研究者詳細 - 藤田　洋史

論文 - 藤田　洋史

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Harderian Gland Development and Degeneration in the Fgf10-Deficient Heterozygous Mouse. 国際誌

Shiori Ikeda, Keita Sato, Hirofumi Fujita, Hitomi Ono-Minagi, Satoru Miyaishi, Tsutomu Nohno, Hideyo Ohuchi

Journal of developmental biology 12 ( 2 ) 2024年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

The mouse Harderian gland (HG) is a secretory gland that covers the posterior portion of the eyeball, opening at the base of the nictitating membrane. The HG serves to protect the eye surface from infection with its secretions. Mice open their eyelids at about 2 weeks of age, and the development of the HG primordium mechanically opens the eye by pushing the eyeball from its rear. Therefore, when HG formation is disturbed, the eye exhibits enophthalmos (the slit-eye phenotype), and a line of Fgf10+/- heterozygous loss-of-function mice exhibits slit-eye due to the HG atrophy. However, it has not been clarified how and when HGs degenerate and atrophy in Fgf10+/- mice. In this study, we observed the HGs in embryonic (E13.5 to E19), postnatal (P0.5 to P18) and 74-week-old Fgf10+/- mice. We found that more than half of the Fgf10+/- mice had markedly degenerated HGs, often unilaterally. The degenerated HG tissue had a melanized appearance and was replaced by connective tissue, which was observed by P10. The development of HGs was delayed or disrupted in the similar proportion of Fgf10+/- embryos, as revealed via histology and the loss of HG-marker expression. In situ hybridization showed Fgf10 expression was observed in the Harderian mesenchyme in wild-type as well as in the HG-lacking heterozygote at E19. These results show that the Fgf10 haploinsufficiency causes delayed or defective HG development, often unilaterally from the unexpectedly early neonatal period.

DOI： 10.3390/jdb12020016

システイニルロイコトリエン受容体CysLTR1は破骨細胞分化と骨吸収に必須ではない

藤田洋史, 服部高子, 久保田聡

日本骨代謝学会学術集会プログラム抄録集 41回 162 - 162 2023年7月

Protrusion of KCNJ13 Gene Knockout Retinal Pigment Epithelium Due to Oxidative Stress–Induced Cell Death

Yuki Kanzaki, Hirofumi Fujita, Keita Sato, Mio Hosokawa, Hiroshi Matsumae, Yuki Morizane, Hideyo Ohuchi

Investigative Opthalmology & Visual Science 63 ( 12 ) 29 - 29 2022年11月

Cysteinyl leukotriene receptor 1 is dispensable for osteoclast differentiation and bone resorption

Hirofumi Fujita, Aoi Ando, Yohei Mizusawa, Mitsuaki Ono, Takako Hattori, Munenori Habuta, Toshitaka Oohashi, Satoshi Kubota, Hideyo Ohuchi

PLOS ONE 17 ( 11 ) e0277307 - e0277307 2022年11月

掲載種別：研究論文（学術雑誌）出版者・発行元：Public Library of Science (PLoS)

Cysteinyl leukotriene receptor 1 (CysLTR1) is a G protein-coupled receptor for the inflammatory lipid mediators cysteinyl leukotrienes, which are involved in smooth muscle constriction, vascular permeability, and macrophage chemokine release. The Cysltr1 gene encoding CysLTR1 is expressed in the macrophage lineage, including osteoclasts, and the CysLTR1 antagonist Montelukast has been shown to suppress the formation of osteoclasts. However, it currently remains unclear whether CysLTR1 is involved in osteoclast differentiation and bone loss. Therefore, to clarify the role of CysLTR1 in osteoclastogenesis and pathological bone loss, we herein generated CysLTR1 loss-of-function mutant mice by disrupting the cysltr1 gene using the CRISPR-Cas9 system. These mutant mice had a frameshift mutation resulting in a premature stop codon (Cysltr1 KO) or an in-frame mutation causing the deletion of the first extracellular loop (Cysltr1^Δ105). Bone marrow macrophages (BMM) from these mutant mice lost the intracellular flux of calcium in response to leukotriene D₄, indicating that these mutants completely lost the activity of CysLTR1 without triggering genetic compensation. However, disruption of the Cysltr1 gene did not suppress the formation of osteoclasts from BMM in vitro. We also demonstrated that the CysLTR1 antagonist Montelukast suppressed the formation of osteoclasts without functional CysLTR1. On the other hand, disruption of the Cysltr1 gene partially suppressed the formation of osteoclasts stimulated by leukotriene D₄ and did not inhibit that by glutathione, functioning as a substrate in the synthesis of cysteinyl leukotrienes. Disruption of the Cysltr1 gene did not affect ovariectomy-induced osteoporosis or lipopolysaccharide-induced bone resorption. Collectively, these results suggest that the CysLT-CysLTR1 axis is dispensable for osteoclast differentiation in vitro and pathological bone loss, while the leukotriene D₄-CysTR1 axis is sufficient to stimulate osteoclast formation. We concluded that the effects of glutathione and Montelukast on osteoclast formation were independent of CysLTR1.

DOI： 10.1371/journal.pone.0277307

Dkk3/REIC Deficiency Impairs Spermiation, Sperm Fibrous Sheath Integrity and the Sperm Motility of Mice. 国際誌

Ruizhi Xue, Wenfeng Lin, Hirofumi Fujita, Jingkai Sun, Rie Kinoshita, Kazuhiko Ochiai, Junichiro Futami, Masami Watanabe, Hideyo Ohuchi, Masakiyo Sakaguchi, Zhengyan Tang, Peng Huang, Yasutomo Nasu, Hiromi Kumon

Genes 13 ( 2 ) 2022年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

The role of Dickkopf-3 (Dkk3)/REIC (The Reduced Expression in Immortalized Cells), a Wnt-signaling inhibitor, in male reproductive physiology remains unknown thus far. To explore the functional details of Dkk3/REIC in the male reproductive process, we studied the Dkk3/REIC knock-out (KO) mouse model. By examining testicular sections and investigating the sperm characteristics (count, vitality and motility) and ultrastructure, we compared the reproductive features between Dkk3/REIC-KO and wild-type (WT) male mice. To further explore the underlying molecular mechanism, we performed RNA sequencing (RNA-seq) analysis of testicular tissues. Our results showed that spermiation failure existed in seminiferous tubules of Dkk3/REIC-KO mice, and sperm from Dkk3/REIC-KO mice exhibited inferior motility (44.09 ± 8.12% vs. 23.26 ± 10.02%, p < 0.01). The Ultrastructure examination revealed defects in the sperm fibrous sheath of KO mice. Although the average count of Dkk3/REIC-KO epididymal sperm was less than that of the wild-types (9.30 ± 0.69 vs. 8.27 ± 0.87, ×106), neither the gap (p > 0.05) nor the difference in the sperm vitality rate (72.83 ± 1.55% vs. 72.50 ± 0.71%, p > 0.05) were statistically significant. The RNA-seq and GO (Gene Oncology) enrichment results indicated that the differential genes were significantly enriched in the GO terms of cytoskeleton function, cAMP signaling and calcium ion binding. Collectively, our research demonstrates that Dkk3/REIC is involved in the process of spermiation, fibrous sheath integrity maintenance and sperm motility of mice.

