+)- and Mg(2+)-free PBS(-), covered by polycarbonate membranes with micropores and exposed to UVC light. With this protocol, localized DSBs are created within subnuclear areas, irrespective of the cell cycle phase. Recruitment of proteins involved in DNANA repair, DNANA damage response, chromatin remodeling and histone modifications can be visualized without any specialized equipment. The quality is the same as that obtained by laser microirradiation or by any other focal irradiation. DSBs become visible within 30 min of UVC irradiation.. |
| 20. |
Aya Ishikawa, Motohiro Yamauchi, Keiji Suzuki, Shunichi Yamashita, Image-based quantitative determination of DNA damage signal reveals a threshold for G2 checkpoint activation in response to ionizing radiation, Genome Integrity, 10.1186/2041-9414-1-10, 1, 1, 10, 2010.08, Background: Proteins involved in the DNA damage response accumulate as microscopically-visible nuclear foci on the chromatin flanking DNA double-strand breaks (DSBs). As growth of ionizing radiation (IR)-induced foci amplifies the ATM-dependent DNA damage signal, the formation of discrete foci plays a crucial role in cell cycle checkpoint activation, especially in cells exposed to lower doses of IR. However, there is no quantitative parameter for the foci which considers both the number and their size. Therefore, we have developed a novel parameter for DNA damage signal based on the image analysis of the foci and quantified the amount of the signal sufficient for G2 arrest.Results: The parameter that we have developed here was designated as SOID. SOID is an abbreviation of Sum Of Integrated Density, which represents the sum of fluorescence of each focus within one nucleus. The SOID was calculated for individual nucleus as the sum of (area (total pixel numbers) of each focus) x (mean fluorescence intensity per pixel of each focus). Therefore, the SOID accounts for the number, size, and fluorescence density of IR-induced foci, and the parameter reflects the flux of DNA damage signal much more accurately than foci number. Using very low doses of X-rays, we performed a "two-way" comparison of SOID of Ser139-phosphorylated histone H2AX foci between G2-arrested cells and mitosis-progressing cells, and between mitosis-progressing cells in the presence or absence of ATM or Chk1/2 inhibitor, both of which abrogate IR-induced G2/M checkpoint. The analysis revealed that there was a threshold of DNA damage signal for G2 arrest, which was around 4000~5000 SOID. G2 cells with <
4000 SOID were neglected by G2/M checkpoint, and thus, the cells could progress to mitosis. Chromosome analysis revealed that the checkpoint-neglected and mitosis-progressing cells had approximately two chromatid breaks on average, indicating that 4000~5000 SOID was equivalent to a few DNA double strand breaks.Conclusions: We developed a novel parameter for quantitative analysis of DNA damage signal, and we determined the threshold of DNA damage signal for IR-induced G2 arrest, which was represented by 4000~5000 SOID. The present study emphasizes that not only the foci number but also the size of the foci must be taken into consideration for the proper quantification of DNA damage signal. © 2010 Ishikawa et al
licensee BioMed Central Ltd.. |
| 21. |
Keiji Suzuki, Motohiro Yamauchi, Yasuyoshi Oka, Masatoshi Suzuki, Shunichi Yamashita, A novel and simple micro-irradiation technique for creating localized DNA double-strand breaks, NUCLEIC ACIDS RESEARCH, 10.1093/nar/gkq226, 38, 12, e129, 2010.07, An ataxia-telangiectasia mutated (ATM)-dependent DNA damage signal is amplified through the interaction of various factors, which are recruited to the chromatin regions with DNA double-strand breaks. Spatial and temporal regulation of such factors is analysed by fluorescence microscopy in combination with laser micro-irradiation. Here we describe a novel and simple technique for micro-irradiation that does not require a laser source. Cells were labelled with BrdU for 48-72 h, covered with porous polycarbonate membranes, and exposed to UVC. All BrdU-labelled cells showed localized foci of phosphorylated ATM, phosphorylated histone H2AX, MDC1 and 53BP1 upon irradiation, showing that these foci were induced irrespective of the cell-cycle phase. They were also detectable in nucleotide excision repair-defective XPA cells labelled with BrdU, indicating that the foci did not reflect an excision repair-related process. Furthermore, an ATM-specific inhibitor significantly attenuated the foci formation, and disappearance of the foci was significantly abrogated in non-homologous end-joining-defective cells. Thus, it can be concluded that micro-irradiation generated DNA double-strand breaks in BrdU-sensitized cells. The present technique should accelerate research in the fields of DNA damage response, DNA repair and DNA recombination, as it provides more chances to perform micro-irradiation experiments without any specific equipment.. |
| 22. |
