研究者業績

落合 和彦

オチアイ カズヒコ  (OCHIAI KAZUHIKO)

基本情報

所属
日本獣医生命科学大学 獣医学部 獣医学科 獣医衛生学研究室 准教授 (DVM. Ph D.)
学位
博士(獣医学)(2005年3月 岐阜大学)

J-GLOBAL ID
201001023189960831
researchmap会員ID
6000021447

外部リンク

研究キーワード

 3

論文

 102
  • Masanori Kobayashi, Akiko Saito, Yoshikazu Tanaka, Masaki Michishita, Masato Kobayashi, Mami Irimajiri, Takeharu Kaneda, Kazuhiko Ochiai, Makoto Bonkobara, Kimimasa Takahashi, Tatsuya Hori, Eiichi Kawakami
    JOURNAL OF VETERINARY MEDICAL SCIENCE 79(4) 719-725 2017年4月  査読有り
    Canine prostate cancer (cPCa) is an untreatable malignant neoplasm resulting in local tissue invasion and distant metastasis. MicroRNAs (miRs) are small non-coding RNAs that function as oncogenes or tumor suppressors. The purpose of this study was to characterize the expression of miRs that are altered in cPCa tissue. The expression levels of 277 mature miRs in prostatic tissue (n=5, respectively) were compared between the non-tumor and tumor groups using real-time PCR. Five miRs (miR-18a, 95, 221, 222 and 330) were up-regulated, but 14 miRs (miR-127, 148a, 205, 299, 329b, 335, 376a, 376c, 379, 380, 381, 411, 487b and 495) were down-regulated specifically in cPCa (P<0.05). These miRs have potential use as early diagnosis markers for cPCa and in miR-based therapy.
  • Hirohiko Okamura, Kaya Yoshida, Hiroyuki Morimoto, Jumpei Teramachi, Kazuhiko Ochiai, Tatsuji Haneji, Akihito Yamamoto
    JOURNAL OF CLINICAL MEDICINE 6(3) 2017年3月  査読有り
    The reversible phosphorylation of proteins plays hugely important roles in a variety of cellular processes, such as differentiation, proliferation, and apoptosis. These processes are strictly controlled by protein kinases (phosphorylation) and phosphatases (de-phosphorylation). Here we provide a brief history of the study of protein phosphorylation, including a summary of different types of protein kinases and phosphatases. One of the most physiologically important serine/ threonine phosphatases is PP2A. This review provides a description of the phenotypes of various PP2A transgenic mice and further focuses on the known functions of PP2A in bone formation, including its role in osteoblast differentiation and function. A reduction in PP2A promotes bone formation and osteoblast differentiation through the regulation of bone-related transcription factors such as Osterix. Interestingly, downregulation of PP2A also stimulates adipocyte differentiation from undifferentiated mesenchymal cells under the appropriate adipogenic differentiation conditions. In osteoblasts, PP2A is also involved in the ability to control osteoclastogenesis as well as in the proliferation and metastasis of osteosarcoma cells. Thus, PP2A is considered to be a comprehensive factor in controlling the differentiation and function of cells derived from mesenchymal cells such as osteoblasts and adipocytes.
  • J. Teramachi, Y. Inagaki, H. Shinohara, H. Okamura, D. Yang, K. Ochiai, R. Baba, H. Morimoto, T. Nagata, T. Haneji
    ORAL DISEASES 23(2) 181-188 2017年3月  査読有り
    ObjectiveIn this study, we aimed to clarify the precise mechanism underlying lipopolysaccharide (LPS)-induced osteoclastogenesis in periodontal disease with a special reference to double-stranded RNA-dependent protein kinase (PKR). Material and MethodsWe dissected the role of PKR in LPS-induced osteoclast differentiation and function using primary mouse bone marrow cells and RAW264.7 pre-osteoclastic cell line. We used a rat experimental periodontitis (PD) model induced by ligature placement with a Porphyromonas gingivalis LPS injection (PD rat) and analyzed the therapeutic effects of C16, a PKR inhibitor, on bone loss in PD rats. ResultsProtein kinase is strongly upregulated and phosphorylated by LPS in the osteoclasts. The inhibition of PKR suppressed LPS-stimulated osteoclast formation and activation. PKR inhibition also suppressed the LPS-mediated activation of NF-B and MAPK, which are critical pathways for osteoclastogenesis. High expressions of PKR were detected in osteoclasts of PD rats, and the treatment with C16 effectively prevented alveolar bone destruction in PD rats. ConclusionsPKR plays a pivotal role in LPS-induced bone loss in PD and, thus, has potential as a therapeutic target for PD.
  • Rei Nakahira, Masaki Michishita, Misaki Kato, Yuki Okuno, Hitoshi Hatakeyama, Hisashi Yoshimura, Daigo Azakami, Kazuhiko Ochiai, Makoto Bonkobara, Kimimasa Takahashi
    JOURNAL OF VETERINARY DIAGNOSTIC INVESTIGATION 29(1) 105-108 2017年1月  査読有り
    A 3-y-old male miniature Dachshund was presented with an similar to 0.8 cm diameter mass in the right mandibular region. Fourteen months later, the mass was 5 x 4 x 3 cm. Grossly, the mass was encapsulated and was homogeneously gray-white on cut surface. Microscopically, the mass was composed of large, round to polygonal tumor cells that were arranged in solid nests and cords separated by a fibrovascular stroma. Tumor cells had large, round, hypochromatic nuclei containing large prominent nucleoli and abundant eosinophilic cytoplasm containing dark blue granules visible with phosphotungstic acid-hematoxylin stain. Metastasis was observed in the mandibular lymph node. Immunohistochemically, tumor cells were positive for CK AE1/AE3, low-molecular-weight CK (CAM5.2), E-cadherin, mitochondria ATPase beta subunit, and S100, but were negative for vimentin, carcinoembryonic antigen, p63, CK14, CD10, and chromogranin A. Ultrastructurally, tumor cells contained numerous mitochondria. Therefore, the tumor was diagnosed as an oncocytic carcinoma of the mandibular gland.
  • Toshinori Omi, Shota Nakazawa, Chihiro Udagawa, Naomi Tada, Kazuhiko Ochiai, Yong Hwa Chong, Yuiko Kato, Hiroko Mitsui, Azusa Gin, Hitomi Oda, Daigo Azakami, Kyoichi Tamura, Toshinori Sako, Takeshi Inagaki, Atsushi Sakamoto, Toshihiko Tsutsui, Makoto Bonkobara, Shuichi Tsuchida, Shigenori Ikemoto
    PLOS ONE 11(10) e0165000 2016年10月  査読有り
    Cat's AB blood group system (blood types A, B, and AB) is of major importance in feline transfusion medicine. Type A and type B antigens are Neu5Gc and Neu5Ac, respectively, and the enzyme CMAH participating in the synthesis of Neu5Gc from Neu5Ac is associated with this cat blood group system. Rare type AB erythrocytes express both Neu5Gc and Neu5Ac. Cat serum contains naturally occurring antibodies against antigens occurring in the other blood types. To understand the molecular genetic basis of this blood group system, we investigated the distribution of AB blood group antigens, CMAH gene structure, mutation, diplotypes, and haplotypes of the cat CMAH genes. Blood-typing revealed that 734 of the cats analyzed type A (95.1%), 38 cats were type B (4.9%), and none were type AB. A family of three Ragdoll cats including two type AB cats and one type A was also used in this study. CMAH sequence analyses showed that the CMAH protein was generated from two mRNA isoforms differing in exon 1. Analyses of the nucleotide sequences of the 16 exons including the coding region of CMAH examined in the 34 type B cats and in the family of type AB cats carried the CMAH variants, and revealed multiple novel diplotypes comprising several polymorphisms. Haplotype inference, which was focused on non-synonymous SNPs revealed that eight haplotypes carried one to four mutations in CMAH, and all cats with type B (n = 34) and AB (n = 2) blood carried two alleles derived from the mutated CMAH gene. These results suggested that double haploids selected from multiple recessive alleles in the cat CMAH loci were highly associated with the expression of the Neu5Ac on erythrocyte membrane in types B and AB of the feline AB blood group system.
  • Daigo Azakami, Eri Onozawa, Masahiro Miyabe, Kazuhiko Ochiai, Masaki Michishita, Taichi Hirano, Yutaka Momota, Katsumi Ishioka, Toshinori Sako
    JOURNAL OF VETERINARY MEDICAL SCIENCE 78(5) 913-916 2016年5月  査読有り
    A 12-year-old female American shorthair cat presented with a one-month history of hematuria and general lethargy. Abdominal ultrasonography revealed complete thickening of the left uterine wall. At a diagnostic laparotomy, a large mass arising from the left uterine horn was discovered, and ovariohysterectomy was performed. Histological diagnosis revealed a T-cell high-grade lymphoma of the uterus. After the ovariohysterectomy, the patient achieved complete remission and was maintained by combination chemotherapy from 14 days after surgery. However, relapse occurred in the urinary bladder wall on day 287, and the patient died of postrenal acute renal failure on day 310. This is the first report of a feline case of primary uterine lymphoma that was treated with ovariohysterectomy followed by systemic chemotherapy.
  • Kazuhiko Ochiai, Masami Morimatsu, Yuiko Kato, Toshina Ishiguro-Oonuma, Chihiro Udagawa, Oumaporn Rungsuriyawiboon, Daigo Azakami, Masaki Michishita, Yuichi Ariyoshi, Hideo Ueki, Yasutomo Nasu, Hiromi Kumon, Masami Watanabe, Toshinori Omi
    ONCOTARGET 7(3) 3273-3286 2016年1月  査読有り筆頭著者
