Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Masatoshi Fujita Last modified date:2019.04.24

Professor / Department of Pharmaceutical Health Care and Sciences / Department of Pharmaceutical Health Care and Sciences / Faculty of Pharmaceutical Sciences

1. Issay Morii, Yukiko Iwabuchi, Sumiko Mori, Masaki Suekuni, Toyoaki Natsume, Kazumasa Yoshida, Nozomi Sugimoto, Masato T. Kanemaki, Masatoshi Fujita, Inhibiting the MCM8-9 complex selectively sensitizes cancer cells to cisplatin and olaparib, Cancer Science, 10.1111/cas.13941, 110, 3, 1044-1053, 2019.03, MCM8 and MCM9 are paralogues of the MCM2-7 eukaryotic DNA replication helicase proteins and play a crucial role in a homologous recombination-mediated repair process to resolve replication stress by fork stalling. Thus, deficiency of MCM8-9 sensitizes cells to replication stress caused, for example, by platinum compounds that induce interstrand cross-links. It is suggested that cancer cells undergo more replication stress than normal cells due to hyperstimulation of growth. Therefore, it is possible that inhibiting MCM8-9 selectively hypersensitizes cancer cells to platinum compounds and poly(ADP-ribose) polymerase inhibitors, both of which hamper replication fork progression. Here, we inhibited MCM8-9 in transformed and nontransformed cells and examined their sensitivity to cisplatin and olaparib. We found that knockout of MCM9 or knockdown of MCM8 selectively hypersensitized transformed cells to cisplatin and olaparib. In agreement with reported findings, RAS- and human papilloma virus type 16 E7-mediated transformation of human fibroblasts increased replication stress, as indicated by induction of multiple DNA damage responses (including formation of Rad51 foci). Such replication stress induced by oncogenes was further increased by knockdown of MCM8, providing a rationale for cancer-specific hypersensitization to cisplatin and olaparib. Finally, we showed that knocking out MCM9 increased the sensitivity of HCT116 xenograft tumors to cisplatin. Taken together, the data suggest that conceptual MCM8-9 inhibitors will be powerful cancer-specific chemosensitizers for platinum compounds and poly(ADP-ribose) polymerase inhibitors, thereby opening new avenues to the design of novel cancer chemotherapeutic strategies..
2. Shinya Watanabe, Hiroki Fujiyama, Takuya Takafuji, Kota Kayama, Masaki Matsumoto, Keiichi I. Nakayama, Kazumasa Yoshida, Nozomi Sugimoto, Masatoshi Fujita, GRWD1 regulates ribosomal protein L23 levels via the ubiquitin-proteasome system, Journal of Cell Science, 10.1242/jcs.213009, 131, 15, 2018.08, Glutamate-rich WD40 repeat-containing 1 (GRWD1) is a Cdt1- binding protein that promotes mini-chromosome maintenance (MCM) loading through its histone chaperone activity. GRWD1 acts as a tumor-promoting factor by downregulating p53 (also known as TP53) via the RPL11-MDM2-p53 axis. Here, we identified GRWD1- interacting proteins using a proteomics approach and showed that GRWD1 interacts with various proteins involved in transcription, translation, DNA replication and repair, chromatin organization, and ubiquitin-mediated proteolysis. We focused on the ribosomal protein ribosomal protein L23 (RPL23), which positively regulates nucleolar stress responses through MDM2 binding and inhibition, thereby functioning as a tumor suppressor. Overexpression of GRWD1 decreased RPL23 protein levels and stability; this effect was restored upon treatment with the proteasome inhibitor MG132. EDD (also known as UBR5), an E3 ubiquitin ligase that interacts with GRWD1, also downregulated RPL23, and the decrease was further enhanced by co-expression of GRWD1. Conversely, siRNA-mediated GRWD1 knockdown upregulated RPL23. Co-expression of GRWD1 and EDD promoted RPL23 ubiquitylation. These data suggest that GRWD1 acts together with EDD to negatively regulate RPL23 via the ubiquitinproteasome system. GRWD1 expression reversed the RPL23- mediated inhibition of anchorage-independent growth in cancer cells. Our data suggest that GRWD1-induced RPL23 proteolysis plays a role in downregulation of p53 and tumorigenesis..