Keiji Suzuki, Maiko Takahashi, Yasuyoshi Oka, Motohiro Yamauchi, Masatoshi Suzuki, Shunichi Yamashita, Requirement of ATM-dependent pathway for the repair of a subset of DNA double strand breaks created by restriction endonucleases, Genome Integrity, 10.1186/2041-9414-1-4, 1, 1, 4, 2010.05, Background: DNA double strand breaks induced by DNA damaging agents, such ionizing radiation, are repaired by multiple DNA repair pathways including non-homologous end-joining (NHEJ) repair and homologous recombination (HR) repair. ATM-dependent DNA damage checkpoint regulates a part of DNA repair pathways, however, the exact role of ATM activity remains to be elucidated. In order to define the molecular structure of DNA double strand breaks requiring ATM activity we examined repair of DNA double strand breaks induced by different restriction endonucleases in normal human diploid cells treated with or without ATM-specific inhibitor.Results: Synchronized G1 cells were treated with various restriction endonucleases. DNA double strand breaks were detected by the foci of phosphorylated ATM at serine 1981 and 53BP1. DNA damage was detectable 2 hours after the treatment, and the number of foci decreased thereafter. Repair of the 3'-protruding ends created by Pst I and Sph I was efficient irrespective of ATM function, whereas the repair of a part of the blunt ends caused by Pvu II and Rsa I, and 5'-protruding ends created by Eco RI and Bam HI, respectively, were compromised by ATM inhibition.Conclusions: Our results indicate that ATM-dependent pathway plays a pivotal role in the repair of a subset of DNA double strand breaks with specific end structures. © 2010 Suzuki et al
licensee BioMed Central Ltd.. |
| 23. |
Hori M, Suzuki K, Udono MU, Yamauchi M, Mine M, Watanabe M, Kondo S, Hozumi Y, Establishment of ponasterone A-inducible the wild-type p53 protein-expressing clones from HSC-1 cells, cell growth suppression by p53 expression and the suppression mechanism, Archives of Dermatological Research, 301, 9, 631-646, 2009.10. |
| 24. |
Yasuhiko Kobayashi, Tomoo Funayama, Nobuyuki Hamada, Tetsuya Sakashita, Teruaki Konishi, Hitoshi Imaseki, Keisuke Yasuda, Masanori Hatashita, Keiichi Takagi, Satoshi Hatori, Keiji Suzuki, Motohiro Yamauchi, Shunichi Yamashita, Masanori Tomita, Munetoshi Maeda, Katsumi Kobayashi, Noriko Usami, Lijun Wu, Microbeam Irradiation Facilities for Radiobiology in Japan and China, JOURNAL OF RADIATION RESEARCH, 10.1269/jrr.09009S, 50, Suppl A, A29-A47, 2009.03, In order to study the radiobiological effects of low dose radiation, microbeam irradiation facilities have been developed in the world. This type of facilities now becomes an essential tool for studying bystander effects and relating signaling phenomena in cells or tissues. This review introduces you available microbeam facilities in Japan and in China, to promote radiobiology using microbeam probe and to encourage collaborative research between radiobiologists interested in using microbeam in Japan and in China.. |
| 25. |
Motohiro Yamauchi, Yasuyoshi Oka, Masashi Yamamoto, Koichi Niimura, Motoyuki Uchida, Seiji Kodama, Masami Watanabe, Ichiro Sekine, Shunichi Yamashita, Keiji Suzuki, Growth of persistent foci of DNA damage checkpoint factors is essential for amplification of G1 checkpoint signaling, DNA REPAIR, 10.1016/j.dnarep.2007.11.011, 7, 3, 405-417, 2008.03, Several DNA damage checkpoint factors form nuclear foci in response to ionizing radiation (IR). Although the number of the initial foci decreases concomitantly with DNA double-strand break repair, some fraction of foci persists. To date, the physiological role of the persistent foci has been poorly understood. Here we examined foci of Ser1981-phosphorylated ATM in normal human diploid cells exposed to 1 Gy of X-rays. While the initial foci size was approximately 0.6 mu m, the one or two of persistent focus (foci) grew, whose diameter reached 1.6 mu m or more in diameter at 24 h after IR. All of the grown persistent foci of phosphorylated ATM colocalized with the persistent foci of Ser139-phosphorylated histone H2AX, MDC1, 53BP1, and NBS1, which also grew similarly. When GO-synchronized normal human cells were released immediately after 1 Gy of X-rays and incubated for 24h, the grown large phosphorylated ATM foci (>= 1.6 mu m) were rarely (av. 0.9%) observed in S phase cells, while smaller foci (These results indicate that the growth of the persistent foci of the DNA damage checkpoint factors plays a pivotal role in G1 arrest, which amplifies G1 checkpoint signals sufficiently for phosphorylating p53 in cells with a limited number of remaining foci. (c) 2007 Elsevier B.V All rights reserved.. |
| 26. |