    REIC/DKK-3 is a tumor suppressor, however, its intracellular physiological functions and interacting molecules have not been fully clarified. Using yeast two-hybrid screening, we found that small glutamine-rich tetratricopeptide repeat-containing protein a (SGTA), known as a negative modulator of cytoplasmic androgen receptor (AR) signaling, is a novel interacting partner of REIC/DKK-3. Mammalian two-hybrid and pull-down assay results indicated that the SGTA-REIC/DKK-3 interaction involved the N-terminal regions of both REIC/DKK-3 and SGTA and that REIC/DKK-3 interfered with the dimerization of SGTA, which is a component of the AR complex and a suppressor of dynein motor-dependent AR transport and signaling. A reporter assay in human prostate cancer cells that displayed suppressed AR signaling by SGTA showed recovery of AR signaling by REIC/DKK-3 expression. Considering these results and our previous data that REIC/DKK-3 interacts with the dynein light chain TCTEX-1, we propose that the REIC/DKK-3 protein interferes with SGTA dimerization, promotes dynein-dependent AR transport and then upregulates AR signaling.
  • Kato Y, Ochiai K, Michishita M, Azakami D, Nakahira R, Morimatsu M, Ishiguro-Oonuma T, Yoshikawa Y, Kobayashi M, Bonkobara M, Kobayashi M, Takahashi K, Watanabe M, Omi T
    Veterinary journal (London, England : 1997) 206(2) 143-148 2015年11月  査読有り責任著者
  • Toshina Ishiguro-Oonuma, Kazuhiko Ochiai, Kazuyoshi Hashizume, Toshihiko Iwanage, Masami Morimatsu
    JAPANESE JOURNAL OF VETERINARY RESEARCH 63(3) 107-114 2015年8月  査読有り
    Nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-kappa B) inhibitor zeta (Nfkbiz) is a nuclear inhibitor of NF-kappa B (I kappa B) protein that is also termed as molecule possessing ankyrin repeats induced by lipopolysaccharide, interleukin-1-inducible nuclear ankyrin repeat protein, or I kappa B zeta; We found previously that disrupting the Nfkbiz gene resulted in atopic dermatitis-like lesions in mice, suggesting an important role for Nfkbiz in the skin. In this study, we examined the cellular function of Nfkbiz in keratinocytes. Immunohistochemical analyses for Ki-67 revealed that Nfkbiz(-/-) keratinocytes were hypoproliferative. In skin from Nfkbiz(-/-) mice, the expression of the keratinocyte differentiation markers K10 and filaggrin were reduced, although that of K14 was unchanged. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay revealed that the frequency of apoptosis was comparable between control and Nfkbiz(-/-) keratinocytes. Interestingly, the subcellular localization of the NF-kappa B subunits and the transcriptional activity of NF-kappa B were not changed in Nfkbiz(-/-) keratinocytes. These findings indicate a novel possible role of Nfkbiz in controlling the proliferation and differentiation of epidermal keratinocytes through NF-kappa B-independent mechanisms.
  • Yasunaga Yoshikawa, Masami Morimatsu, Kazuhiko Ochiai, Toshina Ishiguro-Oonuma, Seiichi Wada, Koichi Orino, Kiyotaka Watanabe
    BMC VETERINARY RESEARCH 11(1) 159 2015年7月  査読有り
    Background: Mammary tumors are the most common tumor type in intact female dogs. Recently, the breast cancer 2 early onset (BRCA2) gene was proposed to be associated with tumorigenesis in dogs. The expression level of BRCA2 is important for its DNA repair function in mammalian cells, and its expression level is linked to tumorigenesis in mammary tissue. However, the expression of canine BRCA2 in mammary tumors is unclear. Results: BRCA2 mRNA levels were compared between seven mammary gland samples and seventeen mammary tumor samples isolated from dogs. The expression level of canine BRCA2 in mammary tumor samples was lower than levels in mammary gland samples. We attempted to identify why the BRCA2 expression level was decreased in mammary tumor samples by promoter sequencing analysis; however, we did not find any mutations in the canine BRCA2 promoter that altered BRCA2 transcription levels. We did detect two types of BRCA2 splice variants in 8 mammary tumor samples. One of the variants induced a frame-shift mutation that could lead to nonsense-mediated mRNA decay, a ubiquitous cellular mechanism that eliminates mRNA containing a premature termination codon. Conclusions: Reduced expression of canine BRCA2 mRNA in mammary tumor samples is a possible mechanism to explain mammary tumor development in dogs. One possible reason for reduced BRCA2 mRNA levels in these tumor samples was nonsense-mediated mRNA decay, not mutations in the BRCA2 promoter region. While it remains unclear why canine BRCA2 expression levels are reduced in mammary tumor samples, this study found that the expression level of BRCA2 was associated with canine mammary tumorigenesis.
  • Kei Fujio, Masami Watanabe, Hideo Ueki, Shun-Ai Li, Rie Kinoshita, Kazuhiko Ochiai, Junichiro Futami, Toyohiko Watanabe, Yasutomo Nasu, Hiromi Kumon
    ONCOLOGY REPORTS 33(4) 1585-1592 2015年4月  査読有り
    Immunotherapy is one of the attractive treatment strategies for advanced prostate cancer. The US Food and Drug Administration (FDA) previously approved the therapeutic vaccine, sipuleucel-T, which is composed of autologous antigen-presenting cells cultured with a fusion protein [prostatic acid phosphatase (PAP) and granulocyte-macrophage colony-stimulating factor (GMCSF)]. Although sipuleucel-T has been shown to prolong the median survival of patients for 4.1 months, more robust therapeutic effects may be expected by modifying the vaccination protocol. In the present study, we aimed to develop and validate a novel vaccination strategy using multiple PAP-fused cytokines for prostate cancer treatment. Using a super gene expression (SGE) system that we previously established to amplify the production of a recombinant protein, significant amounts of PAP-fused cytokines [human GMCSF, interleukin-2 (IL2), IL4, IL7 and mouse GMCSF and IL4] were obtained. We examined the activity of the fusion proteins in vitro to validate their cytokine functions. A significant upregulation of dendritic cell differentiation from monocytes was achieved by PAP-GMCSF when used with the other PAP-fused cytokines. The PAP-fused human IL2 significantly increased the proliferation of lymphocytes, as determined by flow cytometry. We also investigated the in vivo therapeutic effects of multiple PAP-fused cytokines in a mouse prostate cancer model bearing prostate-specific antigen (PSA)- and PAP-expressing tumors. The simultaneous intraperitoneal administration of PAP-GMCSF, -IL2, -IL4 and -IL7 significantly prevented tumor induction and inhibited the tumor growth in the PAP-expressing tumors, yet not in the PSA-expressing tumors. The in vivo therapeutic effects with the multiple PAP-fused cytokines were superior to the effects of PAP-GMCSF alone. We thus demonstrated the advantages of the combined use of multiple PAP-fused cytokines including PAP-GMCSF, and propose a promising prostatic antigen-vaccination strategy to enhance the therapeutic effects.
  • Ishiguro-Oonuma T, Ochiai K, Hashizume K, Morimatsu M
    Biomedical research (Tokyo, Japan) 36(2) 103-107 2015年4月  査読有り
  • Kazuhiko Ochiai, Toshina Ishiguro-Oonuma, Yasunaga Yoshikawa, Chihiro Udagawa, Yuiko Kato, Masami Watanabe, Makoto Bonkobara, Masami Morimatsu, Toshinori Omi
    BIOMEDICAL RESEARCH-TOKYO 36(2) 155-158 2015年4月  査読有り筆頭著者
    Mutations in the breast cancer susceptibility gene BRCA2 leading to the failure of interactions with the recombinase RAD51 are associated with an increased risk of cancer in humans. This interaction depends on the eight BRC repeat (BRC1-8) sequences in BRCA2. We previously reported that canine BRC3 has two polymorphisms (T1425P and K1435R) influencing the interaction with RAD51, and 1435R was identified in mammary tumor dog samples. In this study, we investigated the sequence variations of BRC3 and 4 in 236 dogs of five breeds. Allele frequencies of 1425P and 1435R were 0.063 and 0.314, respectively, and there was no other polymorphism in the sequenced region. A mammalian two-hybrid assay using BRC3-4 sequences demonstrated that 1425P allele reduced the binding strength with RAD51 but 1435R had no effect. These results may provide an insight into the functions of not only individual but also multiple BRC repeats of BRCA2 in dogs.
  • Chihiro Udagawa, Naomi Tada, Junzo Asano, Katsumi Ishioka, Kazuhiko Ochiai, Makoto Bonkobara, Shuichi Tsuchida, Toshinori Omi
    BMC Research Notes 7(1) 904 2014年12月11日  査読有り