DOI： 10.3390/genes13020285

UCP1 expression in the mouse adrenal gland is not upregulated by thermogenic conditions. 国際誌

Hirofumi Fujita, Munenori Habuta, Takako Hattori, Satoshi Kubota, Hiromi Kumon, Hideyo Ohuchi

Biochemical and biophysical research communications 566 184 - 189 2021年8月

記述言語：英語掲載種別：研究論文（学術雑誌）

The uncoupling protein 1 (UCP1) gene is known to be highly expressed in brown adipose tissue (BAT) that functions in thermogenesis. It has been shown that UCP1 mRNA is localized to the mouse adrenal gland, but its significance remains elusive. To explore how UCP1 expression in the adrenal gland is regulated, we generated a reporter knock-in mouse in which the GFP gene was inserted into the UCP1 locus using CRISPR-Cas9 system. Firstly, we confirmed by Western blot analysis UCP1-driven GFP protein expression in interscapular BAT of the knock-in mice kept at 4 °C. Immunohistochemistry showed that GFP protein was detected in the adrenal gland of the knock-in mice. More intense GFP expression was observed in the adrenal medulla than in the cortex of the reporter mice irrespectively of cold exposure. Immunohistochemistry using anti-UCP1 antibody, as well as Western blot analysis verified UCP1 protein expression in the wild-type adrenal medulla. These results suggest that the mouse adrenal gland is a novel organ expressing UCP1 protein and its expression is not upregulated by cold exposure.

DOI： 10.1016/j.bbrc.2021.06.013

副腎髄質におけるUCP1タンパク質発現のUCP1レポーターマウスを用いた検証(UCP1-reporter mice reveal UCP1 protein expression in the adrenal medulla)

Fujita Hirofumi, Habuta Munenori, Hattori Takako, Kubota Satoshi, Kumon Hiromi, Ohuchi Hideyo

The Journal of Physiological Sciences 71 ( Suppl.1 ) 105 - 105 2021年8月

Fgf10-CRISPR mosaic mutants demonstrate the gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung

Munenori Habuta, Akihiro Yasue, Ken-ichi T. Suzuki, Hirofumi Fujita, Keita Sato, Hitomi Kono, Ayuko Takayama, Tetsuya Bando, Satoru Miyaishi, Seiichi Oyadomari, Eiji Tanaka, Hideyo Ohuchi

PLOS ONE 15 ( 10 ) e0240333 - e0240333 2020年10月

Dkk3/REIC, an N-glycosylated Protein, Is a Physiological Endoplasmic Reticulum Stress Inducer in the Mouse Adrenal Gland.

Hirofumi Fujita, Tetsuya Bando, Seiichi Oyadomari, Kazuhiko Ochiai, Masami Watanabe, Hiromi Kumon, Hideyo Ohuchi

Acta medica Okayama 74 ( 3 ) 199 - 208 2020年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

Dickkopf 3 (Dkk3) is a secreted protein belonging to the Dkk family and encoded by the orthologous gene of REIC. Dkk3/REIC is expressed by mouse and human adrenal glands, but the understanding of its roles in this organ is still limited. To determine the functions of Dkk3 in the mouse adrenal gland, we first identified that the mouse Dkk3 protein is N-glycosylated in the adrenal gland as well as in the brain. We performed proteome analysis on adrenal glands from Dkk3-null mice, in which exons 5 and 6 of the Dkk3 gene are deleted. Twodimensional polyacrylamide gel electrophoresis of adrenal proteins from wild-type and Dkk3-null mice revealed 5 protein spots whose intensities were altered between the 2 genotypes. Mass spectrometry analysis of these spots identified binding immunoglobulin protein (BiP), an endoplasmic reticulum (ER) chaperone. To determine whether mouse Dkk3 is involved in the unfolded protein response (UPR), we carried out a reporter assay using ER-stress responsive elements. Forced expression of Dkk3 resulted in the induction of distinct levels of reporter expression, showing the UPR initiated by the ER membrane proteins of activating transcription factor 6 (ATF6) and inositol-requring enzyme 1 (IRE1). Thus, it is possible that Dkk3 is a physiological ER stressor in the mouse adrenal gland.

DOI： 10.18926/AMO/59950

KCNJ13 Gene Deletion Impairs Cell Alignment and Phagocytosis in Retinal Pigment Epithelium Derived from Human-Induced Pluripotent Stem Cells. 国際誌

Yuki Kanzaki, Hirofumi Fujita, Keita Sato, Mio Hosokawa, Hiroshi Matsumae, Fumio Shiraga, Yuki Morizane, Hideyo Ohuchi

Investigative ophthalmology & visual science 61 ( 5 ) 38 - 38 2020年5月

記述言語：英語掲載種別：研究論文（学術雑誌）

Purpose: The purpose of this study was to establish and analyze a cell model of Leber congenital amaurosis type 16 (LCA16), which is caused by mutations in the KCNJ13 gene encoding Kir7.1, an inward-rectifying potassium ion channel. Methods: The two guide RNAs specific to the target sites in the KCNJ13 gene were designed and KCNJ13 knock-out (KO) human-induced pluripotent stem cells (hiPSCs) were generated using the CRISPR/Cas9 system. The KCNJ13-KO hiPSCs were differentiated into retinal pigment epithelial cells (hiPSC-RPEs). The KCNJ13-KO in hiPSC-RPEs was confirmed by immunostaining. Phagocytic activity of hiPSC-RPEs was assessed using the uptake of fluorescently labeled porcine photoreceptor outer segments (POSs). Phagocytosis-related genes in RPE cells were assessed by quantitative polymerase chain reaction. Results: Most of the translated region of the KCNJ13 gene was deleted in the KCNJ13-KO hiPSCs by the CRISPR/Cas9 system, and this confirmed that the Kir7.1 protein was not present in RPE cells induced from the hiPSCs. Expression of RPE marker genes such as BEST1 and CRALBP was retained in the wild-type (WT) and in the KCNJ13-KO hiPSC-RPE cells. However, phagocytic activity and expression of phagocytosis-related genes in the KCNJ13-null hiPSC-RPE cells were significantly reduced compared to those of WT. Conclusions: We succeeded in generating an RPE model of LCA16 using hiPSCs. We suggest that Kir7.1 is required for phagocytosis of POSs by RPE cells and that impaired phagocytosis in the absence of Kir7.1 would be involved in the retinal degeneration found in LCA16.