Motohiro Yamauchi, Keiji Suzuki, Seiji Kodama, Masami Watanabe, Growth of IR-induced foci and G1 checkpoint, International Congress Series, 10.1016/j.ics.2006.10.016, 1299, 235-238, 2007.02, It is well established that several checkpoint-, or repair factors, such as Ser1981-phosphorylated ATM protein, form discrete nuclear foci at the sites of DNA double-strand breaks (DSBs) caused by ionizing radiation (IR). The foci number decreases concurrently with DNA repair, but some fractions of the foci remain long after irradiation. While the mean size of the initial foci was approximately 0.6 μm in diameter 1 h after IR, the foci grew and the mean size reached ∼ 2.0 μm 24 h after irradiation. The percentage of cells with large foci, whose diameter is defined to be 1.6 μm or more, increased dose-dependently. All of the large foci of phosphorylated ataxia-telangiectasia mutated (ATM) colocalized with foci of Ser139-phosphorylated histone H2AX, mediator of DNA damage checkpoint (MDC)1, 53BP1 and Nijmegen breakage syndrome (NBS)1, which also grew similarly to phosphorylated ATM. When G0-synchronized cells were released soon after 1 Gy of X-rays, most of the cells with large phosphorylated ATM foci show no replication protein A (RPA) staining, which is the marker for the S phase cells, while RPA staining was frequently observed in cells with foci less than 1.6 μm in diameter. Furthermore, the percentage of cells with Ser15-phosphorylated p53 increased dependently on focus diameter of phosphorylated ATM, and more than 80% of the cells with large focus showed pan-nuclear staining of phosphorylated p53. These results indicate that the growth of IR-induced foci plays an essential role in amplifying DNA damage checkpoint signals in order to secure cells with a few residual DSBs or misrepaired DSBs against G1-S transition. © 2006 Elsevier B.V. All rights reserved.. |
| 27. |
Keiji Suzuki, Mio Morimoto, Motohiro Yamauchi, Hiromi Yoshida, Seiji Kodama, Kazuhiro Tsukamoto, Masami Watanabe, Non-specific detection of the centrosomes by antibodies recognizing phosphorylated ATM at serine 1981, CELL CYCLE, 5, 9, 1008-1009, 2006.05. |
| 28. |
K Suzuki, H Okada, M Yamauchi, Y Oka, S Kodama, M Watanabe, Qualitative and quantitative analysis of phosphorylated ATM foci induced by low-dose ionizing radiation, RADIATION RESEARCH, 165, 5, 499-504, 2006.05, We examined the formation of phosphorylated ataxia telangiectasia mutated (ATM) foci in exponentially growing normal human diploid cells exposed to low doses of X rays. Phosphorylated ATM foci were detected immediately after irradiation, and the number of foci decreased as the time after irradiation increased. The kinetics of phosphorylated ATM foci was comparable to that of phosphorylated histone H2AX. We found that there were fewer spontaneous phosphorylated ATM foci than that phosphorylated histone H2AX foci. Notably, significant numbers of phosphorylated histone H2AX foci, but not phosphorylated ATM foci, were detected in the S-phase cells. The induction of foci showed a linear dose-response relationship with doses ranging for 10 mGy to 1 Gy, and the average number of phosphorylated ATM foci per gray was approximately 50. The average size of the foci was comparable for the cells irradiated with 20 mGy and I Gy, and there was no significant difference in the kinetics of disappearance of foci, indicating that DNA double-strand breaks are similarly recognized by DNA damage checkpoints and are repaired irrespective of the dose. (c) 2006 by Radiation Research Society.. |
| 29. |
M Yamauchi, K Suzuki, S Kodama, M Watanabe, Abnormal stability of wild-type p53 protein in a human lung carcinoma cell line, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2004.11.174, 330, 2, 483-488, 2005.05, We report here that ectopically expressed wild-type p53 protein showed more than 6 times longer half-life than normal human fibroblasts in NCl-H1299, a widely used cell line derived from non-small cell lung carcinoma lacking the expression of p53 protein. We found no abnormality in the phosphorylation and ubiquitination of p53, and the expression levels of MDM2. Although proteasome activity measured in vitro was not significantly different between the tumor cell line and normal human fibroblasts, proteasome inhibitors, ALLN, MG115, and MG132, did not accumulate p53 protein in the tumor cell line, but did accumulate p53 in normal human cells. These results provide a novel mechanism, by which p53 is stabilized in tumor cells, and they suggest that a mediator should exist between ubiquitinated p53 and proteasome, which may be defective in H1299 cells. (c) 2004 Elsevier Inc. All rights reserved.. |
| 30. |
M Yamauchi, K Suzuki, S Kodama, M Watanabe, Stabilization of alanine substituted p53 protein at Ser15, Thr18, and Ser20 in response to ionizing radiation, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2004.08.175, 323, 3, 906-911, 2004.10, Phosphorylation of p53 at Ser15, Thr18, and Ser20 has been thought to be important for p53 stabilization in response to ionizing radiation. In the present study, we examined the X-ray-induced stabilization of Ala-substituted p53 protein at Ser15, Thr18, and Ser20, whose gene expression was controlled under an ecdyson-inducible promoter. We found that all single-, double-, or triple-Ala-substituted p53 at Ser15, Yhr18, and Ser20 were accumulated in the nucleus similarly to wild-type p53 after X-irradiation. These results indicate that the phosphorylation of p53 at Ser15, Thr18, and Ser20 is not necessarily needed for p53 stabilization in response to ionizing radiation. (C) 2004 Elsevier Inc. All rights reserved.. |
