基本情報

所属

• 獣医学研究院　附属動物病院

職名

• 准教授

学位

• 博士（獣医学）（東京大学）

ホームページURL

https://kaken.nii.ac.jp/d/r/20732902.ja.html

科研費研究者番号

• 20732902

J-Global ID

• 201501084163706568

研究キーワード

• 白血病   腫瘍   獣医内科学   分子生物学   DNAメチル化   エピジェネティクス   

研究分野

• ライフサイエンス / 獣医学 / 臨床獣医学

職歴

• 2023年04月 - 現在 北海道大学 大学院獣医学研究院 附属動物病院 准教授
• 2019年05月 - 2023年03月 北海道大学 大学院獣医学研究院 附属動物病院 特任准教授
• 2015年08月 - 2019年05月 北海道大学 (連合)獣医学研究科 助教
• 2013年12月 - 2015年07月 北海道大学 (連合)獣医学研究科 特任助教
• 2012年11月 - 2013年11月 Temple University Fels Institute for Cancer Research and Molecular Biology Associate Scientist
• 2011年11月 - 2012年10月 Temple University Fels Institute for Cancer Research and Molecular Biology Postdoctoral Fellow
• 2008年04月 - 2011年10月 The University of Texas, MD Anderson Cancer Center Department of Leukemia Postdoctoral Fellow
• 2007年04月 - 2008年03月 国立感染症研究所 血液・安全性研究部 協力研究員

学歴

• 2004年04月 - 2008年03月   東京大学   農学生命科学研究科
• 1998年04月 - 2004年03月   日本大学   生物資源科学部   獣医学科

所属学協会

• 日本エピジェネティクス研究会   日本獣医学会   日本獣医輸血研究会   

研究活動情報

論文

• A de novo nonsense variant in the DMD gene associated with X-linked dystrophin-deficient muscular dystrophy in a cat.

  Nozomu Yokoyama, Yuki Matsumoto, Takahisa Yamaguchi, Kazuki Okada, Ryohei Kinoshita, Genya Shimbo, Hisashi Ukawa, Ryuga Ishii, Kensuke Nakamura, Jumpei Yamazaki, Mitsuyoshi Takiguchi

  Journal of veterinary internal medicine 2024年04月13日 

  BACKGROUND: X-linked dystrophin-deficient muscular dystrophy (MD) is a form of MD caused by variants in the DMD gene. It is a fatal disease characterized by progressive weakness and degeneration of skeletal muscles. HYPOTHESIS/OBJECTIVES: Identify deleterious genetic variants in DMD by whole-genome sequencing (WGS) using a next-generation sequencer. ANIMALS: One MD-affected cat, its parents, and 354 cats from a breeding colony. METHODS: We compared the WGS data of the affected cat with data available in the National Center for Biotechnology Information database and searched for candidate high-impact variants by in silico analyses. Next, we confirmed the candidate variants by Sanger sequencing using samples from the parents and cats from the breeding colony. We used 2 genome assemblies, the standard felCat9 (from an Abyssinian cat) and the novel AnAms1.0 (from an American Shorthair cat), to evaluate genome assembly differences. RESULTS: We found 2 novel high-impact variants: a 1-bp deletion in felCat9 and an identical nonsense variant in felCat9 and AnAms1.0. Whole genome and Sanger sequencing validation showed that the deletion in felCat9 was a false positive because of misassembly. Among the 357 cats, the nonsense variant was only found in the affected cat, which indicated it was a de novo variant. CONCLUSION AND CLINICAL IMPORTANCE: We identified a de novo variant in the affected cat and next-generation sequencing-based genotyping of the whole DMD gene was determined to be necessary for affected cats because the parents of the affected cat did not have the risk variant.
• Use of genome-wide DNA methylation analysis to identify prognostic CpG site markers associated with longer survival time in dogs with multicentric high-grade B-cell lymphoma.

  Yong Bin Teoh, Teita Ishizaki, Yumiko Kagawa, Shoko Yokoyama, Jaroslav Jelinek, Yuki Matsumoto, Hirotaka Tomiyasu, Hajime Tsujimoto, Mitsuyoshi Takiguchi, Jumpei Yamazaki

  Journal of veterinary internal medicine 38 1 316 - 325 2024年 

  BACKGROUND: DNA methylation analysis might identify prognostic CpG sites in CHOP-treated dogs with multicentric high-grade B-cell lymphoma (MHGL) with heterogenous prognosis. OBJECTIVE: To identify prognostic CpG sites of MHGL through genome-wide DNA methylation analysis with pyrosequencing validation. ANIMALS: Test group: 24 dogs. Validation group: 100 dogs. All client-owned dogs were diagnosed with MHGL and treated with CHOP chemotherapy. METHODS: Cohort study. DNA was extracted from lymph node samples obtained via FNA. Genome-wide DNA methylation analysis using Digital Restriction Enzyme Analysis of Methylation (DREAM) was performed on the test group to identify differentially methylated CpG sites (DMCs). Bisulfite pyrosequencing was used to measure methylation status of candidate DMCs in the validation group. Median survival times (MST) were analyzed using Kaplan-Meier (log-rank) product limit method. RESULTS: DREAM analyzed 101 576 CpG sites. Hierarchical clustering of 16 262 CpG sites in test group identified group with better prognosis (MST = 55-477 days vs 10-301 days, P = .007). Volcano plot identified 1371 differentially methylated CpG sites (DMCs). DMC near the genes of FAM213A (DMC-F) and PHLPP1 (DMC-P) were selected as candidates. Bisulfite-pyrosequencing performed on validation group showed group with methylation level of DMC-F < 40% had favorable prognosis (MST = 11-1072 days vs 8-1792 days, P = .01), whereas group with the methylation level combination of DMC-F < 40% plus DMC-P < 10% had excellent prognosis (MST = 18-1072 days vs 8-1792 days, P = .009). CONCLUSION AND CLINICAL IMPORTANCE: Methylation status of prognostic CpG sites delineate canine MGHL cases with longer MST, providing owners with information on expectations of potential improved treatment outcomes.
• Helicobacter pylori infection associated DNA methylation in primary gastric cancer significantly correlates with specific molecular and clinicopathological features.

  Sayumi Tahara, Tomomitsu Tahara, Jumpei Yamazaki, Takuya Shijimaya, Noriyuki Horiguchi, Kohei Funasaka, Toshiro Fukui, Yoshihito Nakagawa, Tomoyuki Shibata, Makoto Naganuma, Tetsuya Tsukamoto, Naoki Ohmiya

  Molecular carcinogenesis 2023年10月17日 

  Helicobacter pylori induces DNA methylation in gastric mucosa, which links to gastric cancer (GC) risk. In contrast, CpG island methylator phenotype (CIMP) is defined as high levels of cancer-specific methylation and provides distinct molecular and clinicopathological features of GC. The association between those two types of methylation in GC remains unclear. We examined DNA methylation of well-validated H. pylori infection associated genes in GC and its adjacent mucosa and investigated its association with CIMP, various molecular subtypes and clinical features. We studied 50 candidate loci in 24 gastric samples to identify H. pylori infection associated genes. Identified loci were further examined in 624 gastric tissue from 217 primary GC, 217 adjacent mucosa, and 190 mucosae from cancer-free subjects. We identified five genes (IGF2, SLC16A2, SOX11, P2RX7, and MYOD1) as hypermethylated in H. pylori infected gastric mucosa. In non-neoplastic mucosa, methylation of H. pylori infection associated genes was higher in patients with GC than those without. In primary GC tissues, higher methylation of H. pylori infection associated genes correlated with CIMP-positive and its related features, such as MLH1 methylated cases. On the other hand, GC with lower methylation of these genes presented aggressive clinicopathological features including undifferentiated histopathology, advanced stage at diagnosis. H. pylori infection associated DNA methylation is correlated with CIMP, specific molecular and clinicopathological features in GC, supporting its utility as promising biomarker in this tumor type.
• Microarchitectures of Barrett's esophagus associated with DNA methylation status.

  Takuya Shijimaya, Tomomitsu Tahara, Jumpei Yamazaki, Sanshiro Kobayashi, Anna Horitani, Yasushi Matsumoto, Naohiro Nakamura, Takashi Okazaki, Yu Takahashi, Takashi Tomiyama, Yusuke Honzawa, Norimasa Fukata, Toshiro Fukui, Makoto Naganuma

  Epigenomics 15 15 759 - 767 2023年08月 

  Aim: DNA methylation is involved in esophageal adenocarcinoma (EAC) and Barrett's esophagus (BE). Microarchitectures of on-neoplastic BE associated with DNA methylation status were examined using magnifying narrow-band imaging (NBI) endoscopy. Patients and methods: Using biopsies from non-neoplastic BE without cancer (n = 66; N group), with EAC (n = 27; ADJ group) and EAC tissue (n = 22; T group), methylation of N33, DPYS, SLC16A12, miR124a3 and miR34bc genes were quantified. Magnifying NBI features of non-neoplastic BE were classified according to their morphologies. Results: The ADJ and T groups presented higher DNA methylation compared with the N group. Magnifying NBI endoscopic features of non-neoplastic BE also correlated with DNA methylation as an independent factor. Conclusion: Microarchitectures of BE visualized by magnifying NBI endoscopy correlated with DNA methylation.
• Comprehensive DNA methylation profiling of Barrett's esophagus and esophageal adenocarcinoma in Japanese patients

  Takuya Shijimaya, Tomomitsu Tahara, Jumpei Yamazaki, Yasushi Matsumoto, Naohiro Nakamura, Yu Takahashi, Takashi Tomiyama, Toshiro Fukui, Tomoyuki Shibata, Makoto Naganuma

  Molecular Carcinogenesis 62 8 1191 - 1200 2023年08月 

  Molecular mechanisms of Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) remain unclear in Japanese patients. Japanese EACs frequently have underlying short length BE: short-segment BE (SSBE), for which, neoplastic potential remains unclear. We performed comprehensive methylation profiling of EAC and BE in Japanese patients, mostly comprised with SSBE. Using three different groups of biopsies obtained from non-neoplastic BE from patients without cancer (n = 50; N group), with EAC (n = 27; ADJ group) and EAC (n = 22; T group), methylation statuses of nine candidate genes (N33, DPYS, SLC16A12, CDH13, IGF2, MLF1, MYOD1, PRDM5, and P2RX7) were examined by the bisulfite pyrosequencing. Reduced representation bisulfite sequencing was performed to characterize the genome-wide methylation status in 32 samples (12 from N, 12 ADJ, and 8 from T groups). In the candidate approach, methylation levels of N33, DPYS, and SLC16A12 were higher in ADJ and T groups compared to that in N group. The ADJ group was an independent factor for higher DNA methylation in non-neoplastic BE. The genome-wide approach demonstrated an increase of hypermethylation from ADJ to T groups relative to N group near the transcription start sites. Among gene groups hypermethylated in ADJ and T groups (n = 645) and T group alone (n = 1438), 1/4 and 1/3 were overlapped with downregulated genes in the microarray data set, respectively. Accelerated DNA methylation is observed in EAC and underlying BE in Japanese patients, mostly comprised with SSBE, highlighting the potential impact of methylation in early carcinogenesis.
• Comprehensive DNA methylation profiling of Barrett's esophagus and esophageal adenocarcinoma in Japanese patients.

  Takuya Shijimaya, Tomomitsu Tahara, Jumpei Yamazaki, Yasushi Matsumoto, Naohiro Nakamura, Yu Takahashi, Takashi Tomiyama, Toshiro Fukui, Tomoyuki Shibata, Makoto Naganuma

  Molecular carcinogenesis 62 8 1191 - 1200 2023年08月 

  Molecular mechanisms of Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) remain unclear in Japanese patients. Japanese EACs frequently have underlying short length BE: short-segment BE (SSBE), for which, neoplastic potential remains unclear. We performed comprehensive methylation profiling of EAC and BE in Japanese patients, mostly comprised with SSBE. Using three different groups of biopsies obtained from non-neoplastic BE from patients without cancer (n = 50; N group), with EAC (n = 27; ADJ group) and EAC (n = 22; T group), methylation statuses of nine candidate genes (N33, DPYS, SLC16A12, CDH13, IGF2, MLF1, MYOD1, PRDM5, and P2RX7) were examined by the bisulfite pyrosequencing. Reduced representation bisulfite sequencing was performed to characterize the genome-wide methylation status in 32 samples (12 from N, 12 ADJ, and 8 from T groups). In the candidate approach, methylation levels of N33, DPYS, and SLC16A12 were higher in ADJ and T groups compared to that in N group. The ADJ group was an independent factor for higher DNA methylation in non-neoplastic BE. The genome-wide approach demonstrated an increase of hypermethylation from ADJ to T groups relative to N group near the transcription start sites. Among gene groups hypermethylated in ADJ and T groups (n = 645) and T group alone (n = 1438), 1/4 and 1/3 were overlapped with downregulated genes in the microarray data set, respectively. Accelerated DNA methylation is observed in EAC and underlying BE in Japanese patients, mostly comprised with SSBE, highlighting the potential impact of methylation in early carcinogenesis.
• Diverse genome-wide DNA methylation alterations in canine hepatocellular tumours.

  Yu Asari, Jumpei Yamazaki, Oo Thandar, Tamami Suzuki, Keisuke Aoshima, Kyosuke Takeuchi, Ryohei Kinoshita, Sangho Kim, Kenji Hosoya, Teita Ishizaki, Yumiko Kagawa, Jaroslav Jelinek, Shoko Yokoyama, Noboru Sasaki, Hiroshi Ohta, Kensuke Nakamura, Mitsuyoshi Takiguchi

  Veterinary medicine and science 2023年07月22日 

  BACKGROUND: Canine hepatocellular tumours (HCTs) are common primary liver tumours. However, the exact mechanisms of tumourigenesis remain unclear. Although some genetic mutations have been reported, DNA methylation alterations in canine HCT have not been well studied. OBJECTIVES: In this study, we aimed to analyse the DNA methylation status of canine HCT. METHODS: Tissues from 33 hepatocellular carcinomas, 3 hepatocellular adenomas, 1 nodular hyperplasia, 21 non-tumour livers from the patients and normal livers from 5 healthy dogs were used. We analysed the DNA methylation levels of 72,367 cytosine-guanine dinucleotides (CpG sites) in all 63 samples. RESULTS AND CONCLUSIONS: Although a large fraction of CpG sites that were highly methylated in the normal liver became hypomethylated in tumours from most patients, we also found some patients with less remarkable change or no change in DNA methylation. Hierarchical clustering analysis revealed that 32 of 37 tumour samples differed from normal livers, although the remaining 5 tumour livers fell into the same cluster as normal livers. In addition, the number of hypermethylated genes in tumour livers varied among tumour cases, suggesting various DNA methylation patterns in different tumour groups. However, patient and clinical parameters, such as age, were not associated with DNA methylation status. In conclusion, we found that HCTs undergo aberrant and diverse patterns of genome-wide DNA methylation compared with normal liver tissue, suggesting a complex epigenetic mechanism in canine HCT.
• Investigation of the therapeutic effects, predictors, and complications of long-term immunosuppressive therapy in dogs with precursor-targeted immune-mediated anemia.