3. Nozomi Sugimoto, Kazumitsu Maehara, Kazumasa Yoshida, Yasuyuki Ohkawa, Masatoshi Fujita, Genome-wide analysis of the spatiotemporal regulation of firing and dormant replication origins in human cells, Nucleic Acids Research, 10.1093/nar/gky476, 46, 13, 6683-6696, 2018.07, In metazoan cells, only a limited number of mini chromosome maintenance (MCM) complexes are fired during S phase, while the majority remain dormant. Several methods have been used to map replication origins, but such methods cannot identify dormant origins. Herein, we determined MCM7-binding sites in human cells using ChIP-Seq, classified them into firing and dormant origins using origin data and analysed their association with various chromatin signatures. Firing origins, but not dormant origins, were well correlated with open chromatin regions and were enriched upstream of transcription start sites (TSSs) of transcribed genes. Aggregation plots of MCM7 signals revealed minimal difference in the efficacy of MCM loading between firing and dormant origins. We also analysed common fragile sites (CFSs) and found a low density of origins at these sites. Nevertheless, firing origins were enriched upstream of the TSSs. Based on the results, we propose a model in which excessive MCMs are actively loaded in a genome-wide manner, irrespective of chromatin status, but only a fraction are passively fired in chromatin areas with an accessible open structure, such as regions upstream of TSSs of transcribed genes. This plasticity in the specification of replication origins may minimize collisions between replication and transcription..
4. Yukari Kondo, Shinichiro Higa, Takeshi Iwasaki, Tomoya Matsumoto, Kazumitsu Maehara, Akihito Harada, Yoshihiro Baba, Masatoshi Fujita, Yasuyuki Ohkawa, Sensitive detection of fluorescence in western blotting by merging images, PLoS One, 10.1371/journal.pone.0191532, 13, 1, 2018.01, The western blotting technique is widely used to analyze protein expression levels and protein molecular weight. The chemiluminescence method is mainly used for detection due to its high sensitivity and ease of manipulation, but it is unsuitable for detailed analyses because it cannot be used to detect multiple proteins simultaneously. Recently, more attention has been paid to the fluorescence detection method because it is more quantitative and is suitable for the detection of multiple proteins simultaneously. However, fluorescence detection can be limited by poor image resolution and low detection sensitivity. Here, we describe a method to detect fluorescence in western blots using fluorescence microscopy to obtain high-resolution images. In this method, filters and fluorescent dyes are optimized to enhance detection sensitivity to a level similar to that of the chemiluminescence method..
5. Yuki Yoshimatsu, Tomomi Nakahara, Katsuyuki Tanaka, Yuki Inagawa, Mako Narisawa-Saito, Takashi Yugawa, Shin-ichi Ohno, Masatoshi Fujita, Hitoshi Nakagama, Tohru Kiyono, Roles of the PDZ-binding motif of HPV 16 E6 protein in oncogenic transformation of human cervical keratinocytes, Cancer Science, doi:10.1111/cas.13264, 10.1111/cas.13264, 2017.05.
6. Mitsunori Higa, Seiichiro Kurashige, Daisuke Kohmon, Kouki Enokitani, Satoko Iwahori, Nozomi Sugimoto, Kazumasa Yoshida, Masatoshi Fujita, TRF2 recruits ORC through TRFH domain dimerization, BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH, 10.1016/j.bbamcr.2016.11.004, 1864, 1, 191-201, 2017.01.
7. Kota Kayama, Shinya Watanabe, Takuya Takafuji, Takahiro Tsuji, Kensuke Hironaka, Masaki Matsumoto, Keiichi Nakayama, Masato Enari, Takashi Kohno, Kouya Shiraishi, Tohru Kiyono, Kazumasa Yoshida, Nozomi Sugimoto, Masatoshi Fujita, GRWD1 negatively regulates p53 via the RPL11-MDM2 pathway and promotes tumorigenesis, EMBO REPORTS, 10.15252/embr.201642444, 18, 1, 123-137, 2017.01, リボソームタンパク質RPL11は、MDM2ユビキチンリガーゼに結合することによりその活性を阻害し、結果としてp53を誘導する。すなわちRPL11はがん抑制因子として機能する。我々は、GRWD1がRPL11と結合しその機能を抑制することにより、がん遺伝子として機能することを発見した。GRWD1のsiRNAによる抑制は、アクチノマイシンDによる核小体ストレスやブレオマイシンによるDSBによって誘導されるp53活性化を促進する。逆に、GRWD1の過剰発現はp53を不安定化し抑制する。また、GRWD1はがん抑制因子RPL11と直接結合することでRPL11によるMDM2抑制を阻害し、結果としてp53誘導に対して抑制的に働く。更に、 GRWD1をHPV16 E7(RBを不活化)、活性化Ras G12Vと共にヒト正常細胞に発現させると、細胞をtransformすることもわかった。 このGRWD1のtransform活性は、RPL11との結合に依存していた。GRWD1のtransforming活性と一致して、p53が野生型のある種のがん患者において、GRWD1の過剰発現は予後不良と強く相関していた。以上から、GRWD1は新たながん(促進)遺伝子として機能していることが明らかとなった。.