    Background: The uncoupling proteins (UCPs) in the mitochondrial inner membrane are members of the mitochondrial anion carrier protein family that play an important role in energy homeostasis. Genetic association studies have shown that human UCP2 and UCP3 variants (SNPs and indels) are associated with obesity, insulin resistance, type 2 diabetes mellitus, and metabolic syndrome. The aim of this study was to examine the genetic association between polymorphisms in UCP2 and UCP3 and metabolic data in dogs. Results: We identified 10 SNPs (9 intronic and 1 exonic) and 4 indels (intronic) in UCP2, and 13 SNPs (11 intronic and 2 exonic) and one indel (exonic) in UCP3, by DNA sequence analysis of 11 different dog breeds (n = 119). An association study between these UCP2 and UCP3 variants and the biochemical parameters of glucose, total cholesterol, lactate dehydrogenase and triglyceride in Labrador Retrievers (n = 50) showed that none of the UCP2 polymorphisms were significantly associated with the levels of these parameters. However, four UCP3 SNPs (intron 1) were significantly associated with total cholesterol levels. In addition, the allele frequencies of two of the four SNPs associated with higher total cholesterol levels in a breed that is susceptible to hypercholesterolemia (Shetland Sheepdogs, n = 30), compared with the control breed (Shiba, n = 30). Conclusion: The results obtained from a limited number of individuals suggest that the UCP3 gene in dogs may be associated with total cholesterol levels. The examination of larger sample sizes and further analysis will lead to increased precision of these results.
  • Tomohisa Tanaka, Hirotake Kasai, Atsuya Yamashita, Kaori Okuyama-Dobashi, Jun Yasumoto, Shinya Maekawa, Nobuyuki Enomoto, Toru Okamoto, Yoshiharu Matsuura, Masami Morimatsu, Noboru Manabe, Kazuhiko Ochiai, Kazuto Yamashita, Kohji Moriishi
    JOURNAL OF VIROLOGY 88(22) 13352-13366 2014年11月  査読有り
    Equine hepacivirus (EHcV) has been identified as a closely related homologue of hepatitis C virus (HCV) in the United States, the United Kingdom, and Germany, but not in Asian countries. In this study, we genetically and serologically screened 31 serum samples obtained from Japanese-born domestic horses for EHcV infection and subsequently identified 11 PCR-positive and 7 seropositive serum samples. We determined the full sequence of the EHcV genome, including the 3' untranslated region (UTR), which had previously not been completely revealed. The polyprotein of a Japanese EHcV strain showed approximately 95% homology to those of the reported strains. HCV-like cis-acting RNA elements, including the stem-loop structures of the 3' UTR and kissing-loop interaction were deduced from regions around both UTRs of the EHcV genome. A comparison of the EHcV and HCV core proteins revealed that Ile(190) and Phe(191) of the EHcV core protein could be important for cleavage of the core protein by signal peptide peptidase (SPP) and were replaced with Ala and Leu, respectively, which inhibited intramembrane cleavage of the EHcV core protein. The loss-of-function mutant of SPP abrogated intramembrane cleavage of the EHcV core protein and bound EHcV core protein, suggesting that the EHcV core protein may be cleaved by SPP to become a mature form. The wild-type EHcV core protein, but not the SPP-resistant mutant, was localized on lipid droplets and partially on the lipid raft-like membrane in a manner similar to that of the HCV core protein. These results suggest that EHcV may conserve the genetic and biological properties of HCV. IMPORTANCE EHcV, which shows the highest amino acid or nucleotide homology to HCV among hepaciviruses, was previously reported to infect horses from Western, but not Asian, countries. We herein report EHcV infection in Japanese-born horses. In this study, HCV-like RNA secondary structures around both UTRs were predicted by determining the whole-genome sequence of EHcV. Our results also suggest that the EHcV core protein is cleaved by SPP to become a mature form and then is localized on lipid droplets and partially on lipid raft-like membranes in a manner similar to that of the HCV core protein. Hence, EHcV was identified as a closely related homologue of HCV based on its genetic structure as well as its biological properties. A clearer understanding of the epidemiology, genetic structure, and infection mechanism of EHcV will assist in elucidating the evolution of hepaciviruses as well as the development of surrogate models for the study of HCV.
  • Kazuhiko Ochiai, Shin-ichi Hayama, Sachie Nakiri, Setsuko Nakanishi, Naomi Ishii, Taiki Uno, Takuya Kato, Fumiharu Konno, Yoshi Kawamoto, Shuichi Tsuchida, Toshinori Omi
    SCIENTIFIC REPORTS 4 5793 2014年7月  査読有り筆頭著者
    In April 2012 we carried out a 1-year hematological study on a population of wild Japanese monkeys inhabiting the forest area of Fukushima City. This area is located 70 km from the Fukushima Daiichi Nuclear Power Plant (NPP), which released a large amount of radioactive material into the environment following the Great East Japan Earthquake of 2011. For comparison, we examined monkeys inhabiting the Shimokita Peninsula in Aomori Prefecture, located approximately 400 km from the NPP. Total muscle cesium concentration in Fukushima monkeys was in the range of 78-1778 Bq/kg, whereas the level of cesium was below the detection limit in all Shimokita monkeys. Compared with Shimokita monkeys, Fukushima monkeys had significantly low white and red blood cell counts, hemoglobin, and hematocrit, and the white blood cell count in immature monkeys showed a significant negative correlation with muscle cesium concentration. These results suggest that the exposure to some form of radioactive material contributed to hematological changes in Fukushima monkeys.
  • J. Guo, D. Yang, H. Okamura, J. Teramachi, K. Ochiai, L. Qiu, T. Haneji
    JOURNAL OF DENTAL RESEARCH 93(5) 508-513 2014年5月  査読有り
    Porphyromonas endodontalis and its main virulence factor, lipopolysaccharide (LPS), are associated with the development of periapical diseases and alveolar bone loss. Calcium hydroxide is commonly used for endodontic therapy. However, the effects of calcium hydroxide on the virulence of P. endodontalis LPS and the mechanism of P. endodontalis LPS-induced bone destruction are not clear. Calcium hydroxide rescued the P. endodontalis LPS-suppressed viability of MC3T3-E1 cells and activity of nuclear factor-kappa B (NF-kappa B) in these cells, resulting in the reduced expression of interleukin-6 and tumor necrosis factor-alpha. In addition, calcium hydroxide inhibited P. endodontalis LPS-induced osteoclastogenesis by decreasing the activities of NF-kappa B, p38, and ERK1/2 and the expression of nuclear factor of activated T-cell cytoplasmic 1 in RAW264.7 cells. Calcium hydroxide also rescued the P. endodontalis LPS-induced osteoclastogenesis and bone destruction in mouse calvaria. Taken together, our present results indicate that calcium hydroxide suppressed bone destruction by attenuating the virulence of P. endodontalis LPS on bone cells.
  • Masami Watanabe, Masakiyo Sakaguchi, Rie Kinoshita, Haruki Kaku, Yuichi Ariyoshi, Hideo Ueki, Ryuta Tanimoto, Shin Ebara, Kazuhiko Ochiai, Junichiro Futami, Shun-Ai Li, Peng Huang, Yasutomo Nasu, Nam-Ho Huh, Hiromi Kumon
    ONCOLOGY REPORTS 31(3) 1089-1095 2014年3月  査読有り
    Gene expression systems with various promoters, including the cytomegalovirus (CMV) promoter, have been developed to increase the gene expression in a variety of normal and cancer cells. In particular, in the clinical trials of cancer gene therapy, a more efficient and robust gene expression system is required to achieve sufficient therapeutic outcomes. By inserting the triple translational enhancer sequences of human telomerase reverse transcriptase (hTERT), Simian virus 40 (SV40) and CMV downstream of the sequence of the BGH polyA, we were able to develop a novel gene expression system that significantly enhances the expression of the genes of interest. We termed this novel gene expression cassette the super gene expression (SGE) system, and herein verify the utility of the SGE cassette for a replication-deficient adenoviral vector. We newly developed an adenoviral vector expressing the tumor suppressor, reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3), based on the CMV promoter-driven SGE system (Ad-SGE-REIC) and compared the therapeutic utility of Ad-SGE-REIC with that of the conventional adenoviral vectors (Ad-CMV-REIC or Ad-CAG-REIC). The results demonstrated that the CMV promoter-SGE system allows for more potent gene expression, and that the Ad-SGE-REIC is superior to conventional adenoviral systems in terms of the REIC protein expression and therapeutic effects. Since the SGE cassette can be applied for the expression of various therapeutic genes using various vector systems, we believe that this novel system will become an innovative tool in the field of gene expression and gene therapy.
  • Noriyuki Horiuchi, Naoyuki Aihara, Hiroshi Mizutani, Shinichi Kousaka, Tsuneyuki Nagafuchi, Mariko Ochiai, Kazuhiko Ochiai, Yoshiyasu Kobayashi, Hidefumi Furuoka, Tetsuo Asai, Koji Oishi
    JOURNAL OF VETERINARY MEDICAL SCIENCE 76(2) 243-248 2014年2月  査読有り