  Mei Sugawara-Suda, Keitaro Morishita, Yuto Iwanaga, Jumpei Yamazaki, Yumiko Kagawa, Nozomu Yokoyama, Noboru Sasaki, Hiroshi Ohta, Kensuke Nakamura, Mitsuyoshi Takiguchi

  The Journal of veterinary medical science 85 7 695 - 701 2023年07月01日 

  Dogs with precursor-targeted immune-mediated anemia (PIMA) are commonly treated with immunosuppressive therapy, but information on predictors of treatment response and response time is limited. Therefore, we retrospectively investigated predictive factors that influenced the treatment response and duration required to observe a response in dogs with PIMA receiving continuous immunosuppressive therapies for more than 105 days. Of 50 client-owned dogs that developed PIMA, 27 were included in this study, of which 18 were responders and 9 were non-responders to immunosuppressive therapies. Sixteen of the 18 responders responded to treatment within 60 days and the remaining 2 responded at 93 and 126 days, respectively. We found that an erythroid-maturation ratio of <0.17 may be a useful predictor for treatment response. In addition, complications of immunosuppressive therapies were investigated further in 50 dogs. Pancreatitis (n=4) and pneumonia (3) occurred over the entire treatment period, and infections such as abscesses (3) tended to be more common in dogs on an extended period of immunosuppressive therapy. These findings may be helpful when planning for the initial treatment and may provide evidence for informed consent about potential comorbidities throughout the treatment course.
• Evaluation of responses to immunosuppressive therapy in dogs with suspected non‐regenerative immune‐mediated anaemia: 11 cases (2012‐2018)

  K. Morishita, M. Sugawara‐Suda, J. Yamazaki, N. Sasaki, K. Nakamura, H. Ohta, M. Takiguchi

  Journal of Small Animal Practice 2023年04月07日
• Transcriptome and proteome analysis of dogs with precursor targeted immune-mediated anemia treated with splenectomy.

  Mei Sugawara-Suda, Keitaro Morishita, Osamu Ichii, Takashi Namba, Keisuke Aoshima, Yumiko Kagawa, Sangho Kim, Kenji Hosoya, Nozomu Yokoyama, Noboru Sasaki, Kensuke Nakamura, Jumpei Yamazaki, Mitsuyoshi Takiguchi

  PloS one 18 5 e0285415  2023年 

  Precursor-targeted immune-mediated anemia (PIMA) in dogs is characterized by persistent non-regenerative anemia and ineffective erythropoiesis, and it is suspected to be an immune-mediated disease. Most affected dogs respond to immunosuppressive therapies; however, some are resistant. In this study, we carried out splenectomy as an alternative therapy for refractory PIMA in dogs, and analyzed gene expression levels in the spleen of dogs with or without PIMA and in serum before and after splenectomy. A total of 1,385 genes were found to express differentially in the spleens from dogs with PIMA compared with healthy dogs by transcriptome analysis, of which 707 genes were up-regulated, including S100A12, S100A8, and S100A9 that are linked directly to the innate immune system and have been characterized as endogenous damage-associated molecular patterns. Furthermore, immunohistochemistry confirmed that S100A8/A9 protein expression levels were significantly higher in dogs with PIMA compared with those in healthy dogs. A total of 22 proteins were found to express differentially between the serum samples collected before and after splenectomy by proteome analysis, of which 12 proteins were up-regulated in the samples before. The lectin pathway of complement activation was identified by pathway analysis in pre-splenectomy samples. We speculated that S100A8/9 expression may be increased in the spleen of dogs with PIMA, resulting in activation of the lectin pathway before splenectomy. These findings further our understanding of the pathology and mechanisms of splenectomy for PIMA.
• 犬尿路上皮癌におけるPDXモデルの樹立

  森下 大暉, 木之下 怜平, 青島 圭佑, 細谷 謙次, 山崎 淳平, 滝口 満喜

  日本獣医学会学術集会講演要旨集 165回 [B2P - 03] (公社)日本獣医学会 2022年09月
• イヌ悪性黒色腫細胞株における放射線照射で生じる上皮間葉転換とDNAメチル化の関与

  田邊 裕晶, 安井 博宣, 山崎 淳平, 木之下 怜平, 山下 晃矢, 加藤 千博, 稲波 修

  日本獣医学会学術集会講演要旨集 165回 [I1A - 14] (公社)日本獣医学会 2022年09月
• ヒストンアセチル化の改変はイヌ血管肉腫細胞に抗腫瘍効果をもたらす

  鈴木 玲海, 青島 圭佑, 山崎 淳平, 小林 篤史, 木村 享史

  日本獣医学会学術集会講演要旨集 165回 [B2P - 08] (公社)日本獣医学会 2022年09月
• Manipulating Histone Acetylation Leads to Antitumor Effects in Hemangiosarcoma Cells.

  Tamami Suzuki, Keisuke Aoshima, Jumpei Yamazaki, Atsushi Kobayashi, Takashi Kimura

  Veterinary and comparative oncology 2022年05月14日 

  Canine hemangiosarcoma (HSA) is a malignant tumor derived from endothelial cells. No effective treatment has yet been developed because of the lack of understanding of its pathogenesis. Histone acetylation, an epigenetic modification, is highly associated with cancer pathogenesis. Manipulating histone acetylation by histone deacetylase inhibitors (HDACi) or bromodomain and extraterminal domain inhibitors (BETi) is one approach to treat various cancers. However, the role of histone acetylation in HSA remains unknown. This study aimed to investigate how histone acetylation functions in HSA pathogenesis using two HDACi, suberanilohydroxamic acid (SAHA) and valproic acid (VPA), and one BETi, JQ1, in vitro and in vivo. Histone acetylation levels were high in cell lines and heterogeneous in clinical cases. SAHA and JQ1 induced apoptosis in HSA cell lines. HSA cell lines treated with SAHA and VPA upregulated inflammatory-related genes and attracted macrophage cell line RAW264 cells, which suggests that SAHA and VPA can affect immune responses. JQ1 stimulated autophagy and inhibited the cell cycle in HSA cell lines. Finally, we demonstrated that JQ1 suppressed HSA tumor cell proliferation in vivo although SAHA and VPA did not affect tumor growth. These results suggest that BETi can be alternative drugs for HSA treatment. Although further research is required, our study indicated that dysregulation of histone acetylation is likely to be involved in HSA malignancy. This article is protected by copyright. All rights reserved.
• Manipulating Histone Acetylation Leads to Adverse Effects in Hemangiosarcoma Cells

  Tamami Suzuki, Keisuke Aoshima*, Jumpei Yamazaki, Atsushi Kobayashi, Takashi Kimura, *Corresponding author

  bioRxiv 2021年12月11日 [査読無し]
  
• Obese status is associated with accelerated DNA methylation change in peripheral blood of senior dogs.

  Jumpei Yamazaki, Shinji Meagawa, Jaroslav Jelinek, Shoko Yokoyama, Noriyuki Nagata, Masashi Yuki, Mitsuyoshi Takiguchi

  Research in veterinary science 139 193 - 199 2021年10月 [査読有り]
   

  Obesity and its associated comorbidities constitute a major and growing health problem worldwide not only involved with people but also dogs and cats. Although few genetic mutations have been associated with obesity in dogs, molecular mechanism remains to be clearly understood. Given the fact that DNA methylation leads to gene expression variability and has plasticity affected by metabolic phenotypes such as obesity in human, the objective of this study is to identify obesity-associated differentially methylated cytosine-phosphate-guanine (CpG) dinucleotide sites in dogs. With genome-wide DNA methylation analysis using next-generation sequencing for blood samples from fourteen Miniature dachshunds with body condition score (BCS) 4-5 and BCS ≥6, over 100,000 sites could be analysed to identify genomic locations of differentially methylated CpG sites. As a result, 191 differentially methylated CpG sites (89 CpG sites were hypermethylated in BCS ≥6 and 102 were hypermethylated in BCS 4-5) were identified. These sites included promoter regions of Kisspeptin receptor (KISS1R) and Calcyphosine 2 (CAPS2) genes which were subsequently validated by bisulfite-pyrosequencing for another set of 157 dog blood samples. KISS1R methylation levels were found to be higher in BCS ≥6 group than BCS 4-5 in senior (>84 months) dogs. Especially male dogs but not female dogs as well as uncastrated male dogs but not castrated male dogs showed this trend. DNA methylation of KISS1R gene will be useful for understanding of comprehensive epigenetic change in obese dogs.
• Genome-wide DNA methylation profile in feline haematological tumours: A preliminary study.

  Jumpei Yamazaki, Jaroslav Jelinek, Shoko Yokoyama, Mitsuyoshi Takiguchi

  Research in veterinary science 140 221 - 228 2021年09月08日 [査読有り]
   

  Although DNA methylation has been analysed in few studies for a limited number of loci in cats with diseases, genome-wide profile of DNA methylation has never been addressed. The hypothesis for this study is that next-generation sequencing with sequential digestion of genomic DNA with SmaI and XmaI enzymes could provide highly quantitative information on methylation levels in cats. Using blood from four healthy control cats and two disease cats as well as three feline lymphoma/leukemia cell lines, approximately 74-94 thousand CpG sites across the cat genome could be analysed. CpG sites in CpG island (CGI) were broadly either methylated or unmethylated in normal blood, while CpG sites in non-CpG islands (NCGI) are largely methylated. Lymphoma cell lines showed thousands of CpG sites with gain of methylation at normally unmethylated CGI sites and loss of methylation at normally methylated NCGI sites. Hypermethylated CpG sites located at promoter regions included genes annotated with 'developmental process' and 'anatomical structure morphogenesis' such as HOXD10. This highly quantitative method would be suitable for studies of DNA methylation changes not only in cancer but also in other common diseases in cats.
• イヌの肝細胞腫瘍におけるDNAメチル化の網羅的解析

  淺利 友, 山崎 淳平, Oo Thander, 細谷 謙次, 金 尚昊, 木之下 怜平, 竹内 恭介, 賀川 由美子, 佐々木 東, 中村 健介, 滝口 満喜

  日本獣医学会学術集会講演要旨集 164回 [HSO - 30] (公社)日本獣医学会 2021年09月
• 非再生性免疫介在性貧血の犬における脾臓摘出術前後の血清中蛋白の網羅的解析

  菅原 芽伊, 山崎 淳平, 森下 啓太郎, 金 尚昊, 細谷 謙次, 佐々木 東, 中村 健介, 滝口 満喜

  日本獣医学会学術集会講演要旨集 164回 [HSO - 60] (公社)日本獣医学会 2021年09月
• DNA methylation landscape of 16 canine somatic tissues by methylation-sensitive restriction enzyme-based next generation sequencing.

  Jumpei Yamazaki, Yuki Matsumoto, Jaroslav Jelinek, Teita Ishizaki, Shingo Maeda, Kei Watanabe, Genki Ishihara, Junya Yamagishi, Mitsuyoshi Takiguchi

  Scientific reports 11 1 10005 - 10005 2021年05月11日 [査読有り]
   

  DNA methylation plays important functions in gene expression regulation that is involved in individual development and various diseases. DNA methylation has been well studied in human and model organisms, but only limited data exist in companion animals like dog. Using methylation-sensitive restriction enzyme-based next generation sequencing (Canine DREAM), we obtained canine DNA methylation maps of 16 somatic tissues from two dogs. In total, we evaluated 130,861 CpG sites. The majority of CpG sites were either highly methylated (> 70%, 52.5-64.6% of all CpG sites analyzed) or unmethylated (< 30%, 22.5-28.0% of all CpG sites analyzed) which are methylation patterns similar to other species. The overall methylation status of CpG sites across the 32 methylomes were remarkably similar. However, the tissue types were clearly defined by principle component analysis and hierarchical clustering analysis with DNA methylome. We found 6416 CpG sites located closely at promoter region of genes and inverse correlation between DNA methylation and gene expression of these genes. Our study provides basic dataset for DNA methylation profiles in dogs.
• Genome-wide DNA methylation analysis of dogs with high lead exposure living near a lead mining area in Kabwe, Zambia.

  Jumpei Yamazaki, Haruya Toyomaki, Shouta M M Nakayama, John Yabe, Kaampwe Muzandu, Jaroslav Jelinek, Shoko Yokoyama, Yoshinori Ikenaka, Mitsuyoshi Takiguchi, Mayumi Ishizuka

  Environmental pollution (Barking, Essex : 1987) 286 117229 - 117229 2021年05月03日 [査読有り]
   

  Lead (Pb) is a heavy metal that has been proven to be toxic to both animals and humans. Genom-wide DNA methylation in domestic dogs exposed to high levels of Pb in Kabwe, Zambia was analyzed in this study. Using next-generation sequencing on samples from 20 domestic dogs (mean blood Pb concentration: 43.6 μg/dL and 7.2 μg/dL in the high and low exposure groups), a digital restriction enzyme analysis of methylation was performed to identify the genomic locations of differentially methylated CpG sites. A validation study on an additional 20 dogs followed (blood Pb concentration: 4.9-29.7 μg/dL). The cluster analysis resolved two broad clusters indicating high and low Pb exposure. The study identified 827 (1.2%) CpG sites with differences in methylation (101 CpG sites were hypermethylated in the low exposure group and 726 were hypermethylated in the high exposure group). The sites corresponded to 26 genes with differentially methylated CpG sites at their promoter regions, including the NGF gene. The methylation of four CpG sites was validated using bisulfite pyrosequencing. The results indicate that aberrant hypermethylation is prevalent in dogs exposed to Pb. The altered DNA methylation of the genes identified in this study contributes to a greater understanding of the epigenetic changes caused by Pb exposure and highlights novel biomarker discoveries across species.
• Long interspersed nucleotide element-1 hypomethylation in canine malignant mucosal melanoma.