DOI： 10.1167/iovs.61.5.38

Dickkopf3 (Dkk3) is required for maintaining the integrity of secretory vesicles in the mouse adrenal medulla. 査読

Habuta M, Fujita H, Sato K, Bando T, Inoue J, Kondo Y, Miyaishi S, Kumon H, Ohuchi H

Cell and tissue research 379 ( 1 ) 157 - 167 2019年10月

Localization of the ultraviolet-sensor Opn5m and its effect on myopia-related gene expression in the late-embryonic chick eye. 査読

Kato M, Sato K, Habuta M, Fujita H, Bando T, Morizane Y, Shiraga F, Miyaishi S, Ohuchi H

Biochemistry and biophysics reports 19 100665 2019年9月

Glutathione accelerates osteoclast differentiation and inflammatory bone destruction. 査読国際誌

Fujita H, Ochi M, Ono M, Aoyama E, Ogino T, Kondo Y, Ohuchi H

Free radical research 53 ( 2 ) 1 - 11 2019年3月

Metabolic abnormalities in adult T-cell leukemia/lymphoma and induction of specific leukemic cell death using photodynamic therapy. 査読

Oka T, Mizuno H, Sakata M, Fujita H, Yoshino T, Yamano Y, Utsumi K, Masujima T, Utsunomiya A

Scientific reports 8 ( 1 ) 14979 2018年10月

Expression analysis of Dickkopf-related protein 3 (Dkk3) suggests its pleiotropic roles for a secretory glycoprotein in adult mouse

Junji Inoue, Hirofumi Fujita, Tetsuya Bando, Yoichi Kondo, Hiromi Kumon, Hideyo Ohuchi

JOURNAL OF MOLECULAR HISTOLOGY 48 ( 1 ) 29 - 39 2017年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPRINGER

Dickkopf-related protein 3 (Dkk3) is the third member of the Dkk gene family and identical to the gene, whose expression was reduced in immortalized cells. Therefore, its another name is reduced expression in immortalized cells. Since the intratumoral introduction of Dkk3 inhibits tumor growth in mouse models of cancers, Dkk3 is likely a tumor suppressor gene. However, the functions of Dkk3 in vivo remain unclear. As the first step to decipher the physiological roles of this gene, we examined the expression pattern of Dkk3 in various tissues from adult mice. In situ hybridization showed that Dkk3 mRNA was detected in the brain, retina, heart, gastrointestinal tract, adrenal glands, thymus, prostate glands, seminal vesicles, testes, and ovaries in a regionally specific manner. Furthermore, we raised anti-mouse Dkk3 antibody and performed immunohistochemistry. Cytoplasmic localization of Dkk3 protein was observed in the cells of the adrenal medulla, while Dkk3 immunoreactivity was observed in the lumen of the stomach and intestine, implying that the Dkk3 protein may be secreted into the lumen of the gastrointestinal tract. These results suggest that Dkk3 has pleiotropic roles for a secretory glycoprotein that acts primarily in the gastrointestinal tract, thymus, endocrine and reproductive organs of the mouse.

DOI： 10.1007/s10735-016-9703-2

抗酸化物質グルタチオンは炎症性骨破壊を促進する

藤田洋史, 大野充昭, 青山絵理子, 大内淑代

日本骨代謝学会学術集会プログラム抄録集 34回 206 - 206 2016年7月

Phytoestrogen Suppresses Efflux of the Diagnostic Marker Protoporphyrin IX in Lung Carcinoma

Hirofumi Fujita, Keisuke Nagakawa, Hirotsugu Kobuchi, Tetsuya Ogino, Yoichi Kondo, Keiji Inoue, Taro Shuin, Toshihiko Utsumi, Kozo Utsumi, Junzo Sasaki, Hideyo Ohuchi

CANCER RESEARCH 76 ( 7 ) 1837 - 1846 2016年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER ASSOC CANCER RESEARCH

One promising method to visualize cancer cells is based on the detection of the fluorescent photosensitizer protoporphyrin IX (PpIX) synthesized from 5-aminolevulinic acid (ALA), but this method cannot be used in cancers that exhibit poor PpIX accumulation. PpIX appears to be pumped out of cancer cells by the ABC transporter G2 (ABCG2), which is associated with multidrug resistance. Genistein is a phytoestrogen that appears to competitively inhibit ABCG2 activity. Therefore, we investigated whether genistein can promote PpIX accumulation in human lung carcinoma cells. Here we report that treatment of A549 lung carcinoma cells with genistein or a specific ABCG2 inhibitor promoted ALA-mediated accumulation of PpIX by approximately 2-fold. ABCG2 depletion and overexpression studies further revealed that genistein promoted PpIX accumulation via functional repression of ABCG2. After an extended period of genistein treatment, a significant increase in PpIX accumulation was observed in A549 cells (3.7-fold) and in other cell lines. Systemic preconditioning with genistein in a mouse xenograft model of lung carcinoma resulted in a 1.8-fold increase in accumulated PpIX. Long-term genistein treatment stimulated the expression of genes encoding enzymes involved in PpIX synthesis, such as porphobilinogen deaminase, uroporphyrinogen decarboxylase, and protoporphyrinogen oxidase. Accordingly, the rate of PpIX synthesis was also accelerated by genistein pretreatment. Thus, our results suggest that genistein treatment effectively enhances ALA-induced PpIX accumulation by preventing the ABCG2-mediated efflux of PpIX from lung cancer cells and may represent a promising strategy to improve ALA-based diagnostic approaches in a broader set of malignancies.

DOI： 10.1158/0008-5472.CAN-15-1484

マウス頭蓋冠におけるリポポリサッカリドによる骨破壊とグルタチオンの効果

藤田洋史, 大野充昭, 青山絵理子, 荻野哲也, 近藤洋一, 大内淑代

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 88回・38回 [1LBA049] - [1LBA049] 2015年12月

Necrotic and apoptotic cells serve as nuclei for calcification on osteoblastic differentiation of human mesenchymal stem cells in vitro

Hirofumi Fujita, Masanao Yamamoto, Tetsuya Ogino, Hirotsugu Kobuchi, Naoko Ohmoto, Eriko Aoyama, Takashi Oka, Tohru Nakanishi, Keiji Inoue, Junzo Sasaki

CELL BIOCHEMISTRY AND FUNCTION 32 ( 1 ) 77 - 86 2014年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-BLACKWELL

A close relationship between cell death and pathological calcification has recently been reported, such as vascular calcification in atherosclerosis. However, the roles of cell death in calcification by osteoblast lineage have not been elucidated in detail. In this study, we investigated whether cell death is involved in the calcification on osteoblastic differentiation of human bone marrow mesenchymal stem cells (hMSC) under osteogenic culture in vitro. Apoptosis and necrosis occurred in an osteogenic culture of hMSC, and cell death preceded calcification. The generation of intracellular reactive oxygen species, chromatin condensation and fragmentation, and caspase-3 activation increased in this culture. A pan-caspase inhibitor (Z-VAD-FMK) and anti-oxidants (Tiron and n-acetylcysteine) inhibited osteogenic culture-induced cell death and calcification. Furthermore, calcification was significantly promoted by the addition of necrotic dead cells or its membrane fraction. Spontaneously dead cells by osteogenic culture and exogenously added necrotic cells were surrounded by calcium deposits. Induction of localized cell death by photodynamic treatment in the osteogenic culture resulted in co-localized calcification. These findings show that necrotic and apoptotic cell deaths were induced in an osteogenic culture of hMSC and indicated that both necrotic and apoptotic cells of osteoblast lineage served as nuclei for calcification on osteoblastic differentiation of hMSC in vitro. Copyright (c) 2013 John Wiley & Sons, Ltd.