    We describe a case of human Becker muscular dystrophy (BMD)-like myopathy that was characterized by the declined stainability of dystrophin at sarcolemma in a pig and the immunostaining for dystrophin on the formalin-fixed, paraffin-embedded (FFPE) tissue. The present case was found in a meat inspection center. The pig looked appeared healthy at the ante-mortem inspection. Muscular abnormalities were detected after carcass dressing as pale, discolored skeletal muscles with prominent fat infiltrations and considered so-called "fatty muscular dystrophy". Microscopic examination revealed following characteristics: diffused fat infiltration into the skeletal muscle and degeneration and regeneration of the remaining skeletal muscle fibers. Any lesions that were suspected of neurogenic atrophy, traumatic muscular degeneration, glycogen storage disease or other porcine muscular disorders were not observed. The immunostaining for dystrophin was conducted and confirmed to be applicable on FFPE porcine muscular tissues and revealed diminished stainability of dystrophin at the sarcolemma in the present case. Based on the histological observations and immunostaining results, the present case was diagnosed with BMD-like myopathy associated with dystrophin abnormality in a pig. Although the genetic properties were not clear, the present BMD-like myopathy implied the occurrence of dystrophinopathy in pigs. To the best of our knowledge, this is the first report of a natural case of myopathy associated with dystrophin abnormalities in a pig.
  • Makoto Ishikawa, Kaya Yoshida, Hirohiko Okamura, Kazuhiko Ochiai, Haruna Takamura, Natsumi Fujiwara, Kazumi Ozaki
    BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR BASIS OF DISEASE 1832(12) 2035-2043 2013年12月  査読有り
    Periodontal diseases are common chronic inflammatory disorders that result in the destruction of tissues around teeth. Many clinical studies suggest that periodontal diseases are risk factors for insulin resistance and diabetic mellitus development. However, the molecular mechanisms by which periodontal diseases regulate the progress of diabetes mellitus remain unknown. In this study, we investigated whether Porphyromonas gingivalis (P.g.), a major pathogen of periodontal diseases, present in the oral cavity, moves to the liver and affects hepatic glycogen synthesis. SNAP26b-tagged P.g. (SNAP-P.g.) was introduced into the oral cavity to induce periodontal disease in 4-week old female Balb/c mice. SNAP-P.g. was detected in the liver extracted from SNAP-P.g.-treated mice using nested PCR analysis. High blood glucose levels tended to promote SNAP-P.g. translocation from the oral cavity to the liver in mice. Periodic acid-Schiff staining suggested that hepatic glycogen synthesis decreased in SNAP-P.g.-treated mice. SNAP-P.g. was also internalized into the human hepatoma cell line HepG2, and this attenuated the phosphorylation of insulin receptor substrate (IRS)-1, Akt and glycogen synthase kinase-3 beta induced by insulin. Insulin-induced glycogen synthesis was suppressed by SNAP-P.g. in HepG2 cells. Our results suggest that P.g. translocation from the oral cavity to the liver may contribute to the progress of diabetes mellitus by influencing hepatic glycogenesis. (C) 2013 Elsevier B.V. All rights reserved.
  • Kazuhiko Ochiai, Masami Watanabe, Daigo Azakami, Masaki Michishita, Yasunaga Yoshikawa, Chihiro Udagawa, Pornphimon Metheenukul, Thippayarat Chahomchuen, Hiroshi Aoki, Hiromi Kumon, Masami Morimatsu, Toshinori Omi
    VETERINARY JOURNAL 197(3) 769-775 2013年9月  査読有り筆頭著者責任著者
    REIC/Dkk-3, a member of the human Dickkopf (Dkk) family, plays a role as a suppressor of growth in several human cancers. In this study, the tumour suppression function of canine REIC/Dkk-3 was investigated. The full-length open reading frame of the canine REIC/Dkk-3 homologue was cloned and the tissue distribution of REIC/Dkk-3 mRNA was determined, along with the subcellular localisation of the REIC/Dkk-3 protein in canine cancer cell lines. Expression of REIC/Dkk-3 was lower in mammary gland tumours and in canine mammary carcinoma cell lines than in normal mammary gland tissue. Overexpression of REIC/Dkk-3 induced apoptosis in canine mammary carcinoma cell lines. These results show that expression of REIC/Dkk-3 is downregulated in canine mammary tumours and that one of the functions of this gene is induction of apoptosis. (C) 2013 Elsevier Ltd. All rights reserved.
  • Shin-ichi Hayama, Sachie Nakiri, Setsuko Nakanishi, Naomi Ishii, Taiki Uno, Takuya Kato, Fumiharu Konno, Yoshi Kawamoto, Shuichi Tsuchida, Kazuhiko Ochiai, Toshinori Omi
    PLOS ONE 8(7) e68530 2013年7月  査読有り
    Following the massive earthquake that struck eastern Japan on March 11, 2011, a nuclear reactor core meltdown occurred at the Fukushima Daiichi Nuclear Power Plant, operated by Tokyo Electric Power Company, and was followed by the release of large amounts of radioactive materials. The objective of this study was to measure the concentration of radiocesium Cs-134 and Cs-137 in the muscle of Japanese monkeys (Macaca fuscata) inhabiting the forest area of Fukushima City and to determine the change in concentration over time as well as the relationship with the level of soil contamination. Cesium concentrations in the muscle of monkeys captured at locations with 100,000-300,000 Bq/m(2) were 6,000-25,000 Bq/kg in April 2011 and decreased over 3 months to around 1,000 Bq/kg. However, the concentration increased again to 2,000-3,000 Bq/kg in some animals during and after December 2011 before returning to 1,000 Bq/kg in April 2012, after which it remained relatively constant. This pattern of change in muscle radiocesium concentration was similar to that of the change in radiocesium concentration in atmospheric fallout. Moreover, the monkeys feed on winter buds and the cambium layer of tree bark potentially containing higher concentrations of radiocesium than that in the diet during the rest of the year. The muscle radiocesium concentration in the monkeys related significantly with the level of soil contamination at the capture locations.
  • Yasunaga Yoshikawa, Kazuhiko Ochiai, Masami Morimatsu, Yu Suzuki, Seiichi Wada, Takahiro Taoda, Satomi Iwai, Seishiro Chikazawa, Koichi Orino, Kiyotaka Watanabe
    PLOS ONE 7(10) e45833 2012年10月  査読有り
    Mammary tumors are the most common tumor type in both human and canine females. Mutations in the breast cancer susceptibility gene, BRCA2, have been found in most cases of inherited human breast cancer. Similarly, the canine BRCA2 gene locus has been associated with mammary tumors in female dogs. However, deleterious mutations in canine BRCA2 have not been reported, thus far. The BRCA2 protein is involved in homologous recombination repair via its interaction with RAD51 recombinase, an interaction mediated by 8 BRC repeats. These repeats are 26-amino acid, conserved motifs in mammalian BRCA2. Previous structural analyses of cancer-associated mutations affecting the BRC repeats have shown that the weakening of RAD51's affinity for even 1 repeat is sufficient to increase breast cancer susceptibility. In this study, we focused on 2 previously reported canine BRCA2 mutations (T1425P and K1435R) in BRC repeat 3 (BRC3), derived from mammary tumor samples. These mutations affected the interaction of canine BRC3 with RAD51, and were considered deleterious. Two BRC3 mutations (K1440R and K1440E), reported in human breast cancer patients, occur at amino acids corresponding to those of the K1435R mutation in dogs. These mutations affected the interaction of canine BRC3 with RAD51, and may also be considered deleterious. The two BRC3 mutations and a substitution (T1430P), corresponding to T1425P in canine BRCA2, were examined for their effects on human BRC3 function and the results were compared between species. The corresponding mutations and the substitution showed similar results in both human and canine BRC3. Therefore, canine BRCA2 may be a good model for studying human breast cancer caused by BRCA2 mutations.
  • Michihiro Nakamura, Aziz Awaad, Koichiro Hayashi, Kazuhiko Ochiai, Kazunori Ishimura
    CHEMISTRY OF MATERIALS 24(19) 3772-3779 2012年10月  査読有り
    Propidium iodide (PI) is a fluorescent nucleic acid dye that contains iodide molecules. Thiol-organosilica particles that were internally functionalized with PI (PI particles) were prepared for X-ray computed tomography (CT) and multicolor fluorescence imaging. PI particles of various sizes were synthesized in a one-pot process. The particles showed unique fluorescent signals and X-ray absorption, with enhancement of the fluorescence intensity of PI located inside organosilica particles. PI particles had multicolor fluorescence, including original fluorescence and near-infrared (NIR). Orally administered PI particles were observed in the gastrointestinal tract (GIT) using fluorescent imaging devices and X-ray CT. An in vivo fluorescence imaging system could detect the NIR fluorescence of PI particles in the GIT specifically. Multipurpose zoom fluorescence microscopy was. used to noninvasively visualize the real-time passage of particles and the movement of the GIT. The passage and distribution of particles over time in the GIT were demonstrated using X-ray CT. A correlation analysis between the fluorescent and X-ray CT data demonstrated the characteristics, limitations, and novel potential for noninvasive functional GIT dual modal imaging.
  • Kaya Yoshida, Hirohiko Okamura, Kazuhiko Ochiai, Yumi Hoshino, Tatsuji Haneji, Masami Yoshioka, Daisuke Hinode, Hideo Yoshida