  Teita Ishizaki, Jumpei Yamazaki, Shinji Meagawa, Nozomu Yokoyama, Keisuke Aoshima, Mitsuyoshi Takiguchi, Takashi Kimura

  Veterinary and comparative oncology 18 4 854 - 860 2020年12月 [査読有り][通常論文]
   

  Canine malignant melanoma is a common cancer with a high mortality rate and is a clinically important disease. DNA methylation has been considered to be a potential tumorigenic mechanism through aberrant DNA methylation at promoter region which represses gene transcription. Global hypomethylation could also facilitate chromosome instability. There are few reports regarding DNA methylation in canine malignant melanoma; therefore, the purpose of this study was to examine DNA methylation status of long interspersed nucleotide element-1 (LINE-1) to be a surrogate marker of genome-wide methylation changes in this disease. We measured levels of DNA methylation of two adjacent cytosine-guanine sites on CpG island (CGI) at the putative promoter of canine LINE-1 sequence by bisulphite-pyrosequencing in 41 canine melanoma patient samples as well as six cell lines compared with normal mucosae. The survival rates were obtained from owners or medical records. We found DNA methylation levels of LINE-1 in normal mucosae were methylated. Interestingly, both melanoma cell lines and clinical melanoma samples showed remarkable hypomethylation. In addition, patients with lower LINE-1 methylation showed worse prognosis than those with higher LINE-1 methylation, though the difference did not reach statistical significance (P = .09). Here, we demonstrate that hypomethylation of LINE-1 is an epigenetically aberrant feature in canine melanoma with possible prognostic value.
• Genome-wide DNA methylation analysis identifies promoter hypermethylation in canine malignant melanoma.

  T Ishizaki, J Yamazaki, J Jelinek, K Aoshima, T Kimura

  Research in veterinary science 132 521 - 526 2020年10月 [査読有り]
   

  Canine malignant melanoma is a common cancer with a high mortality rate. Although previous studies have evaluated various aspects of this tumour, the exact mechanism of tumourigenesis remains unknown. Epigenetic mechanisms, such as DNA methylation, have recently gained attention as aetiological factors for neoplasia in humans. This study aimed to analyse genome-wide DNA methylation patterns in canine malignant melanoma based on next-generation sequencing data. A total of 76,213 CpG sites, including 29,482 sites in CpG islands (CGIs), were analysed using next-generation sequencing of methylation-specific signatures, obtained by sequential digestion with enzymes, to compare normal oral mucosal samples from four healthy dogs, four canine melanoma cell lines (3 oral cavity and 1 skin), and five clinical samples of oral canine melanoma. Malignant melanoma showed increased methylation at thousands of normally unmethylated CpG sites in CGIs and decreased methylation at normally methylated CpG sites in non-CGIs. Interestingly, the promoter regions of 81-393 genes were hypermethylated; 23 of these genes were present in all melanoma cell lines and melanoma clinical samples. Among these 23 genes, six genes with "sequence-specific DNA binding" annotation were significantly enriched, including three Homeobox genes-HMX2, TLX2, and HOXA9-that may be involved in the tumourigenesis of canine malignant melanoma. This study revealed widespread alterations in DNA methylation and a large number of hypermethylated genes in canine malignant melanoma.
• Cholesterol-binding protein TSPO2 coordinates maturation and proliferation of terminally differentiating erythroblasts.

  Benjaporn Kiatpakdee, Kota Sato, Yayoi Otsuka, Nobuto Arashiki, Yuqi Chen, Takuya Tsumita, Wataru Otsu, Akito Yamamoto, Reo Kawata, Jumpei Yamazaki, Yoshikazu Sugimoto, Kensuke Takada, Narla Mohandas, Mutsumi Inaba

  The Journal of biological chemistry 295 23 8048 - 8063 2020年06月05日 [査読有り]
   

  TSPO2 (translocator protein 2) is a transmembrane protein specifically expressed in late erythroblasts and has been postulated to mediate intracellular redistribution of cholesterol. We identified TSPO2 as the causative gene for the HK (high-K+) trait with immature red cell phenotypes in dogs and investigated the effects of the TSPO2 defects on erythropoiesis in HK dogs with the TSPO2 mutation and Tspo2 knockout (Tspo2-/-) mouse models. Bone marrow-derived erythroblasts from HK dogs showed increased binucleated and apoptotic cells at various stages of maturation and shed large nuclei with incomplete condensation when cultured in the presence of erythropoietin, indicating impaired maturation and cytokinesis. The canine TSPO2 induces cholesterol accumulation in the endoplasmic reticulum and could thereby regulate cholesterol availability by changing intracellular cholesterol distribution in erythroblasts. Tspo2-/- mice consistently showed impaired cytokinesis with increased binucleated erythroblasts, resulting in compensated anemia, and their red cell membranes had increased Na,K-ATPase, resembling the HK phenotype in dogs. Tspo2-deficient mouse embryonic stem cell-derived erythroid progenitor (MEDEP) cells exhibited similar morphological defects associated with a cell-cycle arrest at the G2/M phase, resulting in decreased cell proliferation and had a depletion in intracellular unesterified and esterified cholesterol. When the terminal maturation was induced, Tspo2-/- MEDEP cells showed delays in hemoglobinization; maturation-associated phenotypic changes in CD44, CD71, and TER119 expression; and cell-cycle progression. Taken together, these findings imply that TSPO2 is essential for coordination of maturation and proliferation of erythroblasts during normal erythropoiesis.
• 非再生性貧血のミニチュア・ダックスフンド11例に対する脾臓摘出術の治療成績

  菅原 芽伊, 森下 啓太郎, 今井 健友, 山崎 淳平, 佐々木 東, 大田 寛, 細谷 謙次, 滝口 満喜

  SA Medicine 22 3 74 - 75 (株)エデュワードプレス 2020年06月
• Genome-wide DNA methylation analysis in canine gastrointestinal lymphoma.

  Hiroshi Ohta, Jumpei Yamazaki, Jaroslav Jelinek, Teita Ishizaki, Yumiko Kagawa, Nozomu Yokoyama, Noriyuki Nagata, Noboru Sasaki, Mitsuyoshi Takiguchi

  The Journal of veterinary medical science 82 5 632 - 638 2020年05月20日 [査読有り][通常論文]
   

  DNA methylation is the covalent modification of methyl groups to DNA mostly at CpG dinucleotides and one of the most studied epigenetic mechanisms that leads to gene expression variability without affecting the DNA sequence. Genome-wide analysis of DNA methylation identified the signatures that could define subtypes of human lymphoma patients. The objective of this study was to conduct the genome-wide analysis of DNA methylation in dogs with gastrointestinal lymphoma (GIL). Genomic DNA was extracted from endoscopic biopsies from 10 dogs with GIL. We performed Digital Restriction Enzyme Assay of DNA Methylation (DREAM) for genome-wide DNA methylation analysis that could provide highly quantitative information on DNA methylation levels of CpG sites across the dog genome. We successfully obtained data of quantitative DNA methylation level for 148,601-162,364 CpG sites per GIL sample. Next, we analyzed 83,132 CpG sites to dissect the differences in DNA methylation between GIL and normal peripheral blood mononuclear cells (PBMCs). We found 383-3,054 CpG sites that were hypermethylated in GIL cases compared to PBMCs. Interestingly, 773 CpG sites including promoter regions of 61 genes were identified to be commonly hypermethylated in more than half of the cases, suggesting conserved DNA methylation patterns that are abnormal in GIL. This study revealed that there was a large number of hypermethylated sites that are common in most of canine GIL. These abnormal DNA methylation could be involved in tumorigenesis of the canine GIL.
• Dynamic changes in DNA methylation patterns in canine lymphoma cell lines demonstrated by genome-wide quantitative DNA methylation analysis

  J. Yamazaki, J. Jelinek, S. Hisamoto, A. Tsukamoto, M. Inaba

  Veterinary Journal 231 48 - 54 2018年01月01日 [査読有り][通常論文]
   

  DNA methylation is the conversion of cytosine to 5-methylcytosine, leading to changes in the interactions between DNA and proteins. Methylation of cytosine-guanine (CpG) islands (CGIs) is associated with gene expression silencing of the involved promoter. Although studies focussing on global changes or a few single loci in DNA methylation have been performed in dogs with certain diseases, genome-wide analysis of DNA methylation is required to prospectively identify specific regions with DNA methylation change. The hypothesis of this study was that next-generation sequencing with methylation-specific signatures created by sequential digestion of genomic DNA with SmaI and XmaI enzymes can provide quantitative information on methylation levels. Using blood from healthy dogs and cells obtained from canine lymphoma cell lines, approximately 100,000 CpG sites across the dog genome were analysed with the novel method established in this study. CpG sites in CGIs broadly were shown to be either methylated or unmethylated in normal blood, while CpG sites not within CpG islands (NCGIs) were largely methylated. Thousands of CpG sites in lymphoma cell lines were found to gain methylation at normally unmethylated CGI sites and lose methylation at normally methylated NCGI sites. These hypermethylated CpG sites are located at promoter regions of hundreds of genes, such as TWIST2 and TLX3. In addition, genes annotated with ‘Homeobox’ and ‘DNA-binding’ characteristics have hypermethylated CpG sites in their promoter CGIs. Genome-wide quantitative DNA methylation analysis is a sensitive method that is likely to be suitable for studies of DNA methylation changes in cancer, as well as other common diseases in dogs.
• A CpG island methylator phenotype in acute myeloid leukemia independent of IDH mutations and associated with a favorable outcome

  A. D. Kelly, H. Kroeger, J. Yamazaki, R. Taby, F. Neumann, S. Yu, J. T. Lee, B. Patel, Y. Li, R. He, S. Liang, Y. Lu, M. Cesaroni, S. A. Pierce, S. M. Kornblau, C. E. Bueso-Ramos, F. Ravandi, H. M. Kantarjian, J. Jelinek, J-P J. Issa

  LEUKEMIA 31 10 2011 - 2019 2017年10月 [査読有り][通常論文]
   

  Genetic changes are infrequent in acute myeloid leukemia (AML) compared with other malignancies and often involve epigenetic regulators, suggesting that an altered epigenome may underlie AML biology and outcomes. In 96 AML cases including 65 pilot samples selected for cured/not-cured, we found higher CpG island (CGI) promoter methylation in cured patients. Expanded genome-wide digital restriction enzyme analysis of methylation data revealed a CGI methylator phenotype independent of IDH1/2 mutations we term AML-CGI methylator phenotype (CIMP) (A-CIMP+). A-CIMP was associated with longer overall survival (OS) in this data set (median OS, years: A-CIMP+ = not reached, CIMP- = 1.17; P = 0.08). For validation we used 194 samples from The Cancer Genome Atlas interrogated with Illumina 450k methylation arrays where we confirmed longer OS in A-CIMP (median OS, years: A-CIMP+ = 2.34, A-CIMP- = 1.00; P = 0.01). Hypermethylation in A-CIMP+ favored CGIs (OR: CGI/non-CGI = 5.21), and while A-CIMP+ was enriched in CEBPA (P = 0.002) and WT1 mutations (P = 0.02), 70% of cases lacked either mutation. Hypermethylated genes in A-CIMP+ function in pluripotency maintenance, and a gene expression signature of A-CIMP was associated with outcomes in multiple data sets. We conclude that CIMP in AML cannot be explained solely by gene mutations (for example, IDH1/2, TET2), and that curability in A-CIMP+ AML should be validated prospectively.
• Magnifying narrow-band imaging of gastric mucosal morphology predicts the H. pylori-related epigenetic field defect

  Tomomitsu Tahara, Jumpei Yamazaki, Sayumi Tahara, Masaaki Okubo, Tomohiko Kawamura, Noriyuki Horiguchi, Takamitsu Ishizuka, Mitsuo Nagasaka, Yoshihito Nakagawa, Tomoyuki Shibata, Makoto Kuroda, Naoki Ohmiya

  SCIENTIFIC REPORTS 7 1 3090  2017年06月 [査読有り][通常論文]
   

  DNA methylation is associated with "field defect" in the gastric mucosa. To characterize "field defect" morphologically, we examined DNA methylation of non-neoplastic gastric mucosa in relation to their morphology seen by narrow-band imaging (NBI) with magnifying endoscopy. Magnifying NBI of non-neoplastic gastric body was classified as follows: normal-small and round pits with uniform subepithelial capillary networks; type 1-a little enlarged round pits with indistinct subepithelial capillary networks; type 2-remarkably enlarged pits with irregular vessels; and type 3-clearly demarcated oval or tubulovillous pits with bulky coiled or wavy vessels. Methylation of nine candidate genes (MYOD1, SLC16A12, GDNF, IGF2, MIR 124A1, CDH1, PRDM5, RORA and MLF1) were determined by bisulfite pyrosequencing. Infinium HumanMethylation450 array was used to characterize the methylation of >450,000 CpG sites. Mean Z score methylation of nine genes positively correlated with the changes of mucosal patterns from normal to types 1, 2, and 3 (P < 0.0001). Genome-wide analysis showed that development of mucosal patterns correlated with methylation accumulation especially at CpG islands. Genes with promoter CpG islands that were gradually methylated with the development of mucosal patterns significantly enriched the genes involved in zinc-related pathways. The results indicates that gastric mucosal morphology predicts a "field defect" in this tissue type. Accumulation of DNA methylation is associated with "field defect" in the non-neoplastic gastric mucosa. Endoscopic identification of "field defect" has important implications for preventing gastric cancer. Our results suggest that magnifying NBI of gastric mucosal morphology predicts a "field defect" in the gastric mucosa.
• Localization of Toll-like Receptor (TLR) 2 and TLR4 mRNA in the Colorectal Mucosa of Miniature Dachshunds with Inflammatory Colorectal Polyps

  N. Yokoyama, H. Ohta, J. Yamazaki, Y. Kagawa, O. Ichii, N. Khoirun, T. Morita, T. Osuga, S. Y. Lim, N. Sasaki, K. Morishita, K. Nakamura, M. Takiguchi

  JOURNAL OF COMPARATIVE PATHOLOGY 156 2-3 183 - 190 2017年02月 [査読有り][通常論文]
   

  Inflammatory colorectal polyps (ICRPs) are characterized by the formation of multiple or solitary polyps with marked neutrophil infiltration in the colorectal area, and are speculated to be a novel form of breed-specific canine idiopathic inflammatory bowel disease (IBD). In human IBD, toll-like receptor (TLR) 2 and TLR4 have been reported to be involved in the pathogenesis of the disease. The aim of this study was to evaluate the expression of TLR2 and TLR4 mRNA in the colorectal mucosa of dogs with ICRPs by in-situ hybridization using an RNAscope assay. Samples of inflamed colorectal mucosa (n = 5) and non-inflamed mucosa (n = 5) from miniature dachshunds (MDs) with ICRPs and colonic mucosa from healthy beagles (n = 5) were examined. TLR2 and TLR4 hybridization signals were localized to the colorectal epithelium, inflammatory cells and fibroblasts in the inflamed colorectal mucosa of affected dogs. The signals were significantly greater in inflamed colorectal epithelium compared with non-inflamed epithelium of MDs with ICRPs and healthy beagles (P <0.05). These results suggest that increased expression of TLR2 and TLR4 mRNA in the inflamed colorectal mucosa results from not only inflammatory cell infiltration, but also the upregulation of TLR2 and TLR4 mRNA in the colonic epithelium. (C) 2016 Elsevier Ltd. All rights reserved.
• Case report: Congenital methemoglobinemia in a cat with the reduced NADH-cytochrome b5 reductase 3 activity and missense mutations in CYB5R3