8. Masahiro Aizawa, Nozomi Sugimoto, Shinya Watanabe, Kazumasa Yoshida, Masatoshi Fujita, Nucleosome assembly and disassembly activity of GRWD1, a novel Cdt1-binding protein that promotes pre-replication complex formation, BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH, 10.1016/j.bbamcr.2016.08.008, 1863, 11, 2739-2748, 2016.11.
9. Yuki Kuniyoshi, Kazumitsu Maehara, Takashi Iwasaki, Masayasu Hayashi, Yuichiro Semba, Masatoshi Fujita, Yuko Sato, Hiroshi Kimura, Akihito Harada, Ohkawa, Y, Identification of Immunoglobulin Gene Sequences from a Small Read Number of mRNA-Seq Using Hybridomas, PLoS One, 10.1371/journal.pone.0165473, 11, 10, 2016.10.
10. Nozomi Sugimoto, Kazumitsu Maehara, Kazumasa Yoshida, Shuhei Yasukouchi, Satoko Osano, Shinya Watanabe, Masahiro Aizawa, Takashi Yugawa, Tohru Kiyono, Hitoshi Kurumizaka, Yasuyuki Ohkawa, Masatoshi Fujita, Cdt1-binding protein GRWD1 is a novel histone-binding protein that facilitates MCM loading through its influence on chromatin architecture, NUCLEIC ACIDS RESEARCH, 10.1093/nar/gkv509, 43, 12, 5898-5911, 2015.07, 真核細胞染色体DNAはクロマチン構造をとっており、これは一般的に複製や転写に対して阻害的であり、反応にあわせてクロマチン動態を制御する必要がある。一方、DNA複製の最初のステップは、複製開始複合体(pre-RC)形成(ORC/CDC6/Cdt1によるMCM helicaseの装着反応)である。このMCMの装着反応はクロマチン上で高効率で起きる必要があり、その効率の低下はゲノムの不安定性を惹起することが知られている。しかしながら、高効率なMCM装着に必要なクロマチン動態を制御するヒストンシャペロンは不明なままであった。本論文では、我々が新規Cdt1結合蛋白として同定したGRWD1が、CDC6/Cdt1依存性に複製開始点に結合し、MCM装着の促進に働いている事を明らかにした。さらに、GRWD1は、その酸性アミノ酸ドメインを介してヒストンと結合し、ヒストンシャペロン活性を示すことが分かった。以上から、GRWD1はCDC6/Cdt1依存性に複製開始点に結合し、ヌクレオソーム構造を緩めることにより効率よいMCMローディングを促進していると言うモデルが考えられる。.
11. Makoto Ohira, Yuka Iwasaki, Chika Tanaka, Michitaka Kuroki, Naoki Matsuo, Tatsuhiko Kitamura, Masaki Yukuhiro, Hiroyuki Morimoto, Nisha Pang, Tohru Kiyono, Masahide Amemiya, Kozo Tanaka, Kazumasa Yoshida, Nozomi Sugimoto, Takashi Ohshima, Masatoshi Fujita, A novel anti-microtubule agent with carbazole and benzohydrazide structures suppresses tumor cell growth in vivo, Biochimica et Biophysica Acta (BBA) - General Subjects, 10.1016/j.bbagen.2015.04.013, 1850, 1676-1684, 2015.05.