DOI： 10.1002/cbf.2974

Improvement of the Efficacy of 5-aminolevulinic Acid-mediated Photodynamic Treatment in Human Oral Squamous Cell Carcinoma HSC-4

Masanao Yamamoto, Hirofumi Fujita, Naoki Katase, Keiji Inoue, Hitoshi Nagatsuka, Kozo Utsumi, Junzo Sasaki, Hideyo Ohuchi

ACTA MEDICA OKAYAMA 67 ( 3 ) 153 - 164 2013年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：OKAYAMA UNIV MED SCHOOL

Ever since protoporphyrin IX (PpIX) was discovered to accumulate preferentially in cancer cells after 5-aminolevulinic acid (ALA) treatment, photodynamic treatment or therapy (PDT) has been developed as an exciting new treatment option for cancer patients. However, the level of PpIX accumulation in oral cancer is fairly low and insufficient for PDT. Ferrochelatase (FECH) and ATP-binding cassette transporter G2 (ABCG2) are known to regulate PpIX accumulation. In addition, serum enhances PpIX export by ABCG2. We investigated here whether and how inhibitors of FECH and ABCG2 and their combination could improve PpIX accumulation and PDT efficacy in an oral cancer cell line in serum-containing medium. ABCG2 inhibitor and the combination of ABCG2 and FECH inhibitors increased PpIX in the presence of fetal bovine serum (FBS) in an oral cancer cell line. Analysis of ABCG2 gene silencing also revealed the involvement of ABCG2 in the regulation of PpIX accumulation. Inhibitors of FECH and ABCG2, and their combination increased the efficiency of ALA-PDT even in the presence of FBS. ALA-PDT-induced cell death was accompanied by apoptotic events and lipid peroxidation. These results suggest that accumulation of PpIX is determined by the activities of ABCG2 and FECH and that treatment with a combination of their inhibitors improves the efficacy of PDT for oral cancer, especially in the presence of serum.

DOI： 10.18926/AMO/50408

Mitochondrial Localization of ABC Transporter ABCG2 and Its Function in 5-Aminolevulinic Acid-Mediated Protoporphyrin IX Accumulation 査読

Hirotsugu Kobuchi, Koko Moriya, Tetsuya Ogino, Hirofumi Fujita, Keiji Inoue, Taro Shuin, Tatsuji Yasuda, Kozo Utsumi, Toshihiko Utsumi

PLOS ONE 7 ( 11 ) e50082 2012年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PUBLIC LIBRARY SCIENCE

Accumulation of protoporphyrin IX (PpIX) in malignant cells is the basis of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy. We studied the expression of proteins that possibly affect ALA-mediated PpIX accumulation, namely oligopeptide transporter-1 and -2, ferrochelatase and ATP-binding cassette transporter G2 (ABCG2), in several tumor cell lines. Among these proteins, only ABCG2 correlated negatively with ALA-mediated PpIX accumulation. Both a subcellular fractionation study and confocal laser microscopic analysis revealed that ABCG2 was distributed not only in the plasma membrane but also intracellular organelles, including mitochondria. In addition, mitochondrial ABCG2 regulated the content of ALA-mediated PpIX in mitochondria, and Ko143, a specific inhibitor of ABCG2, enhanced mitochondrial PpIX accumulation. To clarify the possible roles of mitochondrial ABCG2, we characterized stably transfected-HEK (ST-HEK) cells overexpressing ABCG2. In these ST-HEK cells, functionally active ABCG2 was detected in mitochondria, and treatment with Ko143 increased ALA-mediated mitochondrial PpIX accumulation. Moreover, the mitochondria isolated from ST-HEK cells exported doxorubicin probably through ABCG2, because the export of doxorubicin was inhibited by Ko143. The susceptibility of ABCG2 distributed in mitochondria to proteinase K, endoglycosidase H and peptide-N-glycosidase F suggested that ABCG2 in mitochondrial fraction is modified by N-glycans and trafficked through the endoplasmic reticulum and Golgi apparatus and finally localizes within the mitochondria. Thus, it was found that ABCG2 distributed in mitochondria is a functional transporter and that the mitochondrial ABCG2 regulates ALA-mediated PpIX level through PpIX export from mitochondria to the cytosol.

DOI： 10.1371/journal.pone.0050082

炎症性サイトカインによる破骨細胞分化とビタミンＥ及びその誘導体の作用

ビタミンE 研究の進歩 15 2012年

Serum-dependent export of protoporphyrin IX by ATP-binding cassette transporter G2 in T24 cells

Tetsuya Ogino, Hirotsugu Kobuchi, Kazuaki Munetomo, Hirofumi Fujita, Masanao Yamamoto, Toshihiko Utsumi, Keiji Inoue, Taro Shuin, Junzo Sasaki, Masayasu Inoue, Kozo Utsumi

MOLECULAR AND CELLULAR BIOCHEMISTRY 358 ( 1-2 ) 297 - 307 2011年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPRINGER

Accumulation of protoporphyrin IX (PpIX) in cancer cells is a basis of 5-aminolevulinic acid (ALA)-induced photodymanic therapy. We studied factors that affect PpIX accumulation in human urothelial carcinoma cell line T24, with particular emphasis on ATP-binding cassette transporter G2 (ABCG2) and serum in the medium. When the medium had no fetal bovine serum (FBS), ALA induced PpIX accumulation in a time- and ALA concentration-dependent manner. Inhibition of heme-synthesizing enzyme, ferrochelatase, by nitric oxide donor (Noc18) or deferoxamine resulted in a substantial increase in the cellular PpIX accumulation, whereas ABCG2 inhibition by fumitremorgin C or verapamil induced a slight PpIX increase. When the medium was added with FBS, cellular accumulation of PpIX stopped at a lower level with an increase of PpIX in the medium, which suggested PpIX efflux. ABCG2 inhibitors restored the cellular PpIX level to that of FBS(-) samples, whereas ferrochelatase inhibitors had little effects. Bovine serum albumin showed similar effects to FBS. Fluorescence microscopic observation revealed that inhibitors of ABC transporter affected the intracellular distribution of PpIX. These results indicated that ABCG2-mediated PpIX efflux was a major factor that prevented PpIX accumulation in cancer cells in the presence of serum. Inhibition of ABCG2 transporter system could be a new target for the improvement of photodynamic therapy.

DOI： 10.1007/s11010-011-0980-5

Effect of risedronate on osteoblast differentiation, expression of receptor activator of NF-κB ligand and apoptosis in mesenchymal stem cells. 査読

Fujita H, Kurokawa K, Ogino T, Ono M, Yamamoto M, Oka T, Nakanishi T, Kobayashi N, Tanaka N, Ogawa T, Suzaki E, Utsumi K, Sasaki J

Basic & clinical pharmacology & toxicology 109 ( 2 ) 78 - 84 2011年8月

Effect of risedronate on osteoblast differentiation, expression of receptor activator of NF-κB ligand and apoptosis in mesenchymal stem cells.