  Akiyoshi Tani, Jumpei Yamazaki, Kensuke Nakamura, Mitsuyoshi Takiguchi, Mutsumi Inaba

  Japanese Journal of Veterinary Research 65 4 201 - 206 2017年 [査読有り][通常論文]
   

  A one-year-old castrated male mixed-breed cat was referred for detailed examination of long-term pale mucous membrane without any clinical episodes that may cause cyanosis. While no causative abnormalities were detected in thoracic radiography and echocardiography, and arterial partial pressure of oxygen was within normal value, methemoglobin concentration of the cat was increased to 30% and cytochrome b5 reductase activity, which converts methemoglobin to hemoglobin, was reduced to 2.0 IU/gHb (13.4 ± 1.7 IU/gHb in control cats, n = 3), indicating congenital methemoglobinemia. Sequence analysis of CYB5R3, which codes cytochrome b5 reductase, showed two missense mutations found in the patient, one of which was predicted to affect protein function.
• Minimal residual disease in canine lymphoma: An objective marker to assess tumour cell burden in remission

  Masahiko Sato, Jumpei Yamazaki, Yuko Goto-Koshino, Asuka Setoguchi, Masashi Takahashi, Kenji Baba, Yasuhito Fujino, Koichi Ohno, Hajime Tsujimoto

  VETERINARY JOURNAL 215 38 - 42 2016年09月 [査読有り][通常論文]
   

  Lymphoma is the most common haematopoietic malignancy in dogs. Since a high proportion of dogs with lymphoma achieve remission soon after initiation of chemotherapy, an objective marker assessing treatment efficacy is required. Following clinical remission, the residual population of tumour cells can be referred to as the minimal residual disease (MRD). MRD traditionally has been detected by cytology and flow cytometry; however, if the burden of malignant cells is low, these methods might not be sufficiently sensitive to detect MRD. As an extension of the development of PCR for antigen receptor gene rearrangements (PARR) in dogs, there has been recent progress in the application of real-time quantitative PCR (RT-qPCR) to canine lymphoma. With the RT-qPCR system, a very high sensitivity (1 cell per 10,000 cells) has been achieved by preparing allele-specific oligonucleotide primers and probes designed from neoplastic clones of each dog. A series of MRD diagnostics studies employing the RT-qPCR system, has revealed its usefulness as a prognostic indicator, an objective marker of treatment efficacy and a predictor of relapse for dogs with lymphoma receiving chemotherapy. Introduction of the MRD monitoring system will provide an innovative scientific tool in the development of superior treatments and monitoring strategies for canine lymphoma. (C) 2016 Elsevier Ltd. All rights reserved.
• Mutation spectrum of TP53 gene predicts clinicopathological features and survival of gastric cancer

  Tomomitsu Tahara, Tomoyuki Shibata, Yasuyuki Okamoto, Jumpei Yamazaki, Tomohiko Kawamura, Noriyuki Horiguchi, Masaaki Okubo, Naoko Nakano, Takamitsu Ishizuka, Mitsuo Nagasaka, Yoshihito Nakagawa, Naoki Ohmiya

  ONCOTARGET 7 27 42252 - 42260 2016年07月 [査読有り][通常論文]
   

  Background and aim: TP53 gene is frequently mutated in gastric cancer (GC), but the relationship with clinicopathological features and prognosis is conflicting. Here, we screened TP53 mutation spectrum of 214 GC patients in relation to their clinicopathological features and prognosis. Results: TP53 nonsilent mutations were detected in 80 cases (37.4%), being frequently occurred as C:G to T:A single nucleotide transitions at 5'-CpG-3' sites. TP53 mutations occurred more frequently in differentiated histologic type than in undifferentiated type in the early stage (48.6% vs. 7%, P=0.0006), while the mutations correlated with venous invasion among advanced stage (47.7% vs. 20.7%, P=0.04). Subset of GC with TP53 hot spot mutations (R175, G245, R248, R273, R282) presented significantly worse overall survival and recurrence free survival compared to others (both P=0.001). Methods: Matched biopsies from GC and adjacent tissues from 214 patients were used for the experiment. All coding regions of TP53 gene (exon2 to exon11) were examined using Sanger sequencing. Conclusion: Our data suggest that GC with TP53 mutations seems to develop as differentiated histologic type and show aggressive biological behavior such as venous invasion. Moreover, our data emphasizes the importance of discriminating TP53 hot spot mutations (R175, G245, R248, R273, R282) to predict worse overall survival and recurrence free survival of GC patients.
• Hypomethylation of TET2 Target Genes Identifies a Curable Subset of Acute Myeloid Leukemia

  Jumpei Yamazaki, Rodolphe Taby, Jaroslav Jelinek, Noel J. M. Raynal, Matteo Cesaroni, Sherry A. Pierce, Steven M. Kornblau, Carlos E. Bueso-Ramos, Farhad Ravandi, Hagop M. Kantarjian, Jean-Pierre J. Issa

  JNCI-JOURNAL OF THE NATIONAL CANCER INSTITUTE 108 2 2016年02月 [査読有り][通常論文]
   

  Background: Acute myeloid leukemia (AML) is curable in a subset of cases. The DNA methylation regulator TET2 is frequently mutated in AML, and we hypothesized that studying TET2-specific differentially methylated CpGs (tet2-DMCs) improves AML classification. Methods: We used bisulfite pyrosequencing to analyze the methylation status of four tet2-DMCs (SP140, MCCC1, EHMT1, and MTSS1) in a test group of 94 consecutive patients and a validation group of 92 consecutive patients treated with cytarabine-based chemotherapy. Data were analyzed with hierarchical clustering, Cox proportional hazards regression, and Kaplan-Meier analyses. All statistical tests were two-sided. Results: In the test cohort, hierarchical clustering analysis identified low levels of tet2-DMC methylation in 31 of 94 (33%) cases, and these had markedly longer overall survival (median survival 72+ vs 14 months, P=.002). Similar results were seen in the validation cohort. tet2-DMC-low status was shown to be an independent predictor of overall survival (hazard ratio= 0.29, P=.0002). In The Cancer Genome Atlas (TCGA) dataset where DNA methylation was analyzed by a different platform, tet2-DMC-low methylation was also associated with improved outcome (median survival= 55 vs 15 months, P=.0003) and was a better predictor of survival than mutations in TET2, IDH1, or IDH2, individually or combined. Conclusions: Low levels of tet2-DMC methylation define a subgroup of AML that is highly curable and cannot be identified solely by genetic and cytogenetic analyses.
• Methylome sequencing for fibrolamellar hepatocellular carcinoma depicts distinctive features

  Gabriel G. Malouf, Tomomitsu Tahara, Valerie Paradis, Monique Fabre, Catherine Guettier, Jumpei Yamazaki, Hi Long, Yue Lu, Noel J-M Raynal, Jaroslav Jelinek, Roger Mouawad, David Khayat, Laurence Brugieres, Eric Raymond, Jean-Pierre J. Issa

  EPIGENETICS 10 9 872 - 881 2015年09月 [査読有り][通常論文]
   

  With the goal of studying epigenetic alterations in fibrolamellar hepatocellular carcinoma (FLC) and establish an associated DNA methylation signature, we analyzed LINE-1 methylation in a cohort of FLC and performed next-generation sequencing of DNA methylation in a training set of pure-FLCs and non-cirrhotic hepatocellular carcinomas (nc-HCC). DNA methylation was correlated with gene expression. Furthermore, we established and validated an epigenetic signature differentiating pure-FLC from other HCCs. LINE-1 methylation correlated with shorter recurrence-free survival and overall survival in resected pure-FLC patients. Unsupervised clustering using CG sites located in islands distinguished pure-FLC from nc-HCC. Major DNA methylation changes occurred outside promoters, mainly in gene bodies and intergenic regions located in the vicinity of liver developmental genes (i.e., SMARCA4 and RXRA). Partially methylated domains were more prone to DNA methylation changes. Furthermore, we identified several putative tumor suppressor genes (e.g., DLEU7) and oncogenes (e.g., DUSP4). While similar to 70% of identified gene promoters gaining methylation were marked by bivalent histone marks (H3K4me3/H3K27me3) in embryonic stem cells, similar to 70% of those losing methylation were marked by H3K4me3. Finally, we established a pure FLC DNA methylation signature and validated it in an independent dataset. Our analysis reveals a distinct epigenetic signature of pure FLC as compared to nc-HCC, with DNA methylation changes occurring in the vicinity of liver developmental genes. These data suggest new options for targeting FLC based on cancer epigenome aberrations.
• TET2 Mutations Affect Non-CpG Island DNA Methylation at Enhancers and Transcription Factor-Binding Sites in Chronic Myelomonocytic Leukemia

  Jumpei Yamazaki, Jaroslav Jelinek, Yue Lu, Matteo Cesaroni, Jozef Madzo, Frank Neumann, Rong He, Rodolphe Taby, Aparna Vasanthakumar, Trisha Macrae, Kelly R. Ostler, Hagop M. Kantarjian, Shoudan Liang, Marcos R. Estecio, Lucy A. Godley, Jean-Pierre J. Issa

  CANCER RESEARCH 75 14 2833 - 2843 2015年07月 [査読有り][通常論文]
   

  TET2 enzymatically converts 5-methylcytosine to 5-hydroxymethylcytosine as well as other covalently modified cytosines and its mutations are common in myeloid leukemia. However, the exact mechanism and the extent to which TET2 mutations affect DNA methylation remain in question. Here, we report on DNA methylomes in TET2 wild-type (TET2-WT) and mutant (TET2-MT) cases of chronic myelomonocytic leukemia (CMML). We analyzed 85,134 CpG sites [28,114 sites in CpG islands (CGI) and 57,020 in non-CpG islands (NCGI)]. TET2 mutations do not explain genome-wide differences in DNA methylation in CMML, and we found few and inconsistent differences at CGIs between TET2-WT and TET2-MT cases. In contrast, we identified 409 (0.71%) TET2-specific differentially methylated CpGs (tet2-DMCs) in NCGIs, 86% of which were hypermethylated in TET2-MT cases, suggesting a strikingly different biology of the effects of TET2 mutations at CGIs and NCGIs. DNA methylation of tet2-DMCs at promoters and nonpromoters repressed gene expression. Tet2-DMCs showed significant enrichment at hematopoietic-specific enhancers marked by H3K4me1 and at binding sites for the transcription factor p300. Tet2-DMCs showed significantly lower 5-hydroxymethylcytosine in TET2-MT cases. We conclude that leukemia-associated TET2 mutations affect DNA methylation at NCGI regions containing hematopoietic-specific enhancers and transcription factor-binding sites. (C)2015 AACR.
• Vital signs monitoring during injectable and inhalant anesthesia in mice

  Atsushi Tsukamoto, Kazuya Serizawa, Reiichiro Sato, Jumpei Yamazaki, Tomo Inomata

  EXPERIMENTAL ANIMALS 64 1 57 - 64 2015年01月 [査読有り][通常論文]
   

  Selecting the appropriate anesthetic protocol for the individual animal is an essential part of laboratory animal experimentation. The present study compared the characteristics of four anesthetic protocols in mice, focusing on the vital signs. Thirty-two male ddY mice were divided into four groups and administered anesthesia as follows: pentobarbital sodium monoanaesthesia; ketamine and xylazine combined (K/X); medetomidine, midazolam, and butorphanol combined (M/M/B); and isoflurane. In each group, rectal temperature, heart rate, respiratory rate, and O-2 saturation (SPO2) were measured, and the changes over time and instability in these signs were compared. The anesthetic depth was also evaluated in each mouse, and the percentage of mice achieving surgical anesthesia was calculated. K/X anesthesia caused remarkable bradycardia, while the respiratory rate and SPO2 were higher than with the others, suggesting a relatively strong cardiac influence and less respiratory depression. The M/M/B group showed a relatively lower heart rate and SPO2, but these abnormalities were rapidly reversed by atipamezole administration. The pentobarbital group showed a lower SPO2, and 62.5% of mice did not reach a surgical anesthetic depth. The isoflurane group showed a marked decrease in respiratory rate compared with the injectable anesthetic groups. However, it had the most stable SPO2 among the groups, suggesting a higher tidal volume. The isoflurane group also showed the highest heart rate during anesthesia. In conclusion, the present study showed the cardiorespiratory characteristics of various anesthetic protocols, providing basic information for selecting an appropriate anesthetic for individual animals during experimentation.
• Effect of midazolam and butorphanol premedication on inhalant isoflurane anesthesia in mice

  Atsushi Tsukamoto, Mami Iimuro, Reiichiro Sato, Jumpei Yamazaki, Tomo Inomata

  Experimental Animals 64 2 139 - 145 2014年12月16日 [査読有り][通常論文]
   

  Isoflurane is a representative inhalant anesthesia used in laboratory animals. However, isoflurane mediates respiratory depression and adverse clinical reactions during induction. In the present study, we established a novel balanced anesthesia method in mice that combined isoflurane anesthesia with midazolam and butorphanol (MB). Thirty-four male C57BL/6J mice received either isoflurane alone or isoflurane with an intra-peritoneal MB premedication (3 mg/kg midazolam and 4 mg/kg butorphanol). The minimum alveolar concentration (MAC) in each group was evaluated. Induction time and adverse clinical reactions were recorded in each group. Core body temperature, heart rate, respiratory rate, and oxygen saturation (SPO2) were assessed before and for 1 h after induction. Premedication with MB achieved a significant reduction in MAC compared with isoflurane monoanesthesia (isoflurane, 1.38 ± 0.15% isoflurane with MB, 0.78 ± 0.10% P< 0.05). Induction time was significantly shortened with MB premedication, and adverse reactions such as excitement or incontinence were observed less frequently. Furthermore, isoflurane anesthesia with MB premedication caused increase of respiratory rates compared to isoflurane monoanesthesia. No significant decrease of SPO2 was observed in MBI anesthesia, while a decrease in SPO2 was apparent with isoflurane monoanesthesia (baseline, 98.3% ± 1.1 10 min after induction, 91.8 ± 6.4% P< 0.05). In conclusion, premedication with MB was effective for the mitigation of respiratory depression induced by isoflurane in mice, with rapid induction and fewer adverse clinical reactions.
• 成人T細胞白血病マウスモデルを用いた癌幹細胞ニッチ形成における破骨細胞の機能解析と破骨細胞を標的とした治療法の開発