    Molecular and Cellular Endocrinology 361(1-2) 99-105 2012年9月25日  査読有り
    Double-stranded RNA-dependent protein kinase (PKR) is involved in various cellular functions. We previously reported that PKR regulates osteoblast differentiation, but the specific mechanisms by which this occurs remain unclear. In this study, we investigated the role of PKR in Glycogen synthase kinase 3β (GSK-3β) regulation of osteoblast differentiation. Lithium chloride (LiCl), a GSK-3β inhibitor, increased GSK-3β phosphorylation in MC3T3-E1 and MG-63 cells. LiCl also inhibited Runx2 and expression of its regulated genes, causing inhibition of Alkaline phosphatase activity and mineralization. LiCl injection to the calvaria in mice suppressed bone formation. Further, GSK-3β phosphorylation was increased in osteoblasts, by Akt-independent mechanisms, in which PKR was constitutively inactivated. A PKR inhibitor, 2-aminopurine, also induced GSK-3β phosphorylation in MC3T3-E1 and MG-63 cells. Further, Runx2 and its regulated genes were inhibited in PKR-inactivated osteoblasts, and differentiation was suppressed through a β-catenin-independent pathway. PKR positively regulates the differentiation of osteoblasts by mediating GSK-3β activity through a β-catenin-independent pathway. © 2012 Elsevier Ireland Ltd.
  • Hideo Ueki, Masami Watanabe, Haruki Kaku, Peng Huang, Shun-Ai Li, Kazuhiko Ochiai, Takeshi Hirata, Hirofumi Noguchi, Hiroshi Yamada, Kohji Takei, Yasutomo Nasu, Yuji Kashiwakura, Hiromi Kumon
    INTERNATIONAL JOURNAL OF ONCOLOGY 41(1) 135-140 2012年7月  査読有り
    A novel transcriptional system was developed that can robustly enhance cancer-specific gene expression. In the system, hTERT promoter-driven gene expression was enhanced by an advanced two-step transcriptional amplification (TSTA). This construct was used to develop a novel system for detection of bladder cancer cells. The current study evaluated the advanced TSTA system by examining the cancer-specific gene transcription in various bladder cancer cell lines. The system significantly enhanced cancer-specific luciferase gene expression in the bladder cancer cell lines in comparison to the previous expression system of one-step or conventional TSTA. The fold gain of the enhancement was significantly correlated to the telomerase activity of the cell lines. A green fluorescent protein (GFP) gene encoding plasmid vector was constructed where hTERT promoter-driving transcription is enhanced by the advanced TSTA to utilize the system for the imaging and detection of viable bladder cancer cells. The advanced TSTA-hTERT-GFP plasmid successfully induced cancer-specific gene expression, showing robust GFP expression in human bladder cancer cell lines, but no visible GFP expression in normal bladder urothelial cells. The control GFP plasm id with a CMV promoter yielded GFP expression in both normal bladder cells and cancer cells. The advanced TSTA-hTERT-GFP plasmid allowed selective visualization of viable human bladder cancer cells in mixed cell culture containing 10- and 100-fold more normal bladder urothelial cells. These findings indicate that the advanced TSTA-hTERT expressional system is a valuable tool for detecting viable bladder cancer cells. The current system can be applied for in vitro detection of bladder cancer cells in urine and other types of cancer cells disseminated in vivo.
  • Keiichiro Kawauchi, Masami Watanabe, Haruki Kaku, Peng Huang, Kasumi Sasaki, Masakiyo Sakaguchi, Kazuhiko Ochiai, Nam-ho Huh, Yasutomo Nasu, Hiromi Kumon
    ACTA MEDICA OKAYAMA 66(1) 7-16 2012年2月  査読有り
    The preclinical safety and therapeutic efficacy of adenoviral vectors that express the REIC/Dkk-3 tumor suppressor gene (Ad-REIC) was examined for use in prostate cancer gene therapy. The Ad-human (h) and mouse (m) REIC were previously demonstrated to induce strong anti-cancer effects in vitro and in vivo, and we herein report the results of two in vivo studies. First, intra-tumor Ad-hREIC administration was examined for toxicity and therapeutic effects in a subcutaneous tumor model using the PC3 prostate cancer cell line. Second, intra-prostatic Ad-mREIC administration was tested for toxicity in normal mice. The whole-body and spleen weights, hematological and serum chemistry parameters, and histological evaluation of tissues from throughout the body were analyzed. Both experiments indicated that there was no significant difference in the examined parameters between the Ad-REIC-treated group and the control (PBS- or Ad-LacZ-treated) group. In the in vitro analysis using PC3 cells, a significant apoptotic effect was observed after Ad-hREIC treatment. Confirming this observation, the robust anti-tumor efficacy of Ad-hREIC was demonstrated in the in vivo subcutaneous prostate cancer model. Based on the results of these preclinical experiments, we consider the adenovirus-mediated REIC/Dkk-3 in situ gene therapy to be safe and useful for the clinical treatment of prostate cancer.
  • Yasunaga Yoshikawa, Masami Morimatsu, Kazuhiko Ochiai, Kento Okuda, Takahiro Taoda, Seishiro Chikazawa, Asako Shimamura, Toshinori Omi, Makoto Bonkobara, Koichi Orino, Kiyotaka Watanabe
    BMC Research Notes 5 173 2012年  査読有り
    Background: Mammary tumors are the most common tumor type in both human and canine females. In women, carriers of mutations in BRCA2, a tumor suppressor gene product, have a higher risk of breast cancer. Canine BRCA2 has also been suggested to have a relationship with mammary tumors. However, clearly deleterious BRCA2 mutations have not been identified in any canine mammary tumors, as appropriate methods to detect mutations or a consensus BRCA2 sequence have not been reported. Findings. For amplification and sequencing of BRCA2, we designed 14 and 20 PCR primer sets corresponding to the BRCA2 open reading frame (ORF) and all 27 exons, respectively, including exon-intron boundaries of the canine BRCA2 regions, respectively. To define the consensus canine BRCA2 ORF sequence, we used established methods to sequence the full-length canine BRCA2 ORF sequence from two ovaries and a testis obtained from individual healthy mongrel dogs and partially sequence BRCA2 genomic sequences in 20-56 tumor-free dogs, each aged over 6 years. Subsequently, we compared these sequences and seven previously reported sequences, and defined the most common base sequences as the consensus canine BRCA2 ORF sequence. Moreover, we established a detection method for identifying splicing variants. Unexpectedly, we also identified novel splicing variants in normal testes during establishment of these methods. Conclusions: The present analysis methods for determining the BRCA2 base sequence and for detecting BRCA2 splicing variants and the BRCA2 ORF consensus sequence are useful for better understanding the relationship between canine BRCA2 mutation status and cancer risk. © 2010 Yoshikawa et al licensee BioMed Central Ltd.
  • K. Ochiai, Y. Yoshikawa, T. Oonuma, Y. Tomioka, K. Hashizume, M. Morimatsu
    VETERINARY JOURNAL 190(2) 293-295 2011年11月  査読有り筆頭著者
    In humans, mutations in the gene for the breast cancer susceptibility protein BRCA2 affect its interactions with the recombinase RAD51 and are associated with an increased risk of cancer. This interaction occurs through a series of eight BRC repeat sequences in BRCA2. A mammalian two-hybrid assay using individual BRC repeats demonstrated that BRC6 did not bind to RAD51, whereas there was strong (BRC1, 2 and 4), intermediate (BRC8), or weak (BRC3, 5 and 7) binding of other BRC repeats to RAD51. In serial deletion mutation experiments, binding strengths were increased when the C-terminal BRC repeat was removed from BRC1-8, BRC1-5 and BRC1-3. These results may provide an insight into the effects of missense or truncation mutations in BRCA2 in canine tumours. (C) 2010 Elsevier Ltd. All rights reserved.
  • Masami Watanabe, Hideo Ueki, Kazuhiko Ochiai, Peng Huang, Yasuyuki Kobayashi, Yasutomo Nasu, Katsumi Sasaki, Haruki Kaku, Yuji Kashiwakura, Hiromi Kumon
    ONCOLOGY REPORTS 26(4) 769-775 2011年10月  査読有り