Fujita H, Kurokawa K, Ogino T, Ono M, Yamamoto M, Oka T, Nakanishi T, Kobayashi N, Tanaka N, Ogawa T, Suzaki E, Utsumi K, Sasaki J

Basic Clin Pharmacol Toxicol. 109 ( 2 ) 78 - 84 2011年8月

α-Tocopheryl succinate induces rapid and reversible phosphatidylserine externalization in histiocytic lymphoma through the caspase-independent pathway 査読

Fujita H, Shiva D, Utsumi T, Ogino T, Ogawa T, Abe K, Yasuda T, Utsumi K, Sasaki J

Mol Cell Biochem. 333 ( 1-2 ) 137 - 149 2010年1月

Mechanism of cell death by 5-aminolevulinic acid-based photodynamic action and its enhancement by ferrochelatase inhibitors in human histiocytic lymphoma cell line U937

Takashi Amo, Noriaki Kawanishi, Masataka Uchida, Hirofumi Fujita, Eri Oyanagi, Toshihiko Utsumi, Tetsuya Ogino, Keiji Inoue, Taro Shuin, Kozo Utsumi, Junzo Sasaki

CELL BIOCHEMISTRY AND FUNCTION 27 ( 8 ) 503 - 515 2009年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-BLACKWELL

Photodynamic therapy (PDT) for tumors is based oil the tumor-selective accumulation of a photosensitizer. protoporphyrin IX (PpIX), followed by irradiation with visible light However, the molecular mechanism of cell death caused by PDT has not been fully elucidated The 5-aminolevulinic acid (ALA)-based photodynamic action (PDA) was dependent oil file accumulation of PpIX, the level of which decreased rapidly by eliminating ALA from the incubation medium in human histiocytic lymphoma U937 cells PDA induced apoptosis characterized by lipid peroxidation. increase in Bak and Bax/Bcl-xL, decrease fit Bid. membrane depolarization, cytochrome c release. caspase-3 activation, phosphandylserine (PS) externalization. PDT-induced cell death seemed to occur predominantly via apoptosis through distribution of PpIX in mitochondria These cell death events were enhanced by ferrochelatase inhibitors These results indicated that ALA-based-PDA induced apoptotic cell death through a mitochondrial pathway and that ferrochelatase inhibitors enhanced the effect of PDT for tumors even at low concentrations of ALA. Copyright (C) 2009 John Wiley & Sons. Ltd

DOI： 10.1002/cbf.1603

Regulation of 5-Aminolevulinic Acid-Mediated Protoporphyrin IX Accumulation in Human Urothelial Carcinomas 査読

Keiji Inoue, Takashi Karashima, Masayuki Kamada, Taro Shuin, Atsushi Kurabayashi, Mutsuo Furihata, Hirofumi Fujita, Kozo Utsumi, Junzo Sasaki

PATHOBIOLOGY 76 ( 6 ) 303 - 314 2009年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：KARGER

Purpose: The purpose of this study was to clarify the regulatory mechanism of protoporphyrin IX (PpIX) synthesis mediated by 5-aminolevulinic acid (ALA) in human urothelial carcinoma (UC), leading to improved accuracy in photodynamic diagnosis and therapy using ALA. Experimental Design: PpIX accumulation in cultured UC cells after incubation for 1-5 h with 0.5-5 mM ALA was analyzed by fluorescence analysis using fluorescence microscopy and flow cytometry technique. Results: PpIX fluorescence mediated by ALA was increased, and the intensity of PpIX fluorescence was time-dependently increased in UC cells compared to noncancerous cells. The distribution of endogenous PpIX fluorescence primarily coincided with mitochondria, and then increased at a specific perinuclear region in the cells during the time of incubation. The ALA-mediated PpIX synthesis in UC cells was suppressed by beta-alanine, an inhibitor of beta-transporters of cell membrane, and carbonyl cyanide p -trifluoromethoxy-phenyl hydrazone, an uncoupler of mitochondrial oxidative phosphorylation. In contrast, the ALA-mediated PpIX accumulation was increased by deferoxamine, an iron chelator, manganese and nitric oxide, which is contributed to PpIX metabolism by inhibiting ferrochelatase activity, generated by a nitric oxide-generating reagent NOC-18. As observed above, ALA-mediated PpIX synthesis in human UC cells was regulated by the process of ALA uptake, ALA conversion to PpIX and metabolism of accumulated PpIX to heme. Conclusions: This shows that the suppression of ferrochelatase increased PpIX accumulation in UC cells using small amount of ALA, thus leading to an improved clinical practicability of photodynamic diagnosis and therapy. Copyright (C) 2009 S. Karger AG, Basel

DOI： 10.1159/000245896

5-Aminolevulinic acid依存性のprotoporphyrine IX蓄積によるガン細胞の診断と治療に関する研究−Tocopherylsuccinateによる増強作用

藤田洋史, 沖村祐弥, 荻野哲也, 保田立二, 阿部浩一, 内海耕慥, 佐々木順造

ビタミンE 研究の進歩 13 2009年

α-Lipoic acid suppresses 6-hydroxydopamine-Induced ROS generation and apoptosis through the stimulation of glutathione synthesis but not by the expression of heme oxygenase-1.

Fujita H, Shiosaka M, Ogino T, Okimura Y, Utsumi T, Sato EF, Akagi R, Inoue M, Utsumi K, Sasaki J

Brain Res 1206 1 - 12 2008年4月

Flow cytometric analysis of Ca2+-induced membrane permeability transition of isolated rat liver mitochondria

Teruo Umegaki, Yuya Okimura, Hirofumi Fujita, Hiromi Yano, Jitsuo Akiyama, Masayasu Inoue, Kozo Utsumi, Junzo Sasaki

Journal of Clinical Biochemistry and Nutrition 42 ( 1 ) 35 - 44 2008年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

The membrane permeability transition (MPT) of mitochondria plays an important role in the mechanism of apoptotic cell death in various cells. Classic type MPT is induced by Ca2+ in the presence of inorganic phosphate and respiratory substrate, and is characterized by various events including generation of reactive oxygen species (ROS), membrane depolarization, swelling, release of Ca2+ and high sensitivity to cyclosporine A. However, the sequence of these events and the effect of antioxidants on their events remain obscure. Flow cytometry is a convenient method to investigate the order of events among various functions occurring in MPT using a limited amount of mitochondria (200 μl of 0.02 mg protein/ml) without contamination by other organelles. Flow cytometric analysis revealed that Ca2+ sequentially induced ROS generation, depolarization, swelling and Ca2+ release in mitochondria by a cyclosporine A-inhibitable mechanism. These results were supported by the finding that Ca2+-induced MPT was inhibited by antioxidants, such as glutathione and N-acetylcysteine. It was also revealed that various inhibitors of Ca2+-induced phospholipase A2 suppressed all of the events associated with Ca2+-induced MPT. These results suggested that ROS generation and phospholipase A2 activation by Ca2+ underlie the mechanism of the initiation of MPT.

