Faculty Profiles - FUJITA Hirofumi

Papers - FUJITA Hirofumi

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

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

PLOS ONE 7 ( 11 ) e50082 2012.11

Language：English Publishing type：Research paper (scientific journal) Publisher：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

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 2011.12

Language：English Publishing type：Research paper (scientific journal) Publisher：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-kappa B Ligand and Apoptosis in Mesenchymal Stem Cells Reviewed

Hirofumi Fujita, Kazuko Kurokawa, Tetsuya Ogino, Mio Ono, Masanao Yamamoto, Takashi Oka, Tohru Nakanishi, Naoya Kobayashi, Noriaki Tanaka, Tomohiro Ogawa, Etsuko Suzaki, Kozo Utsumi, Junzo Sasaki

BASIC & CLINICAL PHARMACOLOGY & TOXICOLOGY 109 ( 2 ) 78 - 84 2011.8

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

Nitrogen-containing bisphosphonates (BPs) are antiresorptive drugs used for the treatment of metabolic bone diseases. Bone marrow stromal cells such as mesenchymal stem cells (MSCs) and MSC-derived osteoblasts that originate from MSCs are known to regulate osteoclast differentiation and activation via the expression of receptor activator of NF-kappa B ligand (RANKL). Although the effects of nitrogen-containing BPs on osteoclasts and osteoblasts have been well investigated, their effects in MSCs have not been clarified. In this study, we investigated the effects of risedronate (RIS), a nitrogen-containing BP, on osteoblast differentiation, RANKL expression and apoptosis in human and rat MSCs. RIS suppressed the formation of mineralized nodules and mRNA expression of differentiation marker genes such as bone sialoprotein and osteocalcin in MSC-derived osteoblasts. The RANKL expression induced by 1,25-(OH)(2) vitamin D(3) was not affected by RIS in human MSC-derived osteoblasts. In addition, treatment with high-concentration RIS induced chromatin condensation, an apoptosis feature, in MSCs. RIS-induced chromatin condensation was suppressed by a pan-caspase inhibitor zVAD-FMK and a cell-permeable isoprenoid analogue geranylgeraniol. These results indicate that RIS suppressed osteoblast differentiation and induced caspase-and isoprenoid depletion-dependent apoptosis and suggest that the antiresorptive effect of RIS is not mediated by a decrease in the RANKL expression in MSC-derived osteoblasts.

DOI： 10.1111/j.1742-7843.2011.00685.x

Effect of Risedronate on Osteoblast Differentiation, Expression of Receptor Activator of NF-kappa B Ligand and Apoptosis in Mesenchymal Stem Cells

Hirofumi Fujita, Kazuko Kurokawa, Tetsuya Ogino, Mio Ono, Masanao Yamamoto, Takashi Oka, Tohru Nakanishi, Naoya Kobayashi, Noriaki Tanaka, Tomohiro Ogawa, Etsuko Suzaki, Kozo Utsumi, Junzo Sasaki

BASIC & CLINICAL PHARMACOLOGY & TOXICOLOGY 109 ( 2 ) 78 - 84 2011.8

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

Nitrogen-containing bisphosphonates (BPs) are antiresorptive drugs used for the treatment of metabolic bone diseases. Bone marrow stromal cells such as mesenchymal stem cells (MSCs) and MSC-derived osteoblasts that originate from MSCs are known to regulate osteoclast differentiation and activation via the expression of receptor activator of NF-kappa B ligand (RANKL). Although the effects of nitrogen-containing BPs on osteoclasts and osteoblasts have been well investigated, their effects in MSCs have not been clarified. In this study, we investigated the effects of risedronate (RIS), a nitrogen-containing BP, on osteoblast differentiation, RANKL expression and apoptosis in human and rat MSCs. RIS suppressed the formation of mineralized nodules and mRNA expression of differentiation marker genes such as bone sialoprotein and osteocalcin in MSC-derived osteoblasts. The RANKL expression induced by 1,25-(OH)(2) vitamin D(3) was not affected by RIS in human MSC-derived osteoblasts. In addition, treatment with high-concentration RIS induced chromatin condensation, an apoptosis feature, in MSCs. RIS-induced chromatin condensation was suppressed by a pan-caspase inhibitor zVAD-FMK and a cell-permeable isoprenoid analogue geranylgeraniol. These results indicate that RIS suppressed osteoblast differentiation and induced caspase-and isoprenoid depletion-dependent apoptosis and suggest that the antiresorptive effect of RIS is not mediated by a decrease in the RANKL expression in MSC-derived osteoblasts.