  水上 拓郎, 滝澤 和也, 栗林 和華子, 平松 竜司, 倉光 球, 山崎 淳平, Hall William, 長谷川 秀樹, 山口 一成, 浜口 功

  日本獣医学会学術集会講演要旨集 156回 194 - 194 (公社)日本獣医学会 2013年08月 [査読有り][通常論文]
  
• Establishment of Five Canine Lymphoma Cell Lines and Tumor Formation in a Xenotransplantation Model

  Saori Umeki, Yasuo Ema, Ryoichi Suzuki, Masahito Kubo, Toshiharu Hayashi, Yasuhiko Okamura, Jumpei Yamazaki, Hajime Tsujimoto, Kenji Tani, Hiroko Hiraoka, Masaru Okuda, Takuya Mizuno

  JOURNAL OF VETERINARY MEDICAL SCIENCE 75 4 467 - 474 2013年04月 [査読有り][通常論文]
   

  Five novel, canine lymphoma cell lines (Ema, CLC, CLK, Nody-1 and UL-1) were established from dogs suffering from lymphoma and characterized in vitro and in vivo. All cell lines, except CLC, were characterized with T-cell phenotypes, by flow cytometric analysis and polymerase chain reaction for antigen receptor rearrangement. Cell proliferation rates and transcriptional levels of MYC, PTEN, KIT and FLT3 varied between each cell line. Intraperitoneal xenotransplantation of Ema, CLC, Nody-1 and UL-1 lymphoma cell lines into NOD/SCID mice induced ascites, intraperitoneal tumors and severe infiltration of lymphoma cells into the pancreas and mesentery. Establishment of novel canine lymphoma cell lines with different characteristics is critical for elucidating the pathophysiology of canine lymphoma and improving current therapies.
• The prognostic significance of minimal residual disease in the early phases of chemotherapy in dogs with high-grade B-cell lymphoma

  Masahiko Sato, Jumpei Yamzaki, Yuko Goto-Koshino, Masashi Takahashi, Yasuhito Fujino, Koichi Ohno, Hajime Tsujimoto

  Veterinary Journal 195 3 319 - 324 2013年03月 [査読有り][通常論文]
   

  The prognostic significance of minimal residual disease (MRD) in the early phases of chemotherapy was examined in 36 dogs with multicentric high-grade B-cell lymphoma. Sequences of immunoglobulin heavy chain (IgH) gene fragments from lymphoma cells were amplified and used to design allele-specific primers and probes for real-time PCR. The dogs were treated with a 6-month modified version of the University of Wisconsin-Madison chemotherapy protocol (UW-25) and evaluated for the MRD level at weeks 6 and 11 of UW-25.Of the 31 dogs that remained on the protocol at week 11, 14 were found to be MRD negative (< 10tumour cells/105 peripheral blood mononuclear cells [PBMCs]), whereas the other 17 were MRD positive (≥10tumour cells/105 PBMCs). The progression-free survival of the dogs with MRD-negative status at week 11 (median, 337days) was significantly longer than that of the MRD-positive dogs at the same time point (median, 196days) (P=0.0002). These results indicate the clinical significance of MRD as a prognostic marker in the early phase of chemotherapy. © 2012 Elsevier Ltd.
• Epigenetic aspects of MDS and its molecular targeted therapy

  Jumpei Yamazaki, Jean-Pierre J. Issa

  INTERNATIONAL JOURNAL OF HEMATOLOGY 97 2 175 - 182 2013年02月 [査読有り][通常論文]
   

  The term "epigenetics" refers to clonally inherited stable variability in gene expression without underlying genetic changes. There are two well-known molecular mechanisms for epigenetic information: DNA methylation and histone modifications. Epigenetic changes have been recognized in the past decade as critical factors for physiological phenomena such as embryogenesis and the differentiation of normal cells. There is recent interest regarding the involvement of aberrant DNA methylation and histone modifications in mediating altered physiology in cancer. MDS is characterized by epigenetic changes, mutations in epigenetic regulators, and response to DNA methylation inhibitors, suggesting that epigenetic changes are unique features of MDS patients. In this article, recent progress in the understanding of MDS epigenetics and epigenetics-based therapies is reviewed.
• The epigenome of AML stem and progenitor cells.

  Yamazaki J, Estecio MR, Lu Y, Long H, Malouf GG, Graber D, Huo Y, Ramagli L, Liang S, Kornblau SM, Jelinek J, Issa JP

  Epigenetics : official journal of the DNA Methylation Society 8 1 92 - 104 2013年01月 [査読有り][通常論文]
  
• Architecture of epigenetic reprogramming following Twist1-mediated epithelial-mesenchymal transition

  Gabriel G. Malouf, Joseph H. Taube, Yue Lu, Tapasree Roysarkar, Shoghag Panjarian, Marcos R. H. Estecio, Jaroslav Jelinek, Jumpei Yamazaki, Noel J-M Raynal, Hai Long, Tomomitsu Tahara, Agata Tinnirello, Priyanka Ramachandran, Xiu-Ying Zhang, Shoudan Liang, Sendurai A. Mani, Jean-Pierre J. Issa

  GENOME BIOLOGY 14 12 R144  2013年 [査読有り][通常論文]
   

  Background: Epithelial-mesenchymal transition (EMT) is known to impart metastasis and stemness characteristics in breast cancer. To characterize the epigenetic reprogramming following Twist1-induced EMT, we characterized the epigenetic and transcriptome landscapes using whole-genome transcriptome analysis by RNA-seq, DNA methylation by digital restriction enzyme analysis of methylation (DREAM) and histone modifications by CHIP-seq of H3K4me3 and H3K27me3 in immortalized human mammary epithelial cells relative to cells induced to undergo EMT by Twist1. Results: EMT is accompanied by focal hypermethylation and widespread global DNA hypomethylation, predominantly within transcriptionally repressed gene bodies. At the chromatin level, the number of gene promoters marked by H3K4me3 increases by more than one fifth; H3K27me3 undergoes dynamic genomic redistribution characterized by loss at half of gene promoters and overall reduction of peak size by almost half. This is paralleled by increased phosphorylation of EZH2 at serine 21. Among genes with highly altered mRNA expression, 23.1% switch between H3K4me3 and H3K27me3 marks, and those point to the master EMT targets and regulators CDH1, PDGFRa and ESRP1. Strikingly, Twist1 increases the number of bivalent genes by more than two fold. Inhibition of the H3K27 methyltransferases EZH2 and EZH1, which form part of the Polycomb repressive complex 2 (PRC2), blocks EMT and stemness properties. Conclusions: Our findings demonstrate that the EMT program requires epigenetic remodeling by the Polycomb and Trithorax complexes leading to increased cellular plasticity. This suggests that inhibiting epigenetic remodeling and thus decrease plasticity will prevent EMT, and the associated breast cancer metastasis.
• マウスモデルを用いたATL癌幹細胞及びそのニッチの解析

  水上 拓郎, 滝澤 和也, 山崎 淳平, 倉光 球, 百瀬 暖佳, 益見 厚子, 長谷川 秀樹, 山口 一成, 浜口 功

  日本獣医学会学術集会講演要旨集 154回 172 - 172 (公社)日本獣医学会 2012年08月 [査読有り][通常論文]
  
• Effects of TET2 mutations on DNA methylation in chronic myelomonocytic leukemia

  Jumpei Yamazaki, Rodolphe Taby, Aparna Vasanthakumar, Trisha Macrae, Kelly R. Ostler, Lanlan Shen, Hagop M. Kantarjian, Marcos R. Estecio, Jaroslav Jelinek, Lucy A. Godley, Jean-Pierre J. Issa

  EPIGENETICS 7 2 201 - 207 2012年02月 [査読有り][通常論文]
   

  TET2 enzymatically converts 5-methyl-cytosine to 5-hydroxymethyl-cytosine, possibly leading to loss of DNA methylation. TET2 mutations are common in myeloid leukemia and were proposed to contribute to leukemogenesis through DNA methylation. To expand on this concept, we studied chronic myelomonocytic leukemia (CMML) samples. TET2 missense or nonsense mutations were detected in 53% (16/30) of patients. In contrast, only 1/30 patient had a mutation in IDH1 or IDH2, and none of them had a mutation in DNMT3A in the sites most frequently mutated in leukemia. Using bisulfite pyrosequencing, global methylation measured by the LINE-1 assay and DNA methylation levels of 10 promoter CpG islands frequently abnormal in myeloid leukemia were not different between TET2 mutants and wild-type CMML cases. This was also true for 9 out of 11 gene promoters reported by others as differentially methylated by TET2 mutations. We found that two non-CpG island promoters, AIM2 and SP140, were hypermethylated in patients with mutant TET2. These were the only two gene promoters (out of 14,475 genes) previously found to be hypermethylated in TET2 mutant cases. However, total 5-methyl-cytosine levels in TET2 mutant cases were significantly higher than TET2 wild-type cases (median = 14.0% and 9.8%, respectively) (p = 0.016). Thus, TET2 mutations affect global methylation in CMML but most of the changes are likely to be outside gene promoters.
• HTLV-1 Taxトランスジェニックマウスを用いたATL癌幹細胞及びそのニッチの同定

  水上 拓郎, 滝沢 和也, 山崎 淳平, 倉光 球, 百瀬 暖佳, 益見 厚子, 長谷川 秀樹, 山口 一成, 浜口 功

  日本獣医学会学術集会講演要旨集 152回 171 - 171 (公社)日本獣医学会 2011年08月 [査読有り][通常論文]
  
• Evaluation of Cytoreductive Efficacy of Vincristine, Cyclophosphamide, and Doxorubicin in Dogs with Lymphoma by Measuring the Number of Neoplastic Lymphoid Cells with Real-Time Polymerase Chain Reaction

  M. Sato, J. Yamazaki, Y. Goto-Koshino, M. Takahashi, Y. Fujino, K. Ohno, H. Tsujimoto

  JOURNAL OF VETERINARY INTERNAL MEDICINE 25 2 285 - 291 2011年03月 [査読有り][通常論文]
   

  Background The cytoreductive efficacy of the individual components of multidrug chemotherapy for canine lymphoma is difficult to evaluate after complete remission. Objectives To compare the cytoreductive efficacy of vincristine (VCR), cyclophosphamide (CPA), and doxorubicin (DXR) in dogs that received a 6-month modified version of the University of Wisconsin-Madison chemotherapy protocol (UW-25). Animals Twenty-nine dogs with high-grade B-cell multicentric lymphoma. Methods Rearranged immunoglobulin heavy chain gene fragments from lymphoma cells were amplified by polymerase chain reaction (PCR) and sequenced to prepare clone-specific primers and probes for real-time PCR. The number of lymphoma cells in peripheral blood was measured from diagnosis to week 11 of UW-25. Results The number of lymphoma cells after the 1st administration of VCR, CPA, and DXR in weeks 1-4 was decreased in 29/29 (100%), 15/29 (51.7%), and 26/27 (96.3%) dogs, respectively. The cytoreductive efficacy of CPA was less than that of VCR and DXR. VCR, CPA, and DXR administered in weeks 6-9 were effective in 5/26 (19.2%), 5/20 (25.0%), and 14/19 (73.7%) dogs, respectively, indicating the sustained cytoreductive efficacy of DXR. CPA nonresponders were heavier and exhibited a shorter 1st remission than CPA responders. Conclusion and Clinical Importance When using UW-25 for treatment of canine lymphoma, CPA was found to have less cytoreductive efficacy than VCR and DXR. Real-time PCR-based quantification of tumor cells is an objective marker of the efficacy of chemotherapeutic agents.
• Increase in Minimal Residual Disease in Peripheral Blood before Clinical Relapse in Dogs with Lymphoma that Achieved Complete Remission after Chemotherapy

  M. Sato, J. Yamazaki, Y. Goto-Koshino, M. Takahashi, Y. Fujino, K. Ohno, H. Tsujimoto

  JOURNAL OF VETERINARY INTERNAL MEDICINE 25 2 292 - 296 2011年03月 [査読有り][通常論文]
   

  Background We developed previously a minimal residual disease (MRD) monitoring system in dogs with lymphoma by exploring a highly sensitive real-time PCR system. Objectives To identify the change in MRD before clinical relapse in dogs with lymphoma that achieved complete remission after chemotherapy. Animals Twenty dogs with multicentric high-grade B-cell lymphoma. Methods MRD levels in peripheral blood mononuclear cells (PBMCs) were measured by real-time PCR amplifying the rearranged immunoglobulin heavy chain gene. MRD measurement and clinical assessment were performed every 2-4 weeks for 28-601 days after completion of chemotherapy. An increase in MRD was defined as an increase by more than 0.5, calculated by log(10)[copy number of MRD per 105 PBMCs], based on the uncertainty level observed in a canine lymphoma cell line. Results During the follow-up period, 15 dogs relapsed in 28-320 days (median, 120 days) after completion of chemotherapy. An increase in MRD was detected 2 weeks or more before relapse in 14 of the 15 dogs, but an increase in MRD before relapse could not be detected in the remaining 1 dog. The time from increased MRD to clinical relapse was 0-63 days (median, 42 days). In contrast, no increase in MRD was detected in 5 dogs that did not experience clinical relapse. Conclusion and Clinical Importance An increase in MRD can be detected before clinical relapse in dogs with lymphoma. Application of early reinduction therapy based on an increase in MRD before clinical relapse may improve treatment outcome in canine lymphoma.
• Monitoring of Minimal Residual Disease (MRD) after Multidrug Chemotherapy and Its Correlation to Outcome in Dogs with Lymphoma: A Proof-of-Concept Pilot Study

  J. Yamazaki, M. Takahashi, A. Setoguchi, Y. Fujino, K. Ohno, H. Tsujimoto

  JOURNAL OF VETERINARY INTERNAL MEDICINE 24 4 897 - 903 2010年07月 [査読有り][通常論文]
   