DOI： 10.3164/jcbn.2008006

Regulation of 5-aminolevulinic acid-dependent protoporphyrin IX accumulations in human histiocytic lymphoma U937 cells

Yuya Okimura, Hirofumi Fujita, Tetsuya Ogino, Keiji Inoue, Toro Shuin, Hiromi Yano, Tatsuji Yasuda, Masayasu Inoue, Kozo Utsumi, Junzo Sasaki

Physiological Chemistry and Physics and Medical NMR 39 ( 1 ) 69 - 82 2007年

記述言語：英語掲載種別：研究論文（学術雑誌）

The aim of the present work is to clarify the mechanism(s) that regulates the accumulation of protoporphyrin IX (PpIX) in human histiocytic lymphoma cell line U937 incubated with 5-aminolevulinic acid (ALA). Biosynthesis and accumulation of PpIX in the cells was determined after incubation with 0.1 ∼ 5 mM ALA using a flow cytometric technique. The synthesized endogenous PpIX was found to localize predominantly in the mitochondrial region of the cells. The ALA-enhanced PpIX synthesis was suppressed by the presence of either β-alanine, a competitive inhibitor of β-transporters on cell membranes, or carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, an uncoupler of mitochondrial oxidative phosphorylation. In contrast, cellular accumulation of PpIX was enhanced by the presence of either deferoxamine (an iron chelater), MnCl2 (a ferrochelatase inhibitor), or Sn-mesoporphyrin (heme oxygenase inhibitor). These results suggest that ALA-enhanced accumulation of PpIX in U937 cells was regulated by cellular uptake and conversion of ALA to PpIX and by degradation of Heme.

Immunoregulation and apoptosis induction of nitric oxide in the human mixed lymphocytes culture

T. Fujiwara, H. Fujita, Y. Okimura, K. Utsumi

TRANSPLANTATION PROCEEDINGS 38 ( 10 ) 3211 - 3213 2006年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE INC

Nitric oxide (NO) is a multifunctional molecule in a variety of physiologic and pathologic processes. Its precise effect on human T lymphocyte responses against alloantigens are not yet fully known, although it has been reported that NO is antiproliferative and can cause apoptosis in several cell types. To address these issues, we analyzed the effects of an NO donor on mixed lymphocyte cultures (MLC) and on apoptosis induction in T lymphocytes activated with alloantigens. NOC 18 was used as an NO donor. The MLC was performed with human peripheral blood mononuclear cells isolated from healthy volunteers. Cell division and interleukin (IL)-2 production were measured with CFSE labeling and an EIA kit, respectively. After cells were incubated with NOC 18 for 24 hours, DNA fragmentation was assessed using the diphenylamine assay. Pre-culture of cells with NOC 18 for 24 hours resulted in significant inhibition of cell proliferation and IL-2 production in MLC. NOC 18 induced DNA fragmentation of cells harvested from an MLC following 7 days of the culture, in a dose-dependent manner, whereas it never exerted any influence on DNA fragmentation of freshly isolated cells. A chemical NO donor, NOC-18, may have immunosuppressive ability when treatment of responder cells occurs before the beginning of the MLC and may induce apoptosis of alloantigen-activated T lymphocytes.

DOI： 10.1016/j.transproceed.2006.10.170

Cell-permeable cAMP analog suppresses 6-hydroxydopamine-induced apoptosis in PC12 cells through the activation of the Akt pathway

Hirofumi Fujita, Tetsuya Ogino, Hirotsugu Kobuchi, Takuzo Fujiwara, Hiromi Yano, Jitsuo Akiyama, Kozo Utsumi, Junzo Sasaki

BRAIN RESEARCH 1113 10 - 23 2006年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

Although cAMP protects neuronal cells from various apoptotic stimulations, its mechanism is not fully elucidated. We report here the molecular mechanism of the 6-hydroxydopamine (6-OHDA)-induced apoptosis of pheochromocytoma PC12 cells and its suppression by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (pCPT-cAMP), which is a membrane permeable cAMP analog. Treatment of PC12 cells with 6-OHDA resulted in the activation of caspases and apoptosis, as detected by chromatin condensation. 6-OHDA also induced superoxide generation, Bid cleavage and mitochondrial membrane depolarization. In addition, Akt phosphorylation that was favorable to cell survival was decreased and p38 MAPK phosphorylation was increased by 6-OHDA. PC12 cell apoptosis was inhibited by pCPT-cAMP, Z-VAD-fmk (a broad-range caspase inhibitor) and tiron (a superoxide scavenger), although PC12 cell apoptosis was not inhibited by cyclosporine A (an inhibitor of mitochondrial membrane permeability transition). Moreover, pCPT-cAMP promoted Akt phosphorylation, but it did not prevent superoxide generation and mitochondrial membrane depolarization. Conversely, LY294002, an inhibitor of Akt upstream molecule PI3-kinase, enhanced 6-OHDA-induced apoptosis. These results indicated that the 6-OHDA-induced apoptosis of PC12 cells was initiated by superoxide generation followed by caspase cascade activation, which was associated with the suppressed Akt phosphorylation and increased p38 phosphorylation. It is likely that pCPT-cAMP prevented the 6-OHDA-induced apoptosis via activation of the PI3-kinase/Akt pathway without any effect on superoxide generation or mitochondrial membrane depolarization. (c) 2006 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.brainres.2006.06.079

4-hydroxy-3,5,3',4'-tetrachlorobiphenyl induced membrane permeability transition in isolated rat liver mitochondria

Hirofumi Fujita, Yuya Okimura, Toshihiko Utsumi, Shigeyuki Kitamura, Hiroaki Kuroki, Takemi Otsuki, Junzo Sasaki, Akihiko Kashiwagi, Kozo Utsumi

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION 38 ( 3 ) 167 - 175 2006年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：JOURNAL CLINICAL BIOCHEMISTRY & NUTRITION

Although much information about the apoptotic and other detrimental actions of polychlorinated biphenyls (PCBs) has been accumulated, the critical role of mitochondrial damage in such toxic action remains to be clarified. We have previously shown that PCBs affect various functions in isolated mitochondria. Recent studies have revealed that mitochondria play an important role in apoptosis through membrane permeability transition (NIPT), and that Ca2+ induces classic MPT characterized by depolarization and swelling of the mitochondria, thereby releasing cytochrome c in a cyclosporin A-sensitive mechanism [Free Rad. Res. 38, 29-35, 2004]. In the present study we investigated the effect of 4-hydroxy3,5,3',4'-tetrachlorobiphenyl (4-OH-TCB) on isolated rat liver mitochondria in order to help clarify the effect of PCBs on MPT. Biochemical analysis revealed that 4-OH-TCB induced calcium release, reactive oxygen species (ROS) generation, depolarization, swelling and cytochrome c release in isolated rat liver mitochondria in a time and concentration dependent manner. These 4-OH-TCB-induced changes in mitochondrial function were found to be inhibited by the presence of cyclosporin A, suggesting an classic type of MPT. It is concluded that 4-OH-TCB induces MPT together with ROS generation, causing apoptosis in certain cells.