    The two-step transcriptional amplification (TSTA) system was previously reported to enhance the tissue-specific gene expression driven by weak promoters, but the enhancement of the gene expression is limited to use in in vitro and in vivo experimental situations. To achieve robust tissue-specific gene expression using the TSTA system, we developed an advanced TSTA system which includes polyglutamines and rat glucocorticoid receptor sequences between the GAL4 and VP16 sequences in the region of the first step of transcription. We evaluated the advanced TSTA system as a method to enhance the human telomerase reverse transcriptase (hTERT) promoter-driving cancer-specific transcription in various cancer cell lines. As a result, the advanced TSTA enhanced cancer-specific luciferase gene expression in all of the examined cancer cell lines, when compared with both the one-step and conventional TSTA systems (an similar to 6- and similar to 17-fold enhancement, respectively). Notably, the enhancement of the hTERT driven expression by the conventional TSTA system was modest and even inferior to the one-step system in several cancer cell lines. We then constructed a luciferase gene encoding the adeno-associated virus vector in which the hTERT promoter-mediated expression was driven by the advanced TSTA or control systems. In an orthotopic liver tumor model, mice were treated with the vector via tail vein injection. An optical imaging device was used to visualize the in vivo luciferase expression in the orthotopic tumor. The advanced TSTA system significantly enhanced the luciferase expression compared with the one-step and conventional TSTA systems (18.0+/-1.0- and 15.9+/-0.85-fold gain, respectively). Therefore, the advanced TSTA system significantly improves hTERT-dependent cancer-specific gene expression both in vitro and in vivo when compared with the previous systems. Since the advanced TSTA method can also be applied to other site-specific gene expression systems using tissue-specific promoters, this approach is expected to become a valuable tool enabling in vivo site-specific targeting in the field of gene therapy and molecular imaging.
  • Kazuhiko Ochiai, Masami Watanabe, Hideo Ueki, Peng Huang, Yasuyuki Fujii, Yasutomo Nasu, Hirofumi Noguchi, Takeshi Hirata, Masakiyo Sakaguchi, Nam-ho Huh, Yuji Kashiwakura, Haruki Kaku, Hiromi Kumon
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 412(2) 391-395 2011年8月  査読有り筆頭著者
    REIC/Dkk-3 is a member of the Dickkopf family proteins known as Wnt-antagonists, and REIC/Dkk-3 expression is downregulated in a broad range of cancer types. REIC/Dkk-3 acts as a tumor suppressor in multiple cancer cell lines by inducing apoptosis through endoplasmic reticulum (ER) stress signaling. However, the intracellular interaction partners of REIC/Dkk-3 have not been fully elucidated. By employing yeast two-hybrid screening, we identified the human dynein light chain, Tctex-1, as a novel interaction partner of REIC/Dkk-3. We further disclosed that the interaction involves the 136-157 amino acid region of REIC/Dkk-3 by using the mammalian two-hybrid system. Interestingly, this binding region of REIC/Dkk-3 with Tctex-1 contains an amino acid sequence motif [-(E) under bar -X-(G) under bar-(R) under bar-(R) under bar -X-(H) under bar-] which was previously reported as the Tctex-1 binding domain of dynein intermediate chain (DIC). Immunocytochemistry demonstrated that both REIC/Dkk-3 and Tctex-1 were localized around the ER of human fibroblasts, and the similar distribution pattern of the proteins suggests that their interaction occurs around the ER. This is the first study showing the interaction of a Dickkopf family protein with a dynein motor complex protein. The link between REIC/Dkk-3 and Tctex-1 may be of significance for understanding the molecular functions of the proteins in ER stress signaling and intracellular dynein motor dynamics, respectively. (C) 2011 Elsevier Inc. All rights reserved.
  • Kazuhiko Ochiai, Yasunaga Yoshikawa, Kumiko Yoshimatsu, Toshina Oonuma, Yukiko Tomioka, Eichi Takeda, Jiro Arikawa, Katsumi Mominoki, Toshinori Omi, Kazuyoshi Hashizume, Masami Morimatsu
    FEBS LETTERS 585(12) 1771-1777 2011年6月  査読有り筆頭著者
    The breast cancer susceptibility protein BRCA2 is essential for recombinational DNA repair. BRCA2 specifically binds to RAD51 via eight BRC repeat motifs and delivers RAD51 to double-stranded DNA breaks. In this study, a mammalian two-hybrid assay and competitive ELISA showed that the interaction between BRC repeat 4 (BRC4) and RAD51 was strengthened by the substitution of a single BRC4 amino acid from valine to isoleucine (V1532I). However, the cancer-associated V1532F mutant exhibited very weak interaction with RAD51. This study used a comparative analysis of BRC4 between animal species to identify V1532 as an important residue that interacts with RAD51. Structured summary of protein interactions: cRAD51 physically interacts with cRAD51 by two hybrid (View interaction) fBRC4 physically interacts with cRAD51 by two hybrid (View interaction) cBRC4 physically interacts with cRAD51 by two hybrid (View interaction) hBRC4 physically interacts with hBRC4 and hRAD51 by competition binding (View Interaction 1, 2) hBRC4 physically interacts with cRAD51 by two hybrid (View interaction) hBRC4 binds to hRAD51 by enzyme linked immunosorbent assay (View interaction) (C) 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • Tomoe Fukumura, Hiroyuki Kose, Chiyo Takeda, Yuko Kurita, Kazuhiko Ochiai, Takahisa Yamada, Kozo Matsumoto
    EXPERIMENTAL ANIMALS 60(2) 125-132 2011年4月  査読有り
    The condition of hyperglycemia results from multiple genetic and environmental factors. In recent years much progress has been made with regards to the search for candidate genes involved in the expression of various common diseases including type 2 diabetes. However less is known about the specific genetic and environmental connections that are important for the development of the disease. In the present study, we used hyperglycemic congenic rats to address this issue. When given a normal diet, two hyperglycemic QTLs (quantitative trait locus), Nidd2/of and Nidd10/of showed mild obesity and/or increased blood glucose in the oral glucose tolerance test. In a double congenic strain possessing both loci, these indices were not significantly different from those of either single congenic strain. In contrast, the double congenic strain fed a high-calorie diet showed significantly greater body weight than the single congenic strains or normoglycemic control rats. Although postprandial glucose levels of the double congenic rat were not further aggravated even on the high fat diet, it was notable that the postprandial insulin levels were drastically elevated. From these results, we constructed a novel model animal especially for the study of prediabetic hyperinsulemia, in which two QTLs and an additional dietary condition are involved. This may help to shed light on the genetic basis and gene-to-diet interaction during the early stage of type 2 diabetes.
  • Udagawa, C, Yong Hwa, C, Shito, M, Kawakami, T, Tada, N, Ochiai, K, Ishioka, K, Tsuchida, S, Omi, T
    ペット栄養学会誌 14(2) 68-75 2011年  査読有り
  • Kai Zhang, Masami Watanabe, Yuji Kashiwakura, Shun-Al Li, Kohei Edamura, Peng Huang, Ken Yamaguchi, Yasutomo Nasu, Yasuyuki Kobayashi, Masakiyo Sakaguchi, Kazuhiko Ochiai, Hiroshi Yamada, Kohji Takei, Hideo Ueki, Nam-Ho Huh, Ming Li, Haruki Kaku, Yanqun Na, Hiromi Kumon
    INTERNATIONAL JOURNAL OF ONCOLOGY 37(6) 1495-1501 2010年12月  査読有り
    The tumor suppressor REIC/Dkk-3 is a secretory protein which was originally identified to be downregulated in human immortalized cells In the present study, we investigated the expression pattern of REIC/Dkk-3 in various cell types to characterize its physiological functions We first examined the expression level of REIC/Dkk-3 in a broad range of cancer cell types and confirmed that it was significantly downregulated in all of the cell types We also examined the tissue distribution pattern in a variety of normal mouse organs Ubiquitous REIC/Dkk-3 protein expression was observed in the organs The expression was abundant in the liver, heart and brain tissue, but was absent in the spleen and peripheral blood mononuclear cells The immunohistochemical analyses revealed that the subcellular localization of REIC/Dkk-3 had a punctate pattern around the nucleus, indicating its association with secretory vesicles In cancer cells stably transfected with REIC/Dkk-3 the protein was predominantly localized to the endoplasmic reticulum (ER) under observation with confocal microscopy Because REIC/ Dkk-3 was found to be abundantly expressed in the acinar epithelial cells of the mouse prostate, we analyzed the effects of recombinant REIC/Dkk-3 protein on the acinar morphogenesis of RWPE-1 cells, which are derived from human normal prostate epithelium Statistically significant acinar growth was observed in the culture condition with 10 mu g/m1 REIC/Dkk-3 protein, implicating the soluble form m prostatic acinar development Current results suggest that REIC/Dkk-3 may play a role in regulating the morphological process of normal tissue architecture through an autocrine and/or paracrine manner
  • Jie Chen, Masami Watanabe, Peng Huang, Masakiyo Sakaguchi, Kazuhiko Ochiai, Yasutomo Nasu, Mamoru Ouchida, Nam-Ho Huh, Kenji Shimizu, Yuji Kashiwakura, Haruki Kaku, Hiromi Kumon
    INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 24(6) 789-794 2009年12月  査読有り