  Background Tumor cell burden in dogs with lymphoma cannot be assessed accurately by diagnostic evaluation during clinical complete remission (CR). Recent advances in polymerase chain reaction (PCR)-based methods enabled us to quantify minimal residual disease (MRD) in canine lymphoma. Hypothesis/Objectives To quantify MRD in dogs with lymphoma treated with multidrug chemotherapy and to correlate it with remission duration after chemotherapy. Animals Seventeen dogs with lymphoma that achieved CR by multidrug chemotherapy. Methods Rearranged immunoglobulin heavy chain or T-cell receptor gamma chain gene fragments from lymphoma cells were PCR amplified and sequenced to prepare clone-specific primers and probes for real-time PCR to quantify MRD. MRD in the peripheral blood was monitored during and at the end of a 25-week multidrug chemotherapy protocol. Correlation between MRD at the end of chemotherapy and remission duration after chemotherapy was analyzed. Results MRD gradually decreased after initiation of multidrug chemotherapy, reached a nadir as low as < 0.019-1.0 cells/mu L at weeks 4-17, and remained low or slightly increased until week 25. MRD at the end of chemotherapy was negatively correlated with remission duration from the end of chemotherapy to relapse. Conclusion and Clinical Importance MRD could be an objective marker to indicate tumor cell burden in dogs with lymphoma even in clinical CR. MRD at the end of chemotherapy could be a prognostic factor to predict remission duration after chemotherapy.
• Identification of cancer stem cells in a Tax-transgenic (Tax-Tg) mouse model of adult T-cell leukemia/lymphoma

  Jumpei Yamazaki, Takuo Mizukami, Kazuya Takizawa, Madoka Kuramitsu, Haruka Momose, Atsuko Masumi, Yasushi Ami, Hideki Hasegawa, William W. Hall, Hajime Tsujimoto, Isao Hamaguchi, Kazunari Yamaguchi

  BLOOD 114 13 2709 - 2720 2009年09月 [査読有り][通常論文]
   

  Adult T-cell leukemia/lymphoma (ATL) is a malignant lymphoproliferative disorder caused by HTLV-I infection. In ATL, chemotherapeutic responses are generally poor, which has suggested the existence of chemotherapy-resistant cancer stem cells (CSCs). To identify CSC candidates in ATL, we have focused on a Tax transgenic mouse (Tax-Tg) model, which reproduces ATL-like disease both in Tax-Tg animals and also after transfer of Tax-Tg splenic lymphomatous cells (SLCs) to nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Using a limiting dilution transplantation, it was estimated that one CSC existed per 10(4) SLCs (0.01%). In agreement with this, we have successfully identified candidate CSCs in a side population (0.06%), which overlapped with a minor population of CD38(-)/CD71(-)/CD117(+) cells (0.03%). Whereas lymphoma did not develop after transplantation of 10(2) SLCs, 10(2) CSCs could consistently regenerate the original lymphoma. In addition, lymphoma and CSCs could also be demonstrated in the bone marrow and CD117(+) CSCs were observed in both osteoblastic and vascular niches. In the CSCs, Tax, Notch1, and Bmi1 expression was down-regulated, suggesting that the CSCs were derived from Pro-T cells or early hematopoietic progenitor cells. Taken together, our data demonstrate that CSCs certainly exist and have the potential to regenerate lymphoma in our mouse model. (Blood. 2009; 114: 2709-2720)
• Quantitative assessment of minimal residual disease (MRD) in canine lymphoma by using real-time polymerase chain reaction

  Jumpei Yamazaki, Kenji Baba, Yuko Goto-Koshino, Asuka Setoguchi-Mukai, Yasuhito Fujino, Koichi Ohno, Hajime Tsujimoto

  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 126 3-4 321 - 331 2008年12月 [査読有り][通常論文]
   

  Lymphoma is the most common hematopoietic malignancy in dogs. Although a large proportion of dogs with lymphoma can achieve clinical remission by initial chemotherapy, most dogs die as a consequence of tumor relapse. We established a quantitative detection system for minimal residual disease (MRD) in canine lymphoma by using real-time polymerase chain reaction (PCR). A canine T-cell lymphoma-derived cell line, namely, UL-1, was used to examine the specificity and sensitivity of the MRD detecting system. Allele-specific oligonucleotide primers and probes were designed based on the sequence of T-cell receptor gamma chain (TCR gamma) gene fragment of UL-1 cells in conjunction with its downstream sequence, which were obtained from the dog genome database. The real-time PCR system for plasmid DNA containing the TCR gamma gene derived from UL-1 cells and the genomic DNA of UL-1 cells revealed that the system was accurate for 10-100,000 copies per reaction and its sensitivity was 1 cell per 10,000 cells. In order to monitor the kinetics of tumor cell number in canine lymphoma, we quantified the level of MRD in the peripheral blood of 7 dogs with lymphoma under chemotherapy. Since the lymphoma cells from the 7 patients were shown to be B-cell origin from the finding of clonal rearrangement of immunoglobulin heavy chain (IgH) gene, allele-specific oligonucleotide primers and probes were prepared based on the sequence of rearranged IgH gene in each case. The number of peripheral blood tumor cells measured by the real-time PCR was comparable to that estimated by conventional hematological examination in 2 cases of stage V lymphoma. MRD in the peripheral blood was detectable in all 7 cases, even in the complete remission (CR) phase. In the 7 lymphoma dogs, changes in the MRD levels of peripheral blood generally paralleled with the changes in the Volumes of lymph nodes. Molecular CR, in which the MRD level was below the detection limit, was not observed in any of these 7 patients under chemotherapy. The MRD level detected by the real-time PCR method described here would be useful for investigating the kinetics Of tumor cell growth and its regression in canine lymphoma patients. (C) 2008 Elsevier B.V. All rights reserved.
• Canine CD20 gene

  R Kano, C Inoiue, H Okano, J Yamazaki, T Takahashi, T Watari, M Tokuriki, A Hasegawa

  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 108 3-4 265 - 268 2005年12月 [査読有り][通常論文]
   

  The human CD20 antigen, a 35 kDa cell surface nonglycosylated hydrophobic phoshpoprotein is expressed consistently on almost all human B-cells, and its monoclonal antibody is used for the therapy on human B-cell lymphoma. In the present study, canine CD20 gene was cloned and sequenced, and the expression of CD20 mRNA was investigated in canine peripheral blood mononuclear cells (PBMCs), and lymph nodes from healthy dogs, and canine lymphoma cells. Using canine cDNA as a template, full-length of canine CD20 gene was sequenced by 5'-RACE and 3'-RACE methods. The full-length of the cDNA sequence of canine CD20 was 1239 bp encoding 297 amino acids. The amino acid sequences of canine CD20 showed 73 and 68% sequence similarities with those of human and mouse, respectively. Canine CD20 was predicted to contain domains of amino acid sequences consisting of two extracellular domains (EM), four transmembrane domains (TM), and three intracellular domains (IC) as in human CD20. Canine CD20 mRNA was detected in PBMCs and lymph node from healthy dogs, and B-cells of canine lymphoma, but not in T-cell lymphoma cells and non-T and non-B-cell lymphoma cells by RT-PCR analysis. From these results, canine CD20 might be targeted for monoclonal antibody therapy against B-cell lymphoma of dogs. (c) 2005 Elsevier B.V. All rights reserved.
• Effect of antineoplastic drugs on the expression of Bcl-2 and Bcl-xL genes in the feline T-cell leukemia cell line

  J Sano, S Nagafuchi, J Yamazaki, K Oguma, R Kano, A Hasegawa

  RESEARCH IN VETERINARY SCIENCE 79 3 197 - 201 2005年12月 [査読有り][通常論文]
   

  The Bcl-2 gene is the first member of a rapidly expanding family of genes that regulate apoptosis. Bcl-2 has been shown to repress cell death triggered by a diverse array of stimuli including chemotherapy and gamma-irradiation. Chemotherapy of feline lymphoma is generally carried out with antineoplastic drugs, which are reported to induce apoptosis in tumor cells. However, the precise apoptotic signals, induced by chemotherapeutic drugs against feline tumors have not been fully characterized. Therefore, we have evaluated the expression of Bcl-2 and Bcl-xL in FT-1 upon in vitro treatment with these drugs. In the present study, full length of feline Bcl-xL gene was sequenced, and the expressions of Bcl-2 and Bcl-xL mRNAs in feline lymphoma cell line (FT-1) cultured with doxorubicin, prednisolone or vincristine were investigated. Feline Bcl-xL clone was 1163 base pairs in length and encoded 233 amino acids. The predicted amino acid sequence was 99.1%, 98.7%, 96.1%, 97.4%, 97.0% and 97.9% homologous to predicted Bcl-xL of dog, human, mouse, pig, rat and sheep, respectively. The levels of Bcl-2 transcripts at 24 h incubation in FT-1 stimulated with doxorubicin (0.3 mu g/ml), prednisolone (0.2 mu g/ml) and vincristine (5 ng/ml) were increased to about 41.0-, 62.0- and 11.1-fold to those in non-stimulated FT-1, respectively. Oil the other hand, the level of Bcl-xL transcripts at 24 h incubation in FT-1 stimulated by doxorubicin and prednisolone were significantly increased about 4.2- and 5.8-folds to the controls and inducible level of Bcl-xL by vincristine was decreased about 0.35-folds. (c) 2005 Elsevier Ltd. All rights reserved.
• Acute monocytic leukaemia in a cat

  N Nagashima, R Kano, A Hirai, J Yamazaki, C Inoue, M Hisasue, PF Moore, A Hasegawa

  VETERINARY RECORD 157 12 347 - 349 2005年09月 [査読有り][通常論文]
   

  A three-year-old cat with lymphadenopathy, non-regenerative anaemia and marked leucocytosis (171(.)3 x 10(9) white blood cells/l) was diagnosed with monocytic leukaemia and treated with a combination of anticancer drugs. A number of mature and immature monocyte-like cells were detected in the peripheral blood and bone marrow; they proved to be monocytic cells by cytochemical examination and an analysis of their cell surface phenotype, indicating that the cat suffered from acute myeloid leukaemia, subclassified as monocytic leukaemia (MS). Treatment with cytarabine, doxorubicin, vincristine and prednisolone greatly reduced the number of blast cells in the cat's peripheral blood and bone marrow. The cat was in partial remission for 67 days and survived for 95 days after it was first examined.
• Expression of apoptosis-related gene mRNAs in feline T-cells infected with feline immunodeficiency virus (FIV)

  J Yamazaki, N Hasebe, S Nagafuchi, K Baba, H Tsujimoto, R Kano, A Hasegawa

  VETERINARY MICROBIOLOGY 101 1 1 - 8 2004年06月 [査読有り][通常論文]
   

  In the present study, full length of feline bax,. bcl-2, bcl-xL and caspase 3 genes were sequenced and the expression of, these mRNAs were also investigated in FIV-infected lymphocytes. The full length cDNA sequence of bax (646 bp), bcl-2 (1423 bp), bcl-xL (1163 bp) and caspase 3 genes (1208 bp) contained a single open reading frame of 579 bp coding 193 amino acids, 708 bp coding 236 amino acids, 702bp coding 234,amino acids and 834 bp coding 278 amino acids, respectively. Number of apoptotic Kumi-1 cells gradually increased after FIV infection and approximately 70% were apoptotic and 30% were viable in the cells infected with FIV after 8-day incubation, though approximately 80% were non-apoptotic and 20% were dead in non-infected cells. The expression of bcl-2 mRNA in lymphocytes of established cell line was increased by FIV. The amounts of mRNAs of bax, caspase 3 and bcl-xL in FIV-infected cells were not different from those in uninfected control cells. (C) 2004 Elsevier B.V. All rights reserved.

書籍

• 猫の治療ガイド2020 : 私はこうしている

  山崎淳平 (担当:分担執筆範囲:輸血療法 輸血副作用)
  Eduward press 2020年08月 (ISBN: 9784866711225) xvi, 963, 23p
• 犬の治療ガイド2020 : 私はこうしている

  山崎淳平 (担当:分担執筆範囲:輸血療法 輸血副作用)
  株式会社EDUWARD Press 2020年08月

講演・口頭発表等

• イヌのがんにおける異常DNAメチル化解析  [招待講演]

  山崎淳平

  遺伝研研究会「コンパニオンアニマルのゲノム医療」 2020年12月 シンポジウム・ワークショップパネル（指名）
• イヌ腫瘍の新たなメカニズム～異常DNAメチル化～  [招待講演]

  山崎淳平

  第16回日本獣医内科学アカデミー学術大会（JCVIM2020） 2020年02月
• AML患者の治療奏功群を規定するTET2特異的可変メチル化領域の同定  [招待講演]

  山崎淳平

  第42回日本分子生物学会年会 2019年12月
• イヌを用いたトランスレーショナル・エピジェネティクス研究  [招待講演]

  山崎淳平

  日本人類遺伝学会第64回大会 2019年11月
• エピジェネティクス 臨床応用の新たな可能性  [招待講演]

  山崎 淳平

  第14回日本獣医内科学アカデミー学術大会（JCVIM2018） 2018年02月
• 犬のリンパ腫における微小残存病変（MRD）  [招待講演]

  山崎 淳平

  第12回日本獣医がん学会 2015年01月
• リンパ腫の微小残存病変(MRD)定量システム  [招待講演]

  山崎 淳平

  第7回日本獣医内科学アカデミー学術大会（JCVIM2011） 2011年02月
• リンパ腫症例における微小残存病変（MRD）定量でわかること  [招待講演]

  山崎 淳平

  第4回日本獣医内科学アカデミー学術大会（JCVIM2007） 2007年08月

その他活動・業績

• イヌ血管肉腫におけるヒストンラクチル化とグルタミン代謝の役割の解析

  鈴木玲海, 青島圭佑, 山崎淳平, 安井博宣, 平島一輝, 木村享史 がんと代謝研究会プログラム&抄録集 9th 2023年
• イヌ血管肉腫におけるDNAメチル化異常とDNAメチル化阻害による抗腫瘍効果

  鹿又崚, 青島圭佑, 青島圭佑, 鈴木玲海, 山崎淳平, 山崎淳平, 木村享史 日本獣医学会学術集会講演要旨集 166th 2023年
• イヌ血管肉腫におけるヒストンラクチル化およびグルタミン代謝の役割の解析

  鈴木玲海, 青島圭佑, 山崎淳平, 安井博宣, 平島一輝, 木村享史 日本癌学会学術総会抄録集(Web) 82nd 2023年
• 犬尿路上皮癌におけるPDXモデルの樹立

  森下 大暉, 木之下 怜平, 青島 圭佑, 細谷 謙次, 山崎 淳平, 滝口 満喜 日本獣医学会学術集会講演要旨集 165回 [B2P -03] 2022年09月
• イヌの肝細胞腫瘍におけるDNAメチル化の網羅的解析

  淺利 友, 山崎 淳平, Oo Thander, 細谷 謙次, 金 尚昊, 木之下 怜平, 竹内 恭介, 賀川 由美子, 佐々木 東, 中村 健介, 滝口 満喜 日本獣医学会学術集会講演要旨集 164回 [HSO -30] 2021年09月
• 非再生性免疫介在性貧血の犬における脾臓摘出術前後の血清中蛋白の網羅的解析