DOI： 10.3164/jcbn.38.167

アポトーシスとミトコンドリア -神経細胞を加味して-

藤田洋史, 小渕浩嗣, 内海耕慥

Clinical Neuroscience 24(6), 643-646 2006年

Mechanism and characteristics of stimuli-dependent ROS generation in undifferentiated HL-60 cells

S Muranaka, H Fujita, T Fujiwara, T Ogino, EF Sato, J Akiyama, Imada, I, M Inoue, K Utsumi

ANTIOXIDANTS & REDOX SIGNALING 7 ( 9-10 ) 1367 - 1376 2005年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：MARY ANN LIEBERT, INC

It has been widely believed that undifferentiated human promyelocytic leukemia cells (HL-60) have no ability to generate reactive oxygen species (ROS) responding to stimuli. We report here that undifferentiated HL-60 cells possess NADPH oxidase and that generation of superoxide can be measured using a highly sensitive chemiluminescence dye, L-012. Five subunits of NADPH oxidase, namely, gp91(phox), p22(phox), p67(phox), p47(phox), and Rac 2, were detected in undifferentiated HL-60 cells by immunoblotting analysis. The contents of these NADPH oxidase components in the cells were increased with the differentiation induced by phorbol myristate acetate (PMA), except for p22(phox). Messenger RNAs of these subunits were also detected by the RT-PCR method, and their expressions increased except that of p22(phox) with the differentiation induced by PMA. Kinetic analysis using L-012 revealed that HL-60 cells generated substantial amounts of ROS by various stimulants, including formylmethionyl-leucyl-phenylaianine, PMA, myristic acid, and a Ca2+ ionophore, A23187. Both diphenyleneiodonium (an inhibitor of FAD-dependent oxidase) and apocynin (a specific inhibitor of NADPH oxidase) suppressed this stimuli-dependent ROS generation. Genistein, staurosporine, uric acid, and sodium azide inhibited the ROS generation in undifferentiated HL-60 cells in a similar way to that in undifferentiated neutrophils. These results suggested that the mechanism of ROS generation in undifferentiated HL-60 cells is the same as that in primed neutrophils.

DOI： 10.1089/ars.2005.7.1367

Involvement of Ras/extracellular signal-regulated kinase, but not Akt pathway in risedronate-induced apoptosis of U937 cells and its suppression by cytochalasin B 査読

H Fujita, T Utsumi, S Muranaka, T Ogino, H Yano, J Akiyama, T Yasuda, K Utsumi

BIOCHEMICAL PHARMACOLOGY 69 ( 12 ) 1773 - 1784 2005年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD

Although risedronate, a nitrogen containing bisphosphonate (BPs), strongly inhibits bone resorption by enhanced apoptosis of osteoclasts, its mechanism remained unclear. In this study, we investigated the molecular mechanism of risedronate-induced apoptosis of U937 cells, with a focus on extracellular signal-regulated kinase 1/2 (ERK 1/2) and protein kinase B (Akt) pathways, mitochondria-mediated apoptosis, and the effect of disruption of the actin cytoskeleton. Risedronate facilitated the relocation of Ras from membrane to cytosol through inhibited isoprenylation. Accordingly, risedronate suppressed the phosphorylation of ERK 1/2, a downstream survival signaling kinase of Ras, affected the intracellular distribution of Bcl-xL, and induced the mitochondrial membrane depolarization, cytochrome c release, activated caspase cascade and DNA fragmentation. The risedronate-induced apoptosis was effectively suppressed with cyclosporine A plus trifluoperazine, potent inhibitors of mitochondrial membrane permeability transition (MPT). The risedronate-induced apoptosis was independent of Akt, another cAMP-dependent survival signaling kinase. Risedronate facilitated dephosphorylation of Bad at Ser112, an ERK phosphorylation site, but not at Ser136, an Akt phosphorylation site. All of these apoptosis-related changes induced by risedronate were strongly suppressed by cytochalasin B, an inhibitor of actin filament polymerization. These results indicate that risedronate-induced apoptosis in U937 cells involves Ras/ERK, but not Akt signaling pathway, and is dependent on MPT, and that disruption of the actin cytoskeleton inhibits the risedronate-induced apoptosis at its early step. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bcp.2005.03.006

A possible site of superoxide generation in the complex I segment of rat heart mitochondria

ST Ohnishi, T Ohnishi, S Muranaka, H Fujita, H Kimura, K Uemura, K Yoshida, K Utsumi

JOURNAL OF BIOENERGETICS AND BIOMEMBRANES 37 ( 1 ) 1 - 15 2005年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPRINGER/PLENUM PUBLISHERS

We searched for possible sites of superoxide generation in the complex I segment of the respiratory chain by studying both forward and reverse electron transfer reactions in isolated rat heart mitochondria. Superoxide production was monitored by measuring the release of hydrogen peroxide from mitochondria with a fluorescence spectrophotometer using the Amplex red/horseradish peroxidase system. In the forward electron transfer, a slow superoxide production in the presence of glutamate and malate was enhanced by both rotenone and piericidin A (specific inhibitors at the end of the complex I respiratory chain). Both diphenileneiodonium and ethoxyformic anhydride (inhibitors for respiratory components located upstream of the respiratory chain) inhibited the enhancement by rotenone and piericidin A.
In contrast, in reverse electron transfer driven by ATP, both diphenileneiodonium and ethoxyformic anhydride enhanced the superoxide production. Piericidin A also increased superoxide production. Rotenone increased it only in the presence of piericidin A. Our results suggest that the major site of superoxide generation is not flavin, but protein-associated ubisemiquinones which are spin-coupled with iron-sulfer cluster N2.

DOI： 10.1007/s10863-005-4117-y

The response of human T lymphocytes against alloantigens following fas-mediated activation-induced cell death

T Fujiwara, H Fujita, S Muranaka, K Utsumi, S Kusaka, K Hamazaki

TRANSPLANTATION PROCEEDINGS 37 ( 1 ) 46 - 48 2005年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE INC

We examined the response of T lymphocytes activated with specific alloantigens following Fas-mediated apoptosis; using a mixed lymphocyte culture (MLC) system. Cells obtained from an MLC after 6 or 7 days of culture were incubated for are additional 24 hours in the presence or absence of the agonistic monoclonal antibody (MoAb), 701, or the antagonistic MoAb, ZB4. We assessed DNA fragmentation/specific cytotoxiy of the MoAb-treated cells. Cells harvested after 4 days of culture were sensitive to apoptosis induced by 701 with maximum DNA fragmentation observed on day 6. ZB4 slightly inhibited apoptosis of the cells compared with controls. The simultaneous addition of recombinant interleukin-2 (rIL-2) with the MoAbs significantly inhibited DNA fragmentation in control and ZB4-treated cells, but had little effect on the 7C11-treated cells. Control and ZB4-treated MLC cells showed cytotoxic activities against specific target cells, namely > 10%. In contrast, the 7C11-treated cells showed < 5% cytotoxicity. Although the addition of rIL-2 increased specific percentage cytotoxicity of control and ZB4-treated cells, it had little effect on the specific cytotoxic activity of the 7C11-treated MLC cells. These results suggest that specific cytotoxic T lymphocytes may be eliminated via apoptosis mediated by the Fas/Fas ligand system.