    The reduced expression in immortalized cells (REIC)/Dickkopf (Dkk)-3. a member of the Dkk gene family, is a tumor suppressor in a broad range of cancers. REIC/Dkk-3 transfected stable clones of mouse prostate cancer RM9 cells (RM9-REIC) and the empty vector-transfected control clone cells (RM9-EV) were established. Clones were used to evaluate the anti-cancer effects and a proteomics analysis of REIC/Dkk-3 continuous expression was performed. The RM9-REIC cells show a feeble appearance and the cell membrane shows irregular buds known as blebs. In vitro cell proliferation was significantly suppressed in RM9-REIC clones in comparison to the control. The apoptosis assay was done under standard culture conditions and RM9-REIC showed a higher incidence of apoptosis. The RM9-EV and RM9-REIC cells were orthotopically implanted into a C57BL/6 mouse prostate. After 2 weeks, the tumor growth was significantly inhibited in RM9-REIC cells in comparison to the control. Two-dimensional gel electrophoresis was used to examine the modification of protein expression by the gene transfection. The analysis with mass spectrometry disclosed that expression of peroxiredoxin-l, GST-P1. transgelin-2, MRP-L12, ARD, GRP78 and Sorcin were increased and eEF1A-1 and cyclophilin-40 protein were decreased in RM9-REIC cells. Therefore, REIC/Dkk-3 stable transfectants show a reduction of malignancy in mouse prostate cancer RM9 cells in vitro and in vivo. The result of the proteomics analysis might provide important clues to clarify the anti-cancer molecular mechanism of REIC/Dkk-3 gene transfer.
  • Masakiyo Sakaguchi, Ken Kataoka, Fernando Abarzua, Ryuta Tanimoto, Masami Watanabe, Hitoshi Murata, Swe Swe Than, Kaoru Kurose, Yuji Kashiwakura, Kazuhiko Ochiai, Yasutomo Nasu, Hiromi Kumon, Nam-ho Huh
    JOURNAL OF BIOLOGICAL CHEMISTRY 284(21) 14236-14244 2009年5月  査読有り
    We previously showed that the tumor suppressor gene REIC/Dkk-3, when overexpressed by an adenovirus (Ad-REIC), exhibited a dramatic therapeutic effect on human cancers through a mechanism triggered by endoplasmic reticulum stress. Adenovirus vectors show no target cell specificity and thus may elicit unfavorable side effects through infection of normal cells even upon intra-tumoral injection. In this study, we examined possible effects of Ad-REIC on normal cells. We found that infection of normal human fibroblasts (NHF) did not cause apoptosis but induced production of interleukin (IL)-7. The induction was triggered by endoplasmic reticulum stress and mediated through IRE1 alpha, ASK1, p38, and IRF-1. When Ad-REIC-infected NHF were transplanted in a mixture with untreated human prostate cancer cells, the growth of the cancer cells was significantly suppressed. Injection of an IL-7 antibody partially abrogated the suppressive effect of Ad-REIC-infected NHF. These results indicate that Ad-REIC has another arm against human cancer, an indirect host-mediated effect because of overproduction of IL-7 by mis-targeted NHF, in addition to its direct effect on cancer cells.
  • Masami Watanabe, Yuji Kashiwakura, Peng Huang, Kazuhiko Ochiai, Junichiro Futami, Shun-Ai Li, Munenori Takaoka, Yasutomo Nasu, Masakiyo Sakaguchi, Nam-Ho Huh, Hiromi Kumon
    INTERNATIONAL JOURNAL OF ONCOLOGY 34(3) 657-663 2009年3月  査読有り
    The REIC/Dkk-3 gene has been reported to be a tumor suppressor and the expression is significantly down-regulated in a broad range of cancer cell types. The protein is secretory, but the physiological function remains unclear. This study demonstrated that recombinant REIC/Dkk-3 protein induced the differentiation of human CD14(+) monocytes into a novel cell type ((REIC/Dkx-3)Mo). (REIC/Dkk-3)Mo resembles immature dendritic cells generated with IL-4 and GM-CSF. Both these cell populations exhibit similar proportions of CD11c(+), CD40(+), CD86(+) and HLA-DR(+) cells and endocytic capacity, but (REIC/Dkk-3)Mo is negative for CD1a antigen. An analysis of the signal transducers and activators of transcription (STAT) pathways revealed that REIC/Dkk-3 induces phosphorylation of STAT 1 and STAT 3. Furthermore, intratumoral administration of REIC/Dkk-3 protein significantly suppressed tumor growth with CD11c(+) and CD8(+) (dendritic and killer T cell marker, respectively) cell accumulation and enhanced anticancer cytolytic activity of splenocytes. These data indicated a cytokine-like role of REIC/Dkk-3 protein in monocyte differentiation that might be exploited therapeutically.
  • K. Kawasaki, M. Watanabe, M. Sakaguchi, Y. Ogasawara, K. Ochiai, Y. Nasu, H. Doihara, Y. Kashiwakura, N-h Huh, H. Kumon, H. Date
    CANCER GENE THERAPY 16(1) 65-72 2009年1月  査読有り
    The overexpression of reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3), a tumor suppressor gene, induced apoptosis in human prostatic and testicular cancer cells. The aim of this study is to examine the potential of REIC/Dkk-3 as a therapeutic target against breast cancer. First, the in vitro apoptotic effect of Ad-REIC treatment was investigated in breast cancer cell lines and the adenovirus-mediated overexpression of REIC/Dkk-3 was thus found to lead to apoptotic cell death in a c-Jun-NH(2)-kinase (JNK) phosphorylaion-dependent manner. Moreover, an in vivo apoptotic effect and MCF/Wt tumor growth inhibition were observed in the mouse model after intratumoral Ad-REIC injection. As multidrug resistance (MDR) is a major problem in the chemotherapy of progressive breast cancer, the in vitro effects of Ad-REIC treatment were investigated in terms of the sensitivity of multidrug-resistant MCF7/ADR cells to doxorubicin and of the P-glycoprotein expression. Ad-REIC treatment in MCF7/ADR cells also downregulated P-glycoprotein expresssion through JNK activation, and sensitized its drug resistance against doxorubicin. Therefore, not only apoptosis induction but also the reversal of anticancer drug resistance was achieved using Ad-REIC. We suggest that REIC/Dkk-3 is a novel target for breast cancer treatment and that Ad-REIC might be an attractive agent against drug-resistant cancer in combination with conventional antineoplastic agents.
  • Yuji Kashiwakura, Kazuhiko Ochiai, Masami Watanabe, Fernando Abarzua, Masakiyo Sakaguchi, Munenori Takaoka, Ryuta Tanimoto, Yasutomo Nasu, Nam-ho Hub, Hiromi Kumon
    CANCER RESEARCH 68(20) 8333-8341 2008年10月  査読有り
    REIC/Dickkopf-3 (Dkk-3), a tumor suppressor gene, has been investigated in gene therapy studies. Our previous study suggested that REIC/Dkk-3-induced apoptosis mainly resulted from phosphorylation of c-Jun-NH2 kinase (JNK) in prostate cancer cells. However, the precise mechanisms, especially the molecular mechanisms regulating JNK phosphorylation, remain unclear. In this study, we investigated the mechanisms participating in JNK phosphorylation in the context of a refractory cancer disease, malignant mesothelioma (MM). Adenovirus-mediated overexpression of REIC/Dkk-3 induced apoptosis mainly through JNK activation in immortalized MM cells (211H cells). Interestingly, transcriptional down-regulation of inhibition of differentiation-1 (Id-1) was detected in REIC/Dkk-3-overexpressed 211H cells. Moreover, restoration of Id-1 expression antagonized REIC/Dkk-3-induced JNK phosphorylation and apoptosis. Mutagenesis experiments with the 2.1-kb human Id-1 promoter revealed that activating transcription factor 3 (ATF3) and Smad interaction, with their respective binding motifs, was essential for REIC/Dkk-3-mediated suppression of Id-1 promoter activity. ATF3 activation was probably induced by endoplasmic reticulum stress. Finally, we showed strong antitumor effects from REIC/Dkk-3 gene transfer into the pleural cavity in an orthotopic MM mouse model. Relative to control tumor tissue, REIC/Dkk-3-treated tumor tissue showed down-regulated expression of Id-1 mRNA, enhanced expression of phosphorylated JNK, and an increased number of apoptotic cells. In summary, we first showed that both ATF3 and Smad were crucially and synergistically involved in down-regulation of Id-1, which regulated JNK phosphorylation in REIC/Dkk-3-induced apoptosis. Thus, gene therapy with REIC/Dkk-3 may be a promising therapeutic tool for MM. [Cancer Res 2008;68(20):8333-41]
  • Fernando Abarzua, Yuji Kashiwakura, Munenori Takaoka, Masami Watanabe, Kazuhiko Ochiai, Masakiyo Sakaguchi, Takao Iwawaki, Ryuta Tanimoto, Yasutomo Nasu, Nam-ho Huh, Hiromi Kumon
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 375(4) 614-618 2008年10月  査読有り
    Overexpression of REIC/Dkk-3 (a tumor suppressor gene) induces cancer cell apoptosis through endoplasmic reticulum (ER) stress. Therefore, the identification of the portion of REIC/Dkk-3 that causes ER stress may be essential for the development of cancer treatment based on REIC/Dkk-3. Here, we made several truncated forms of REIC/Dkk-3 and investigated their therapeutic potentials against prostate cancer. Among three truncated forms, a variant comprising the N-terminal 78 amino acid region of REIC/Dkk-3 ((1-78) REIC/Dkk-3) most strongly induced ER stress and apoptosis in human prostate cancer cells (PC3). For in vivo gene expression, we coupled a biodegradable polymer with naked DNA, which attained robust trans-gene expression in PC3-derived subcutaneous tumor. In therapeutic experiments, we demonstrated that multiple direct injections of polymer-conjugated (1-78)REIC/Dkk-3 plasmid provoke ER stress and significantly reduced the subcutaneous tumor volume compared with the control group. We suggest this non-viral strategy may be an effective alternative to viral gene therapy. (C) 2008 Elsevier Inc. All rights reserved.