  菅原 芽伊, 山崎 淳平, 森下 啓太郎, 金 尚昊, 細谷 謙次, 佐々木 東, 中村 健介, 滝口 満喜 日本獣医学会学術集会講演要旨集 164回 [HSO -60] 2021年09月
• エピジェネティック治療

  山崎淳平 Veterinary Oncology 2020年04月 [査読無し][招待有り]
  
• 輸血前検査と血液型

  山崎淳平 Veterinary Board 2020年01月 [査読無し][招待有り]
  
• 過去3年間の輸血後副反応に関する回顧的研究

  細田 千尋, 森下 啓太郎, 山崎 淳平, 高田 健介, 細谷 謙次, 金 尚昊, 佐々木 東, 大田 寛, 滝口 満喜 北海道獣医師会雑誌 63 (8) 337 -337 2019年08月 [査読無し][通常論文]
  
• 第13回日本獣医内科学アカデミー学術大会「研究発表」JVM賞受賞研究 犬におけるDNAメチル化のゲノムワイド解析法の樹立

  久本 真也, 山崎 淳平, 稲葉 睦 獣医畜産新報 70 (7) 504 -506 2017年07月
• A CpG island methylator phenotype in acute myeloid leukemia independent of IDH mutations and associated with a favorable outcome

  Andrew D. Kelly, Heike Kroeger, Jumpei Yamazaki, Rodolphe Taby, Frank Neumann, Sijia Yu, Justin T. Lee, Rong He, Shoudan Liang, Yue Lu, Matteo Cesaroni, Sherry A. Pierce, Steven M. Kornblau, Carlos E. Bueso-Ramos, Farhad Ravandi, Hagop M. Kantarjain, Jaroslav Jelinek, Jean-Pierre J. Issa CANCER RESEARCH 76 2016年07月 [査読無し][通常論文]
  
• Magnifying Narrow-Band Imaging of Gastric Mucosal Morphology Correlates With H. pylori Related Epigenetic Field Defect

  Tomomitsu Tahara, Jumpei Yamazaki, Tomoyuki Shibata, Masaaki Okubo, Tomohiko Kawamura, Noriyuki Horiguchi, Takamitsu Ishizuka, Naoko Nakano, Mitsuo Nagasaka, Yoshihito Nakagawa, Naoki Ohmiya GASTROENTEROLOGY 150 (4) S359 -S360 2016年04月 [査読無し][通常論文]
  
• Genome-wide methylation analysis reveals an independently validated CpG island methylator phenotype associated with favorable prognosis in acute myeloid leukemia.

  Andrew D. Kelly, Heike Kroeger, Jumpei Yamazaki, Rodolphe Taby, Frank Neumann, Justin T. Lee, Rong He, Shoudan Liang, Yue Lu, Matteo Cesaroni, Sherry A. Pierce, Steven M. Kornblau, Carlos E. Bueso-Ramos, Farhad Ravandi, Hagop M. Kantarjian, Jean-Pierre J. Issa, Jaroslav Jelinek CLINICAL CANCER RESEARCH 21 2015年09月 [査読無し][通常論文]
  
• ミニチュア・ダックスフンドの炎症性結直腸ポリープ症例の結直腸粘膜におけるToll-like receptor 2,4の局在解析

  横山 望, 大田 寛, 賀川 由美子, 山崎 淳平, 市居 修, Khoirun Nisa, 森田 智也, 大菅 辰幸, 佐々木 東, 森下 啓太郎, 中村 健介, 滝口 満喜 日本獣医学会学術集会講演要旨集 158回 402 -402 2015年08月 [査読無し][通常論文]
  
• 犬のリンパ腫における微小残存病変（MRD)

  山崎淳平 Veterinary Oncology 2015年 [査読無し][招待有り]
  
• CpG hypermethylation marks potentially curable acute myeloid leukemia

  Jaroslav Jelinek, Heike Kroeger, Jumpei Yamazaki, Rodolphe Taby, Frank Neumann, Justin T. Lee, Rong He, Shoudan Liang, Yue Lu, Matteo Cesaroni, Sherry A. Pierce, Steven M. Kornblau, Carlos E. Bueso-Ramos, Farhad Ravandi-Kashani, Hagop M. Kantarjian, Jean-Pierre J. Issa CANCER RESEARCH 74 (19) 2014年10月 [査読無し][通常論文]
  
• An siRNA screen identifies CHD4 as a target for epigenetic therapy

  Yasuyuki Okamoto, Jumpei Yamazaki, Takahiro Sato, Matteo Cesaroni, Woonbok Chung, Judith Garriga, Jaroslav Jelinek, Richard A. Katz, Jean-Pierre Issa CANCER RESEARCH 74 (19) 2014年10月 [査読無し][通常論文]
  
• The Osteoclast Targeting Therapy In Bone Metastasis For a Mouse Model Of Adult T Cell Leukemia

  Takuo Mizukami, Kazuya Takizawa, Jumpei Yamazaki, Wakako Kuribayashi, Madoka Kuramitsu, Ryuji Hiramatsu, William W. Hall, Hideki Hasegawa, Kazunari Yamaguchi, Isao Hamaguchi BLOOD 122 (21) 2013年11月 [査読無し][通常論文]
  
• Cancer Drivers Affected by Aberrant DNA Methylation in MDS and AML

  Jaroslav Jelinek, Shoudan Liang, Frank Neumann, Rong He, Yue Lu, Jumpei Yamazaki, Rodolphe F. Taby, Louis Ramagli, Marcos R. Estecio, Jean-Pierre Issa BLOOD 118 (21) 747 -748 2011年11月 [査読無し][通常論文]
  
• Limited Effect of TET2 Mutations on Promoter DNA Methylation in Chronic Myelomonocytic Leukemia

  Jumpei Yamazaki, Rodolphe F. Taby, Aparna Vasanthakumar, Trisha Macrae, Kelly R. Ostler, Lanlan Shen, Hagop M. Kantarjian, Marcos R. Estecio, Jaroslav Jelinek, Lucy Godley, Jean-Pierre Issa BLOOD 118 (21) 599 -599 2011年11月 [査読無し][通常論文]
  
• Methylation status of histone H3 Lysine 4 in normal melanocytes is a major determinant of DNA hypermethylation in melanoma

  Hai Long, Yue Lu, Jumpei Yamazaki, Na Yoon, Marcos R. H. Estecio, Jaroslav Jelinek, Shoudan Liang, Jean-Pierre J. Issa CANCER RESEARCH 71 2011年04月 [査読無し][通常論文]
  
• 犬のリンパ腫における微小残存病変(MRD)解析とその臨床的有用性

  山崎淳平 小動物腫瘍臨床 2011年 [査読無し][招待有り]
  
• Genome-Wide Epigenetic Analysis of Cancer Stem Cells (CSCs) In Acute Myeloid Leukemia

  Jumpei Yamazaki, Marcos R. Estecio, Jaroslav Jelinek, David Graber, Yue Lu, Louis Ramagli, Shoudan Liang, Steven M. Kornblau, Jean-Pierre Issa BLOOD 116 (21) 1497 -1497 2010年11月 [査読無し][通常論文]
  
• Estimating frequency of cancer stem cells in a mouse model of adult T-cell leukemia/lymphoma

  Jumpei Yamazaki, Takuo Mizukami, William W. Hall, Isao Hamaguchi BLOOD 115 (10) 2118 -2118 2010年03月 [査読無し][通常論文]
  
• HTLV-1 Taxトランスジェニックマウスを用いたATL様腫瘍幹細胞の同定と解析

  滝澤 和也, 水上 拓郎, 山崎 淳平, 倉光 球, 百瀬 暖佳, 益見 厚子, 本嶋 藍, 鶴野 親是, 大隈 和, 長谷川 秀樹, 浜口 功, 山口 一成 臨床血液 50 (9) 1170 -1170 2009年09月 [査読無し][通常論文]
  
• 犬リンパ腫の微小残存病変定量によるウィスコンシン大学多剤併用プロトコール期間中に使用される各抗癌剤の効果の比較

  佐藤雅彦, 山崎淳平, 後藤裕子, 高橋雅, 藤野泰人, 大野耕一, 辻本元 獣医畜産新報 (1056) 2009年
• イヌのNHLにおける微小残存病変の検討

  山崎淳平, 馬場健司, 瀬戸口明日香, 藤野泰人, 大野耕一, 辻本元 臨床血液 48 (9) 952 2007年09月30日 [査読無し][通常論文]
  

特許

• 特願WO2017070189A1:Hypomethylation of tet2 target genes for identifying a curable subgroup of acute myeloid leukemia  

  Jean-Pierre J. Issa, Jumpei Yamazaki

受賞

• 2017年02月 第13回 日本獣医内科学アカデミー学術大会（JCVIM 2017） 研究アワード（文永堂出版 JVM賞）
   イヌにおけるDNAメチル化ゲノムワイド解析法の樹立 

  受賞者: 久本真也;山崎淳平;稲葉睦
• 2008年03月 日本獣医学会 第145回日本獣医学会 大会長賞
   イヌのリンパ腫における微小残存病変についての研究 

  受賞者: 山崎淳平
• 2006年09月 The 16th European College of Veterinary Internal Medicine Oral Abstract Presentation Award
   Quantification of minimal residual disease (MRD) using real-time polymerase chain reaction in canine lymphoma. 

  受賞者: 山崎淳平

共同研究・競争的資金等の研究課題

• 外来性の化学物質（xenobiotics）に対する哺乳類の適応進化/変化

  日本学術振興会：科学研究費助成事業

  研究期間 : 2021年04月 -2026年03月 

  代表者 : 石塚 真由美, 早川 卓志, 武田 一貴, 川合 佑典, 山崎 淳平, 池中 良徳, 中山 翔太

   

  我々はこれまでの研究により、高次の動物が外来性の化学物質（Xenobiotics）への「適応」ために、化学物質の代謝や排泄など、解毒のカスケードに沿って薬物代謝酵素などの一連の多様性を発展させてきたとの仮説を立てた。化学物質感受性には多様性があり、解毒代謝酵素の解析から、この多様性獲得の主要因は食餌由来の化学物質である可能性を報告した。本研究では食性の観点および重度環境汚染域に棲息する動物を中心に、哺乳類が日常的に曝露される化学物質にどのように適応してきたのか、多様な種を用いてその進化を機能面から明らかにする。これまでの研究で構築してきた多様な動物試料を用いた研究体制を生かし、食性のユニークな動物種を対象としたウェットな実験や、データベースを用いた網羅的解析により、外来性の化学物質に対する動物の「適応」と「共存」メカニズムを明らかにする。
  今年度は、食肉目クマ科、スカベンジャー種（腐肉食類）等について、ゲノムデータベースを用いた遺伝子解析を行った。外来化学物質の代謝を担う第I相反応酵素シトクロムP450、第II相反応のグルクロン酸転移酵素や硫酸転移酵素について、種間比較を行い、食性との関連性について解析した。特に硫酸転移酵素についてはこれまで分子進化と食性に関するデータは少なく、研究成果については、現在論文を作成している。野生げっ歯類、トカゲ、一部の食肉性哺乳類について、次世代シークエンサーを用いて、エクソーム解析およびトランスクリプトームを行っている。また農薬や重金属の環境汚染地域に生活・棲息している野生げっ歯類及びヒトについてメタボローム解析を行った。現在データを解析中であるが、野生げっ歯類についてはDDT高濃度汚染域に棲息している野生げっ歯類のステロイド代謝への影響が認められた。現在、野生げっ歯類をDDT環境汚染レベル下で世代飼育し、フィールドデータの検証を行っている。
• 尿のリンパ組織発達誘導を中心とする腎盤－腎臓病態軸

  日本学術振興会：科学研究費助成事業

  研究期間 : 2021年04月 -2025年03月 

  代表者 : 市居 修, 岡村 匡史, 昆 泰寛, 西邑 隆徳, 矢吹 映, 山崎 淳平, 中村 鉄平, 細谷 実里奈, 堀野 太郎

   

  ヒトと伴侶動物では、個体の高齢化に伴い慢性腎臓病(CKD)症例が増えている。CKDは糸球体や尿細管間質の慢性炎症を主体とし、難治性である。近年、腎臓内に形成された誘導性 リンパ組織による慢性炎症の増悪と遷延が問題視されている。我々は腎盤(腎臓から尿を受ける嚢状構造、腎盂)に尿路関連リンパ組織(UTALT/UTALS)を発見し、その発達がCKD進行と強く相関することを見出した。本研究では、疾患モデル動物や伴侶動物・ヒト症例の精査を基軸に、CKDにおけるUTALS発達の意義、特に腎臓の慢性炎症との病態連関を解明する。さらに、UTALTが発達する理由として“CKD時の尿が導く腎盤上皮バリアの脆弱化とそれに続く尿の腎盤侵入”を証明し、尿の新たな存在意義“リンパ組織の発達誘導”を提唱する。 これまでヒトとマウスのUTALSは、移行上皮直下でT細胞、B細胞やマクロファージ等の免疫細胞で構成され、膠原線維や細網線維を含むことを明らかにした。また、腎盤UTALSでは、CCL・CXCLケモカインおよびその受容体遺伝子が発現しており、これらケモカインは主に腎盤間質に発現していた。腎炎モデルマウス(MRL/lpr)のUTALSは顕著に発達し、CCL・CXCLケモカインの発現は腎病理スコア(糸球体傷害、尿細管間質傷害、誘導性リンパ組織形成)と有意に正の相関にあった。また、MRL/lprの腎盤上皮は形態や細胞間接着分子(Occludin、ZO-1)の発現を変化させ、腎盤上皮バリア異常が示唆された。さらに色素の尿路逆行性投与実験において、MRL/lprでは色素が腎盤腔から腎盤組織に漏れ出た。今後、尿とUTALSの関係をさらに詳細に解析する。 腎盤を巻き込む腎臓病の重篤化は深刻であり、死亡率も高い。本研究では、UTALSを中心とした腎盤－腎臓病態軸の解明から、難治性疾患CKDの治療戦略に新たな道を切り拓く。
• 異常DNAメチル化をターゲットとした腫瘍の微小残存病変検出法と超早期診断法の確立

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2021年04月 -2025年03月 

  代表者 : 山崎 淳平

   