12. Satoko Iwahori, Daisuke Kohmon, Junya Kobayashi, Yuhei Tani, Takashi Yugawa, Kenshi Komatsu, Tohru Kiyono, Nozomi Sugimoto, Masatoshi Fujita, ATM regulates Cdt1 stability during the unperturbed S phase to prevent re- replication, CELL CYCLE, 10.4161/cc.27274, 13, 3, 471-481, 2014.02, ATMはDNA損傷応答、特にDNA二重鎖切断(DSB)応答において中心的な役割を持っているPI3キナーゼ関連キナーゼである。その変異は、毛細血管拡張性小脳失調症(AT)の原因となる。しかし、ATMはDSB誘導時のみならず、通常の細胞周期においても何らかの機能を持っている可能性が考えられていた。Cdt1はDNA複製開始に必須の因子であり、ORC/CDC6と協調してMCMヘリカーゼをクロマチンにloadすることにより、複製開始複合体を形成する。一方、Cdt1は細胞周期S期になると分解抑制され、この制御は複製を1回のみ正確に行うために極めて重要である。本研究で我々は、ATMがこのCdt1のS期の分解制御に関与していることを明らかにした。ATMは、恐らくAkt-SCF-Skp2の経路を介して、Cdt1の適切な分解を促進している。本研究はATMの新規機能を明らかにすると共に、ATの病態理解を進める上でも重要であると考えられる。.
13. Takashi Yugawa, Koichiro Nishino, Shin-ichi Ohno, Tomomi Nakahara, Masatoshi Fujita, Naoki Goshima, Akihiro Umezawa, Tohru Kiyono, Noncanonical NOTCH signaling limits self-renewal of human epithelial and induced pluripotent stem cells through ROCK activation, Molecular and Cellular Biology, 10.1128/MCB.00577-13, 33, 22, 4434-4447, 2013.11.
14. Naoki Nishimoto, Masanori Watanabe, Shinya Watanabe, Nozomi Sugimoto, Takashi Yugawa, Tsuyoshi Ikura, Osamu Koiwai, Tohru Kiyono, Masatoshi Fujita, Heterocomplex formation by Arp4 and beta-actin is involved in the integrity of the Brg1 chromatin remodeling complex, JOURNAL OF CELL SCIENCE, 10.1242/jcs.104349, 125, 16, 3870-3882, 2012.08, 筋肉の主要な蛋白質として知られているアクチンは、細胞運動や細胞増殖においても重要な役割を演じている。最近、このアクチンが細胞核内にも存在し、転写制御や細胞周期制御を通して細胞増殖制御に必須の役割を演じていることが示唆されつつあるが、その詳細は不明である。また、核内にはアクチンと相同性を持つアクチン関連蛋白質Arp4も存在するが、同様に機能や制御の詳細は不明なままであった。本研究では、アクチンとArp4が複合体(おそらく二量体)を形成することにより、幾つかの重要なクロマチンリモデリング因子複合体、HAT(ヒストンアセチル化酵素)複合体およびc-myc転写因子複合体(主要ながん遺伝子の一つ)の構成成分となり、それらの複合体の機能制御に関与していることを明らかにした。.
15. Sugimoto N, Yugawa T, Iizuka M, Kiyono T and Fujita M., Chromatin remodeler Sucrose Non-Fermenting 2 Homolog (SNF2H) is recruited onto DNA replication origins through interaction with Cdc10 protein-dependent transcript 1 (Cdt1) and promotes pre-replication complex formation., J. Biol. Chem. , 286: 39200-39210, 2011.11, DNA複製開始反応制御機構の解明は、細胞増殖制御の理解と言う点のみならず、新規抗がん剤開発を考える上でも重要である。Cdt1はORC/CDC6と協調して、複製ヘリカーゼMCM複合体を染色体へloadingする。Cdt1は強くこの反応を促進する。このCdt1によるMCM loading反応の促進において、ATP依存性クロマチンリモデリング因子SNF2Hが重要な役割を演じている事を明らかにし報告した。すなわち、(1)SNF2HはCdt1と物理的に相互作用し、これらに依存して複製開始点に結合する。(2)SNF2Hの複製開始点への結合はG1期で増加し、G2/M期で減少する。(3)SNF2HをsiRNAで抑制すると複製開始点でのMCM loadingが抑制される。以上から、SNF2HはCdt1との結合を介して複製開始点に集積し、ヌクレオソームの構造を制御することでMCMのクロマチン結合を制御しているとの仮説が考えられる。.
16. Zushi Y, Narisawa-Saito M, Noguchi K, Yoshimatsu Y, Yugawa T, Egawa N, Fujita M, Urade M and Kiyono T., An in vitro multistep carcinogenesis model for both HPV-positive and –negative human oral squamous cell carcinoma., Am. J. Cancer Res. , 1: 869-881 , 2011.08, a.
17. Yugawa T, Narisawa-Saito M, Yoshimatsu Y, Haga K, Ohno S, Egawa N, Fujita M and Kiyono, T., DeltaNp63alpha repression of the Notch1 gene supports the proliferative capacity of normal human keratinocytes and cervical cancer cells., Cancer Res. , 70: 4032-4044, 2010.05.