  • Yasunaga Yoshikawa, Masami Morimatsu, Kazuhiko Ochiai, Masashi Nagano, Yukiko Tomioka, Nobuo Sasaki, Kazuyoshi Hashizume, Toshihiko Iwanaga
    AMERICAN JOURNAL OF VETERINARY RESEARCH 69(10) 1323-1328 2008年10月  査読有り
    Objective-To establish novel polymorphic markers for analysis of loss of heterozygosity (LOH), so as to study the possible involvement of BRCA2 in mammary tumors obtained from dogs. Sample Population-Blood samples, mammary gland specimens, or mammary tumors from 3 tumor-bearing dogs and 10 tumor-free dogs. Procedures-Nucleotide sequence analysis was performed with a DNA autosequencer. Loss of heterozygosity analysis was performed for markers established in the present study. The expression level of canine BRCA2 was quantified by real-time PCR analysis. Results-3 novel microsatellite markers with high heterozygosity rates (> 50%) were established, and the previously reported marker for canine BRCA2 gene locus was improved. These markers were used for the analysis of DNA from formalin-fixed and paraffin-embedded samples. By use of these markers, LOH in canine BRCA2 was identified as a result of recombination. In mammary tumor DNA that corresponded to the LOH-positive dog, the level of canine BRCA2 expression was decreased compared with that of nonneoplastic mammary gland tissue; the open reading frame contained 4 missense variations, 1 insertion variation, and 1 silent variation, some of which were localized to functional domains. Conclusions and Clinical Relevance-3 novel polymorphic markers were developed for LOH analysis of canine BRCA2 and identified a dog with LOH with some variations in the functional domains. These markers could be useful for assessing the relevance of BRCA2 variation in mammary tumors of dogs.
  • Toshina Oonuma, Masami Morimatsu, Kazuhiko Ochiai, Toshihiko Iwanaga, Kazuyoshi Hashizume
    JOURNAL OF VETERINARY MEDICAL SCIENCE 69(3) 279-284 2007年3月  査読有り
    Molecule possessing ankyrin-repeats induced by lipopolysaccharide (MAIL) is a nuclear I kappa B protein that is also known as interleukin-1-inducible nuclear ankyrin repeat protein and inhibitor of nuclear factor kappa B zeta (I kappa B zeta). We previously observed that MAIL-deficient mice were affected by atopic dermatitis-like skin lesions and demonstrated the importance of MAIL in the skin. In this study, we investigated MAIL expression in mouse keratinocytes. MAIL mRNA was constitutively expressed in the skin epidermis. MAIL expression was also confirmed in primary keratinocytes and the PAM212 keratinocyte cell line. The inhibitors of nuclear factor kappa B (NF kappa B)-Bay 11-7082 and the I kappa B alpha M supersuppressor-considerably downregulated MAIL expression in the keratinocytes. Immunoreactivity for NF-kappa B components was localized in the cytoplasm and nucleus of normal unstimulated keratinocytes. The expression level of MAIL in the skin did not change following lipopolysaccharide (LPS) administration to mice. Interestingly, in accordance with the in vivo findings, the MAIL expression level did not change following LPS stimulation even in primary keratinocytes; however, MAIL expression was strongly increased by interleukin-1 stimulation. These results collectively suggest that the constitutive expression of MAIL in keratinocytes is controlled, at least in part, by NF-kappa B and that there may be LPS-specific repressive mechanisms that inhibit MAIL induction.
  • Yoshikawa, Y, Morimatsu, M, Ochiai, K, Nagano, M, Yamane, Y, Tomizawa, N, Sasaki, N, Hashizume, K
    J. Vet. Med. Sci. 67(10) 1013-1017 2005年  査読有り
  • Yoshikawa, Y, Morimatsu, M, Ochiai, K, Nagano, M, Yamane, Y, Tomizawa, N, Sasaki, N, Hashizume, K
    Biomed Res. 26(3) 109-116 2005年  査読有り
  • K Ochiai, M Morimatsu, Y Yoshikawa, B Syuto, K Hashizume
    BIOMEDICAL RESEARCH-TOKYO 25(6) 269-275 2004年12月  査読有り筆頭著者
    In humans and mice, the interaction between the breast cancer susceptibility protein, BRCA2, and RAD51 recombinase is essential for DNA repair by homologous recombination, the failure of this process can predispose to cancer. Cells with mutated BRCA2 are hypersensitive to ionizing radiation (IR) and exhibit defective DNA repair. Using yeast and mammalian two-hybrid assays, we demonstrate that canine Rad51 protein interacts specifically with the C-terminus of canine Brca2. In support of the biological significance of this interaction, we found that radiation-induced focus formation of Rad51 in COS-7 cells was compromised by forced expression of the C-terminus of canine Brca2. A similar result was obtained for the murine C-terminus. These data suggest that the C-terminal domain of canine Brca2 functions to bind Rad51 and that this domain contributes to the IR-induced assembly of the Rad51 complex in vivo.
  • Koichi Ushizawa, Chandana B. Herath, Kanako Kaneyama, Satoshi Shiojima, Akira Hirasawa, Toru Takahashi, Kei Imai, Kazuhiko Ochiai, Tomoyuki Tokunaga, Yukio Tsunoda, Gozoh Tsujimoto, Kazuyoshi Hashizume
    Reproductive Biology and Endocrinology 2(2) 77 2004年11月24日  査読有り
    Background: After fertilization, embryo development involves differentiation, as well as development of the fetal body and extra-embryonic tissues until the moment of implantation. During this period various cellular and molecular changes take place with a genetic origin, e.g. the elongation of embryonic tissues, cell-cell contact between the mother and the embryo and placentation. To identify genetic profiles and search for new candidate molecules involved during this period, embryonic gene expression was analyzed with a custom designed utero-placental complementary DNA (cDNA) microarray. Methods: Bovine embryos on days 7, 14 and 21, extra-embryonic membranes on day 28 and fetuses on days 28 were collected to represent early embryo, elongating embryo, pre-implantation embryo, post-implantation extra-embryonic membrane and fetus, respectively. Gene expression at these different time points was analyzed using our cDNA microarray. Two clustering algorithms such as k-means and hierarchical clustering methods identified the expression patterns of differentially expressed genes across pre-implantation period. Novel candidate genes were confirmed by real-time RT-PCR. Results: In total, 1,773 individual genes were analyzed by complete k-means clustering. Comparison of day 7 and day 14 revealed most genes increased during this period, and a small number of genes exhibiting altered expression decreased as gestation progressed. Clustering analysis demonstrated that trophoblast-cell-specific molecules such as placental lactogens (PLs), prolactin-related proteins (PRPs), interferon-tau, and adhesion molecules apparently all play pivotal roles in the preparation needed for implantation, since their expression was remarkably enhanced during the pre-implantation period. The hierarchical clustering analysis and RT-PCR data revealed new functional roles for certain known genes (dickkopf-1, NPM, etc) as well as novel candidate genes (AW464053, AW465434, AW462349, AW485575) related to already established trophoblast-specific genes such as PLs and PRPs. Conclusions: A large number of genes in extra-embryonic membrane increased up to implantation and these profiles provide information fundamental to an understanding of extraembryonic membrane differentiation and development. Genes in significant expression suggest novel molecules in trophoblast differentiation. © 2004 Ushizawa et al licensee BioMed Central Ltd.
  • 落合和彦, 森松正美, 吉川泰永, 宇賀聡, 首藤文榮, 橋爪一善
    獣医生化学 41(2) 51-59 2004年  査読有り筆頭著者
  • BH Lee, K Yoshimatsu, A Maeda, K Ochiai, M Morimatsu, K Araki, M Ogino, S Morikawa, J Arikawa
    VIRUS RESEARCH 98(1) 83-91 2003年12月  査読有り
    We performed yeast two-hybrid screening of a human kidney cell cDNA library to study the biological role of the hantavirus nucleocapsid protein (NP). We found that Seoul virus (SEOV) and Hantaan virus (HTNV) NPs were associated with small ubiquitin-like modifier (SUMO)-1-interacting proteins PIAS 1, PIASxbeta, HIPK2, CHD3, and TTRAP, which interacted with the SUMO-1 conjugating enzyme (Ubc-9) and SUMO-1 in the yeast two-hybrid assay. Interactions between the HIPK2, CHD3, and TTRAP proteins and SEOV NP were also shown in a mammalian two-hybrid assay. However, there was no interaction between PIAS proteins and NP, which was probably due to the inhibitory effect of PIAS on transcription in the mammalian two-hybrid assay. Nevertheless, a co-expression experiment suggested the existence of a PIAS-NP interaction in the cytoplasm. The region spanning amino acids 100-125 of SEOV NP, which represents a critical region for NP-NP polymerization. was found to be responsible for the interaction with SUMO-1-related molecules in both the yeast and mammalian two-hybrid assays. These results add to the information on interactions of hantavirus NP and host cellular proteins. (C) 2003 Elsevier B.V. All rights reserved.

MISC

 33

講演・口頭発表等

 81

担当経験のある科目(授業)

 6

共同研究・競争的資金等の研究課題

 13

産業財産権

 2