DOI： 10.1016/j.transproceed.2004.12.244

羅布麻抽出物（ベネトロン）の抗酸化作用とその解析査読

藤田洋史, 沖村祐弥, 内海俊彦

医学と薬学 54 491 - 503 2005年

Molecular mechanism of diclofenac-induced apoptosis of promyelocytic leukemia: Dependency on reactive oxygen species, Akt, Bid, cytochrome c, and caspase pathway

A Inoue, S Muranaka, H Fujita, T Kanno, H Tamai, K Utsumi

FREE RADICAL BIOLOGY AND MEDICINE 37 ( 8 ) 1290 - 1299 2004年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD

Nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in a variety of cells, but the mechanism of this effect has not been fully elucidated. We report that diclofenac, a NSAID, induces growth inhibition and apoptosis of HL-60 cells through modulation of mitochondrial functions regulated by reactive oxygen species (ROS), Akt, caspase-8, and Bid. ROS generation occurs in an early stage of diclofenac-induced apoptosis preceding cytochrome c release, caspase activation, and DNA fragmentation. N-Acetyl-L-CySteine, an antioxidant, suppresses ROS generation, Akt inactivation, caspase-8 activation, and DNA fragmentation. Cyclic AMP, an inducer of Akt phosphorylation, suppresses Akt inactivation, Bid cleavage, and DNA fragmentation. LY294002, a PI3 kinase inhibitor, enhances Akt inactivation and DNA fragmentation. Ac-IETD-CHO, a caspase-8 inhibitor, suppresses Bid cleavage and DNA fragmentation. z-VAD-fmk, a universal caspase inhibitor, but not cyclosporin A (CsA), an inhibitor of mitochondrial membrane permeability transition, suppresses DNA fragmentation. These results suggest the sequential mechanism of diclofenac-induced apoptosis of HL-60 cells: ROS generation suppresses Akt activity, thereby activating caspase-8, which stimulates Bid cleavage and induces cytochrome c release and the activation of caspase-9 and -3 in a CsA-insensitive mechanism. Furthermore, we found that 2-methoxyestradiol (2-ME), a superoxide dismutase inhibitor, significantly enhances diclofenac-induced apoptosis; that is, diclofenac combined with 2-ME may have therapeutic potential in the treatment of human leukemia. (C) 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.freeradbiomed.2004.07.003

Involvement of ceramide in the mechanism of Cr(VI)-induced apoptosis of CHO cells

S Muranaka, T Kanno, H Fujita, H Kobuchi, J Akiyama, T Yasuda, K Utsumi

FREE RADICAL RESEARCH 38 ( 6 ) 613 - 621 2004年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：TAYLOR & FRANCIS LTD

Mitochondria reduce Cr(VI) to Cr(V) with concomitant generation of reactive oxygen species, thereby exhibiting cytotoxic effects leading to apoptosis in various types of cells. To clarify the mechanism by which Cr(VI) induces apoptosis, we examined the effect of Cr(VI) on Chinese hamster ovary (CHO) cells. Cr(VI) increased cellular levels of ceramide by activating acid sphingomyelinase (ASMase) and inhibiting the phosphorylation of pleckstrin homology domain-containing protein kinase B (Akt). Cr(VI) also induced cyclosporin A- and trifluoperazine-sensitive depolarization of mitochondria and activated caspase-3, 8 and 9, thereby causing fragmentation of cellular DNA. The presence of desipramine, an inhibitor of ASMase, and membrane permeable pCPT-cAMP suppressed the Cr(VI)-induced activation of caspases and DNA fragmentation. These results suggested that accumulation of ceramide play an important role in the Cr(VI)-induced apoptosis of CHO cells through activation of mitochondrial membrane permeability transition.

DOI： 10.1080/10715760410001694035

アクトネル（リセドロネート）によるU937細胞のアポトーシスとその機構査読

藤田洋史, 菅野智子, 内海俊彦, 村中式部, 藤原拓造, 内海耕慥

医学と薬学 51 697 - 710 2004年

Mitochondrial swelling and cytochrome c release: Sensitivity to cyclosporin A and calcium 査読

Tomoko Kanno, Hirofumi Fujita, Shikibu Muranaka, Hiromi Yano, Toshihiko Utsumi, Tamotsu Yoshioka, Masayasu Inoue, Kozo Utsumi

Physiological Chemistry and Physics and Medical NMR 34 ( 2 ) 91 - 102 2002年

記述言語：英語掲載種別：研究論文（学術雑誌）

The opening of mitochondrial membrane permeability transition (MPT) pores, which results in a cyclosporin A (CsA)-sensitive and Ca 2+-dependent dissipation of the membrane potential (delta psi) and swelling (classical MPT), has been postulated to play an important role in the release of cytochrome c (Cyt.c) and also in apoptotic cell death. Recently, it has been reported that CsA-insensitive or Ca 2+-independent MPT can be classified as non-classic MPT. Therefore, we studied the effects of apoptosis-inducing agents on mitochondrial functions with respect to their CsA-sensitivity and Ca 2+-dependency. CsA-sensitive mitochondrial swelling, depolarization, and the release of Ca 2+ and Cyt.c were induced by low concentrations of arachidonic acid, triiodothyronine (T 3), or 6-hydroxdopamine but not by valinomycin and high concentrations of the fatty acid or T 3. Fe 2+/VADP and 2,2,-azobis-(2-amidinopropane) dihydrochloride (AAPH) induced swelling of mitochondria and the release of Ca 2+ and Cyt.c were not coupled with depolarization or CsA-sensitivity while dibucaine-induced swelling occurred without depolarization, Cyt.c-release or by a CsA-sensitive mechanism. A protonophoric FCCP and SF-6847 induced depolarization and Ca 2+-release occurred in a CsA-insensitive manner and failed to stimulate the release of Cyt.c. These results indicate that ambient conditions of mitochondria can greatly influence the state of membrane stability and that Cyt.c release may occur not only via a CsA-sensitive MPT but also by way of a CsA-insensitive membrane deterioration.

CHARACTERIZATION AND EXPRESSION OF CDNA-ENCODING COPROPORPHYRINOGEN OXIDASE FROM A PATIENT WITH HEREDITARY COPROPORPHYRIA 査読

H FUJITA, M KONDO, S TAKETANI, N NOMURA, K FURUYAMA, R AKAGI, T NAGAI, M TERAJIMA, RA GALBRAITH, S SASSA

HUMAN MOLECULAR GENETICS 3 ( 10 ) 1807 - 1810 1994年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：OXFORD UNIV PRESS UNITED KINGDOM

Hereditary coproporphyria (HCP) is an acute hepatic porphyria with autosomal dominant inheritance, but with a variable degree of clinical expression. Molecular cloning, sequencing and expression of the defective gene for coproporphyrinogen oxidase (CPO) in a patient with HCP were carried out. Enzyme assays revealed that CPO activity in EBV-transformed lymphoblastoid cells from the proband and one of her sisters was approximate to 50% of normal. Nucleotide sequence analysis of CPO cDNAs isolated from the proband's cells demonstrated three base substitutions, and three accompanying amino acid substitutions. An A514 --> C transition causing a Asn172 --> His substitution occurred in one allele, while two other transitions, G265 --> A and G580 --> A, caused Gly89 --> Ser and Val194 --> lle substitutions, respectively, in the other allele. The A514 --> C and the G580 --> A transitions are known genetic polymorphisms. Transfection of CPO cDNA into Escherichia coli demonstrated that cDNA with the G265 --> A transition produced a protein with less than 5% of normal enzyme activity. These findings indicate that the G265 --> A transition, involving the highly conserved glycine residue at the 89th position, is responsible for the CPO defect in the patient and accounts for the partial deficiency of CPO activity in this pedigree.

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