  イヌ悪性黒色腫に特異的なDNAメチル化変化部位6カ所を選別、バイサルファイトシークエンス用のPCRアッセイを構築した。血漿中セルフリーDNAからのDNAメチル化検出が可能がどうか検討を行うため、イヌ悪性黒色腫症例の保存血漿よりセルフリーDNAを抽出後、PCRを行い期待通りの増幅産物を確認した。本PCR産物を次世代シークエンスによる解析に進めるため、異なる症例および異なる日付ごとにバーコードプライマーを用いてライブラリ作製を行った。その後、次世代シークエンスによって得られたfastqファイルについてTrim-galoreによるアダプター除去、bismarkによるイヌゲノムの仮想バイサルファイト処理を行ったデータへのマッピング、各CpGサイトにおけるメチル化／非メチル化数のカウントを行った。その結果、最低でもカバレッジが10,000、最高で1,000,000得られた、つまり0.01%から0.0001%のメチル化を定量可能であることが判明した。また、この際の次世代シークエンスによる解析のコストは数万円であったため、同時に解析したサンプル数が10以上であることを考慮すると、定量とコストに見合った結果が得られることが確認できた。 DNAメチル化定量に関する結果においては、まずその信頼性のチェックのために血漿中セルフリーDNAと並行して腫瘍細胞そのものを同方法によってメチル化を定量したところ、当然のことながら正常細胞には見られない異常なメチル化が検出可能であった。次に血漿セルフリーDNAを用いたDNAメチル化定量では、0.01%-0.2%ほどのメチル化が検出された。本メチル化量が病状と相関するか、サンプル数を増やしている。
• 副腎－肝臓連関によるイヌ肝細胞癌発症メカニズムの解明

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2020年04月 -2023年03月 

  代表者 : 滝口 満喜, 村上 正晃, 山崎 淳平, 池中 良徳

   

  犬の肝細胞癌における、ステロイドホルモン産生過剰を起点とする発がん仮説の検証を目指し、前年度に引き続いた検討を行った。2年目となる本年度は肝細胞癌・副腎皮質機能亢進症罹患犬における血中ステロイドホルモン・代謝産物のプロファイリング、ならびに肝臓組織でのエピゲノム変異の解析を遂行した。 ステロイドプロファイリングでは前年度に確立した測定系を発展拡大させ、新たに6種の副腎皮質ホルモンと4種の代謝産物を合わせた、合計19種類のステロイドプロファイル解析が可能となった。この新たな測定系を用いて、肝細胞癌36症例、副腎皮質機能亢進症15症例、併発11症例、コントロール19症例を対象として、血液中のステロイドプロファイルを解析した。しかし、血液中のステロイドプロファイルに疾患特異性、もしくは疾患群間での差が認められなかった。 DNAメチル化の解析では、実験的ステロイドホルモン誘発性脂肪肝、副腎皮質腫機能亢進症併発肝細胞癌および肝細胞癌単独症例の腫瘍組織と腫瘍近傍組織の、合計3種類の肝臓組織を用い、多段階発癌仮説の検証を試みた。その結果、脂肪肝・近傍組織・腫瘍組織と段階的なメチル化変化が存在し、腫瘍においてメチル化レベルの高い遺伝子が6、低い遺伝子が12、抽出できた。 現時点では血中ステロイドプロファイルに肝細胞癌と副腎皮質機能亢進症との関連を示唆する血中ステロイドプロファイリングは見つかっていない。一方で、ステロイド肝から腫瘍への段階的なDNAメチル化レベルの変化が存在することが明らかになった。犬の肝細胞癌においてステロイドが発症に役割を果たしていることは十分に予想され、本年度の成果を複合的かつ詳細に解析することで、多段階発がん説の検証が進む。
• 魚類における自然免疫記憶の誘導機構の解明

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2020年04月 -2023年03月 

  代表者 : 瀧澤 文雄, 末武 弘章, 山崎 淳平

   

  単球・マクロファージは、自然免疫応答の中心的な白血球であり、病原体に対する初期応答の中枢を担っている。サイトカインであるコロニー刺激因子１（CSF1）は、単球・マクロファージの増殖、分化、および生存に関わる因子であり、我々はこれまでにトラフグのCSF1遺伝子の単離に成功している。そこで、トラフグCSF1組換えタンパク質を用いて、マクロファージの培養を試みるためにトラフグの分泌型CSF1bの組換えタンパク質の大量作製および精製を行った。 血液や腎臓の白血球から単球・マクロファージを分離するために、従来行われているプラスチックフラスコに対する接着法を試みた。接着した細胞のうち、6割ほどがマクロファージであり、白血球中のマクロファージの濃度を高めることには成功した。しかし、遺伝子発現などによりマクロファージの特性を正確に測定するためには、マクロファージをより高純度に精製する必要である。これまでに、トラフグマクロファージのマーカーとなる分子候補をいくつか発見できているため、これら分子およびセルソーターを利用してマクロファージの精製を試みている。 魚類のマクロファージは、主に血液や腎臓に存在するが、魚類特有の器官である鰾にもマクロファージのマーカー遺伝子が発現していることが確認でき、ウイルス感染によりこれらマーカー遺伝子の発現量の増加が認められた。以上から、血液やリンパ組織に限らず、粘膜組織においてもマクロファージが感染防御に関わることが分かった。
• イヌの新規ゲノムワイドDNAメチル化解析：腫瘍性疾患特異的DNAメチル化の同定

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2018年04月 -2021年03月 

  代表者 : 山崎 淳平, 市居 修

   

  イヌの悪性黒色腫、消化器型リンパ腫および肝細胞癌におけるエピジェネティクス特にDNAメチル化に関し、次世代シークエンサーを用いて新たな知見を得ることによってイヌの腫瘍疾患メカニズムを解明することにつなげるため、令和元年度は以下の項目について検討を行った。 イヌの口腔粘膜型悪性黒色腫28症例と正常犬の口腔内粘膜組織４種類をサンプルとして用いた。 DNAメチル化の状態によって共通の認識部位(CCCGGG)の切断形式が異なる２種類の制限酵素(SmaI, XmaI)を用いてDNAを切断し、次世代シークエンサーを利用して、ゲノム上に存在するCpGサイトのDNAメチル化の定量解析を行った。各サンプルにおいて、約130,000～180,000箇所のCpGサイトにおけるDNAメチル化の定量解析が可能であった。次に、全てのサンプルに共通して得られた約43,000CpGサイトのDNAメチル化データを用いた相関解析およびクラスタリング解析によって、悪性黒色腫症例と正常粘膜組織群の間にDNAメチル化状態の明らかな違いが認められた。また、興味深いことに、脱メチル化に焦点をあてた詳細な解析において悪性黒色腫症例が２群に分類されること、またより脱メチル化が強い群が脱メチル化が弱い群に比べ、予後が悪い傾向があることが判明した。これについて、ヒトの腫瘍における脱メチル化が報告されているLINE1配列におけるDNAメチル化の解析を行ったところ、脱メチル化の程度によって予後と相関している傾向が認められた。 また、消化器型リンパ腫10症例、肝細胞癌12症例についても現在DREAM法による検討を行ったところ、正常肝細胞との明らかな差が見出されている。
• 核内受容体を介した免疫記憶形成機構の解明とワクチン療法への応用

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2017年04月 -2021年03月 

  代表者 : 高田 健介, 稲葉 睦, 山崎 淳平

   

  過去に感染した病原体が再度体内に侵入すると、免疫系はより素早く強力に応答する。この現象は免疫記憶と呼ばれ、ワクチンに応用されてきた。しかし、免疫記憶の詳細な成立機構は未だ明らかにされていない。免疫記憶の本体は抗原特異的な応答の後、体内で長期間維持される記憶リンパ球であり、記憶Tリンパ球の分化機構を解明することは、細胞性免疫記憶を誘導可能なワクチンの開発基盤となる。記憶Tリンパ球の分化には、脂質代謝が重要な役割を果たすことが近年明らかとなってきた。本研究は、RORファミリー核内受容体が代謝を競合的制御することで、記憶Tリンパ球の分化を担うという仮説のもと、免疫記憶機構の解明を目的とする。さらに、これらの核内受容体に特異的な合成リガンドを用いて、ワクチン療法に応用することを目指す。
  本研究ではこれまでに、当該核内受容体を介したシグナルが、in vitroにおいて活性化CD8Tリンパ球の生存に影響を与えることをあきらかにした。さらに、この生存への効果が核内受容体のコレルテロール代謝制御機能に関連することを網羅的遺伝子発現解析から明らかにした。以上の実験結果について、現在論文を作成中であり、近日中に専門誌に投稿する予定である。さらに、当該核内受容体の欠損あるいは合成リガンドの生体内投与がCD8Tリンパ球の感染応答に与える影響について、予備的な検討を行ったところ、細胞の増殖と生存、表現型に影響が認められた。
• DNAメチル化解析による種雄牛由来精子の新規評価法開発

  公益財団法人 高橋産業経済研究財団：研究助成

  研究期間 : 2019年 -2020年 

  代表者 : 山崎 淳平
• 犬のリンパ腫におけるDNAメチル化情報を用いた予後予測への応用

  アニコム キャピタル株式会社：

  研究期間 : 2019年07月 

  代表者 : 山崎淳平
• TRIM-SUMO-プロテアソーム経路：新たな膜蛋白質異常認識・分解機構の実証

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2016年04月 -2019年03月 

  代表者 : 稲葉 睦, 高田 健介, 山崎 淳平

   

  アニオン交換輸送体バンド3変異体R664X AE1の小胞体関連分解(ERAD)は、Ub化非依存性である。本研究では、それがSUMO化にも非依存性であり、かつプロテアソーム系への関与が知られるTRIM28等のTRIMファミリータンパク質に関与が認められず、いわゆるTRIM-SUMO-11S経路は関与しないことが明らかになった。さらに、R664X AE1の分解は、そのN末端側細胞質ドメインの構造とディスロコンDerlinsの作用が不可欠なこと、26S (20S + 19S)プロテアソームが働くことが示された。
• TET2によるエンハンサー特異的DNA脱メチル化機構の解明

  公益財団法人 住友電工グループ：社会貢献基金

  研究期間 : 2018年 -2019年 

  代表者 : 山崎 淳平
• TET2によるエンハンサー特異的DNA脱メチル化機構の解明

  公益財団法人金原一郎記念医学医療振興財団：

  研究期間 : 2018年 -2019年 

  代表者 : 山崎 淳平
• DNAメチル化網羅解析法による種雄牛の受胎性の評価

  公益財団法人伊藤記念財団：研究助成

  研究期間 : 2018年 -2019年 

  代表者 : 山崎 淳平
• TET2が司るDNA脱メチル化メカニズムの解明

  公益財団法人 上原記念生命科学財団：研究奨励金

  研究期間 : 2017年 -2019年 

  代表者 : 山崎 淳平
• イヌのゲノムワイドDNAメチル化情報基盤の構築と多角的健康増進への応用

  アニコム先進医療研究所株式会社：

  研究期間 : 2018年12月 

  代表者 : 山崎淳平
• DNA脱メチル化酵素TET2の結合因子同定による難治性血液疾患メカニズムの解明

  公益信託 永尾武難病研究基金：

  研究期間 : 2017年 -2018年 

  代表者 : 山崎 淳平
• 網羅的DNAメチル化解析が同定する肥満によるエピジェネティックな変化

  クリニカルニュートリション研究会：スカラーシッププログラム

  研究期間 : 2017年 -2018年 

  代表者 : 山崎 淳平, 山下 青空
• DNAメチル化解析を用いたウシの精液性状変化の検出

  公益財団法人 北海道科学技術総合振興センター：若手研究人材育成事業

  研究期間 : 2017年 -2018年 

  代表者 : 山崎 淳平
• DNAメチル化解析による環境中鉛汚染水の毒性メカニズム解明

  公益財団法人 クリタ水・環境科学振興財団：国内研究助成

  研究期間 : 2017年 -2018年 

  代表者 : 山崎 淳平
• 新規DNAメチル化網羅探索法を用いた牛の精液性状の評価法開発

  公益財団法人 伊藤記念財団：研究助成

  研究期間 : 2017年 -2018年 

  代表者 : 山崎 淳平
• DNA脱メチル化酵素TET2によるエンハンサー領域特異的制御機構の解明

  公益財団法人 武田科学振興財団：医学系研究奨励（基礎）

  研究期間 : 2016年 -2018年 

  代表者 : 山崎 淳平
• ユビキチン非依存性タンパク質小胞体分解におけるTRIM-SUMO-11Sプロテアソーム経路の解明

  文部科学省：科学研究費補助金 基盤研究（Ｂ）（一般）

  研究期間 : 2016年 -2018年 

  代表者 : 稲葉 睦
• ＴＳＰＯ２による時期特異的小胞体コレステロール蓄積と赤芽球の成熟制御

  日本学術振興会：科学研究費助成事業

  研究期間 : 2015年04月 -2017年03月 

  代表者 : 稲葉 睦, 山﨑 淳平, 佐藤 耕太

   

  成熟異常を呈する犬のHK赤血球表現型は、コレステロール代謝への関与が想定されるTSPO2遺伝子の変異により生じる。本研究では、細胞形態、細胞表面抗原、ヘモグロビン合成、細胞周期、遺伝子発現等に焦点を置き、後期赤芽球の成熟・脱核とTSPO2の関係を犬骨髄由来赤芽球、赤芽球系培養細胞を用いて解析した。その結果、TSPO2が、後期赤芽球におけるコレステロール代謝の変動を介して細胞増殖、細胞周期、ヘモグロビン合成や脱核の時期を調節し得ることが示された。
• イヌのリンパ腫におけるDNAメチル化のゲノムワイド解析―機序解明と臨床応用―

  日本学術振興会：科学研究費助成事業 若手研究(B)

  研究期間 : 2015年04月 -2017年03月 

  代表者 : 山崎 淳平

   

  イヌリンパ腫細胞株5株に関し、次世代シークエンサーによる解析を行ったところ、約100,000CpGサイトのDNAメチル化の定量的な解析が可能であった。また、正常血液サンプルとリンパ腫細胞株の区別が可能であった。さらに、各々のリンパ腫細胞株において1000～4000個の高メチル化部位、および3000~10,000個の低メチル化部位が発見された。これらのうち、特に高メチル化部位の近傍には腫瘍に関連すると考えられる遺伝子が認められた。 イヌのリンパ腫自然発症例24例についても同様の解析・検討を行ったところ、DNAメチル化のグループ分けが予後因子となる可能性が示された。
• イヌのリンパ腫自然発症例におけるDNAメチル化のゲノムワイド解析

  北海道大学：北海道大学総長室事業推進経費（公募型）「若手研究者自立支援（A）」

  研究期間 : 2014年04月 -2015年03月 

  代表者 : 山崎淳平

大学運営

委員歴

• 2020年04月 - 現在   日本獣医輸血研究会   運営委員

社会貢献活動

• 運営委員

  期間 : 2020年04月01日 - 現在

  役割 : 運営参加・支援

  主催者・発行元 : 日本獣医輸血研究会
• 輸血前検査の適応と限界

  期間 : 2018年02月28日

  役割 : 講師

  主催者・発行元 : 北海道大学 動物医療センター