DOI： 10.1111/j.1742-7843.2011.00685.x

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

Hirofumi Fujita, Daisuke Shiva, Toshihiko Utsumi, Tetsuya Ogino, Tomohiro Ogawa, Koichi Abe, Tatsuji Yasuda, Kozo Utsumi, Junzo Sasaki

MOLECULAR AND CELLULAR BIOCHEMISTRY 333 ( 1-2 ) 137 - 149 2010.1

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER

Phosphatidylserine (PS) externalization is a key feature of apoptotic cell death and plays an important role in clearance of apoptotic cells by phagocytes. PS externalization during apoptosis is generally an irreversible event mediated by caspase activation and is accompanied by other apoptotic events. We report here that an apoptosis inducer alpha-tocopheryl succinate (TOS) can induce PS externalization that is independent of apoptosis and reversible in the absence of fetal bovine serum (FBS) in histiocytic lymphoma U937 cells. In the presence of FBS, TOS induced PS externalization via a caspase-dependent mechanism accompanied by mitochondrial depolarization, cell shrinkage, increase of caspase-3 activity, and chromatin condensation. In contrast, in the absence of FBS, TOS induced the rapid PS externalization which was not accompanied by other apoptotic events. The PS externalization was reversible by removing TOS and was not involved in Ca(2+)-dependent scramblase activation and thiol oxidation of aminophospholipid translocase. A similar PS externalization was also induced by cholesteryl hemisuccinate (CS), the other succinate ester. These results suggested that the mechanism of TOS- and CS-induced PS externalization in the absence of FBS was different from it occurring during typical apoptosis.

DOI： 10.1007/s11010-009-0214-2

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 2009.12

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed

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

PATHOBIOLOGY 76 ( 6 ) 303 - 314 2009

Language：English Publishing type：Research paper (scientific journal) Publisher：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

alpha-lipoic acid suppresses 6-hydroxydoparnine-induced ROS generation and apoptosis through the stimulation of glutathione synthesis but not by the expression of heme oxygenase-1

Hirofumi Fujita, Masahiko Shiosaka, Tetsuya Ogino, Yuya Okimura, Toshihiko Utsumi, Eisuke F. Sato, Reiko Akagi, Masayasu Inoue, Kozo Utsumi, Junzo Sasaki

BRAIN RESEARCH 1206 1 - 12 2008.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

We previously reported that the generation of reactive oxygen species (ROS) is the initial event in cell death induced by 6-hydroxydopamine (6-OHDA), an experimental model of Parkinsonism. Since recent studies suggested the important role of antioxidant activity of alpha-lipoic acid (LA) in the suppression of apoptosis of various types, we studied the effect on 6-OHDA-induced apoptosis of PC12 cells. Biochemical analysis revealed that LA suppressed the 6-OHDA-induced ROS generation, increase of caspase-like activity and chromatin condensation. The suppression of 6-OHDA-induced apoptosis by LA required pre-incubation of PC12 cells with LA for 12-24 h. LA increased the intracellular levels of heme oxygenase-1 (HO-1) and glutathione (GSH) and stimulated the expression of GSH synthesis-related genes such as cystine/glutamate antiporter and gamma-glutamylcysteine synthetase (gamma-GCS). However, Sn-mesoporphyrin IX, an inhibitor of HO-1, did not attenuate the LA-induced suppression of apoptosis. In contrast, buthionine sulfoximine, an inhibitor of gamma-GCS, attenuated the LA-induced suppression of ROS generation and chromatin condensation. in addition, a transcription factor Nrf2, which regulates the expression of antioxidant enzymes such as gamma-GCS, translocated to the nucleus by LA. These results suggested that LA suppressed the 6-OHDA induced-apoptosis by the increase in cellular glutathione through stimulation of the GSH synthesis system but not by the expression of HO-1. (C) 2008 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.brainres.2008.01.081

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 2008.1

Language：English Publishing type：Research paper (scientific journal)

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

Language：English Publishing type：Research paper (scientific journal)

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 2006.12

Language：English Publishing type：Research paper (scientific journal) Publisher：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 2006.10

Language：English Publishing type：Research paper (scientific journal) Publisher：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 2006.5

Language：English Publishing type：Research paper (scientific journal) Publisher：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

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 2005.9

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed

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

BIOCHEMICAL PHARMACOLOGY 69 ( 12 ) 1773 - 1784 2005.6

Language：English Publishing type：Research paper (scientific journal) Publisher：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 2005.2

Language：English Publishing type：Research paper (scientific journal) Publisher：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 2005.1

Language：English Publishing type：Research paper (scientific journal) Publisher：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

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