18. Yoshida K, Sugimoto N, Iwahori S, Yugawa T, Narisawa-Saito M, Kiyono T, Fujita M., CDC6 interaction with ATR regulates activation of a replication checkpoint in higher eukaryotic cells., J. Cell Sci., 123: 225-235, 2010.01.
19. Iizuka M, Takahashi Y, Mizzen C A, Cook R G, Fujita M, Allis C D, Frierson Jr H F, Fukusato T and Smith M M. , Histone acetyltransferase Hbo1: catalytic activity, cellular abundance, and links to primary cancers., Gene, 436: 108-114, 2009.05.
20. Sugimoto N, Yoshida K, Tatsumi Y, Yugawa T, Narisawa-Saito M, Waga S, Kiyono T, Fujita M., Redundant and differential regulation of multiple licensing factors ensures prevention of rereplication in normal human cells. , J. Cell Sci. , 122: 1184-1191, 2009.04.
21. Sasaki R, Narisawa-Saito M, Yugawa T, Fujita M, Tashiro H, Katabuchi H and Kiyono T., Oncogenic transformation of human ovarian surface epithelial cells with defined cellular oncogenes., Carcinogenesis, 30: 423-431, 2009.03.
22. Sugimoto N, Kitabayashi I, Osano S, Tatsumi Y, Yugawa T, Narisawa-Saito M, Matsukage A, Kiyono T, Fujita, M., Identification of novel human Cdt1-binding proteins by a proteomics approach: Proteolytic regulation by APC/C-Cdh1., Mol. Biol. Cell , 19: 1007-1021, 2008.03.
23. Tatsumi Y, Ezura K, Yoshida K, Yugawa T, Narisawa-Saito M, Kiyono T, Ohta S, Obuse C, Fujita M., Involvement of human ORC and TRF2 in pre-replication complex assembly at telomeres, Genes Cells , 13: 1045-1059, 2008.10.
24. Mizushina Y, Takeuchi T, Hada T, Maeda N, Sugawara F, Yoshida H, Fujita M., The inhibitory action of SQDG (sulfoquinovosyl diacylglycerol) from spinach on Cdt1-geminin interaction., Biochimie , 90: 947-956, 2008.06.
25. Narisawa-Saito M, Yoshimatsu Y, Ohno S, Yugawa T, Egawa N, Fujita M, Hirohashi S, and Kiyono T., An in vitro multistep carcinogenesis model for human cervical cancer., Cancer Res. , 68: 5699-5705, 2008.07.
26. Mizushina Y, Takeuchi T, Takagusagi Y, Yonezwa Y, Mizuno T, Yanagi K, Imamoto N, Sugawara F, Sakaguchi K, Yoshida H and Fujita M., Coenzyme Q10 as a potent compound that inhibits Cdt1-geminin interaction., Biochim. Biophy. Acta , 1780: 203-213, 2008.02.
27. Tatsumi Y, Sugimoto N, Yugawa T, Narisawa-Saito M, Kiyono T, Fujita M., Deregulation of Cdt1 induces chromosomal damage without rereplication and leads to chromosomal instability., J. Cell Sci. , 119: 3128-3140, 2006.08.
28. Yugawa, T, Handa, K, Narisawa-Saito, M, Ohno, S, Fujita, M, Kiyono, T, Regulation of Notch1 gene expression by p53 in epithelial cells, MOLECULAR AND CELLULAR BIOLOGY, 10.1128/MCB.02119-06, 27, 10, 3732-3742, 2007.05.
29. Nishitani H, Sugimoto N, Roukos V, Nakanishi Y, Saijo M, Obuse C, Tsurimoto T, Nakayama K-I, Nakayama K, Fujita M, Lygerous Z, Nishimoto T., Two E3 ubiquitin ligases, SCF-Skp2 and DDB1-Cul4, target human Cdt1 for proteolysis., EMBO J. , 25: 1126-1136, 2006.03.
30. Sugimoto N, Tatsumi Y, Tsurumi T, Matsukage A, Kiyono T, Nishitani H, Fujita M. , Cdt1 phosphorylation by cyclin A-dependent kinases negatively regulates its function without affecting geminin binding., J Biol. Chem. , 10.1074/jbc.M313175200, 279, 19, 19691-19697, 279: 19691-19697, 2004.05.