Faculty Profiles - OKAMURA Hirohiko

Papers - OKAMURA Hirohiko

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Transcription factor NF-Y regulates mdr1 expression through binding to inverted CCAAT sequence in drug-resistant human squamous carcinoma cells Reviewed

H Okamura, K Yoshida, E Sasaki, H Morimoto, T Haneji

INTERNATIONAL JOURNAL OF ONCOLOGY 25 ( 4 ) 1031 - 1037 2004.10

Language：English Publishing type：Research paper (scientific journal) Publisher：PROFESSOR D A SPANDIDOS

In this study, the expression and transcriptional regulation of the multidrug resistance-1 (MDR1) gene in multidrug-resistant SCCTF cells and -sensitive SCCKN cells derived from human squamous carcinoma were investigated. RT-PCR revealed that mdr1 mRNA was highly expressed in SCCTF cells while it was under the limit of detection in SCCKN cells. With an electrophoretic mobility shift assay using the mdr1 promoter region, a DNA-protein complex was detected strongly in SCCTF cells, but weakly in SCCKN cells. Incubation of the DNA-protein complex with an anti-NF-Y antibody caused a supershift in the migration to a position near the origin of the gel. Chromatin immunoprecipitation assay with an anti-NF-Y antibody showed that NF-Y binds to mdr1 promoter in SCCTF cells. The mdr1 promoter region including its NF-Y binding sequence was cloned into the luciferase reporter plasmid pGL3-basic vector, and this vector was used to transfect SCCTF and SCCKN cells. The luciferase assay showed that the inverted CCAAT sequence in the mdr1 promoter region is involved in the positive regulation of mdrl promoter activity. NF-YA protein was expressed at higher levels in SCCTF cells than that in SCCKN cells. Hoechst dye staining also showed that MDR1 protein acts more effectively as an efflux pump in SCCTF cells than that in SCCKN cells.

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Double-stranded RNA mediates selective gene silencing of protein phosphatase type 1 delta isoform in HEK-293 cells Reviewed

H Morimoto, H Okamura, K Yoshida, S Kitamura, T Hanejl

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY 19 ( 4 ) 327 - 331 2004.8

Language：English Publishing type：Research paper (scientific journal) Publisher：TAYLOR & FRANCIS LTD

The reversible phosphorylation of proteins mediates cellular signals in eukaryotic cells. RNA interference inhibits the expression of genes and proteins in a sequence-specific manner and provides a tool to study the functions of target molecules. The effect of RNA interference on protein phosphatase isoforms in HEK-293 cells was examined. Protein phosphatase 1 delta (PP1delta) sequence-specific double-stranded RNA (dsRNA) inhibited mRNA and protein expression of the PP1delta. This RNA interference did not affect the expression of alpha and gamma1 isoforms of PP1. Transfection of antisense RNA specific for PP1delta also suppressed the expression of PP1delta. It was further demonstrated by an in vitro RNA cleavage assay that extracts of HEK-293 cells catalyzed the processing of dsRNA. This cell line had much stronger mRNA expression of Dicer, an RNase III-like enzyme, than did human osteoblastic MG63 cells. The present results show that RNA interference is a useful tool to distinguish between PP1 isoforms.

DOI： 10.1080/14756360410001704452

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Okadaic acid stimulates expression of Fas receptor and Fas ligand by activation of nuclear factor kappa-B in human oral squamous carcinoma cells Reviewed

M Fujita, K Goto, K Yoshida, H Okamura, H Morimoto, S Kito, J Fukuda, T Haneji

ORAL ONCOLOGY 40 ( 2 ) 199 - 206 2004.2

Language：English Publishing type：Research paper (scientific journal) Publisher：PERGAMON-ELSEVIER SCIENCE LTD

In the present study, we used western blot and RT-PCR analysis to examine the expression of proteins and mRNAs of Fas receptor and Fas ligand in human oral squamous carcinoma SCC-25 cells treated with okadaic acid. Treatment with okadaic acid enhanced the expression of proteins and mRNAs of both Fas receptor and Fas ligand in SCC-25 cells. The amount of IkappaB-alpha in whole cell lysates decreased, while the level of NF-kappaB in nucleus increased, in the okadaic acid-treated cells. Okadaic acid-treatment also alters the cellular localization of NF-kappaB, from cytoplasm to nuclei. To investigate the activation of NF-kappaB in okadaic acid-treated SCC-25 cells, we performed electrophoretic mobility get shift assay using nuclear extracts and the consensus oligonucleotide for NF-kappaB DNA binding site. The binding of nuclear proteins to the oligonucleotide of NF-kappaB increased when the cells had been treated with 20 nM okadaic acid for 4 h. We transfected the cells with pFLF1, which has the promoter region of Fas receptor gene containing NF-kappaB binding site. A luciferase reporter gene assay demonstrated that the activity in the cells transfected with pFLF1 and treated with 20 nM okadaic acid increased in a time-dependent manner and that the activity was more than three-fold over that in the control cells. Our results suggest that NF-kappaB activated at early stages in the okadaic acid-treated SCC-25 cells stimulated the promoter activity of Fas receptor in the cells leading to the apoptotic death of these cells. (C) 2003 Elsevier Ltd. All rights reserved.

DOI： 10.1016/S1368-8375(03)00152-0

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薬剤耐性扁平上皮癌細胞におけるアポトーシスと転写調節因子 Reviewed

岡村裕彦

徳島大学大学院歯学研究科 17 ( 1 ) 123 - 136 2004

Transblot and cytochemical identification of avidin-interacting proteins in mitochondria of cultured cells Reviewed

E Sasaki, Y Okamoto, K Yoshida, H Okamura, K Shimizu, F Nasu, H Morimoto, T Haneji

HISTOCHEMISTRY AND CELL BIOLOGY 120 ( 4 ) 327 - 333 2003.10

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

Cell lysates prepared from 3T3-L1 cells were analyzed by western blotting using the avidin-biotin complex system and anti-Bax antibody. The antibody interacted with bands of proteins with estimated molecular weights of 120, 74, 72, and 25 kDa. However, only the 25-kDa band was detected with the anti-Bax antibody when the direct immunoblotting method was used. Peroxidase-conjugated avidin interacted with the 120-, 74-, and 72-kDa bands. This interaction was not limited to 3T3-L1 cells, because peroxidase-avidin also interacted with these three proteins in MC3T3-E1, YROS, Saos-2, MG63, SCCKN, and SCCTF cells although the staining intensity was different in each cell type. Avidin-peroxidase also interacted with these three proteins in the mitochondria-containing fractions prepared from 3T3-L1 cells. FITC-streptavidin was also localized in mitochondria in the cultured cells. The localization of avidin/streptavidin-interacting proteins in mitochondria was confirmed by using double staining with FITC-streptavidin and Mito-tracker.

DOI： 10.1007/s00418-003-0572-x

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Cleavage of nucleolin and argyrophilic nucleolar organizer region associated proteins in apoptosis-induced cells Reviewed

S Kito, K Shimizu, H Okamura, K Yoshida, H Morimoto, M Fujita, Y Morimoto, T Ohba, T Haneji

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 300 ( 4 ) 950 - 956 2003.1

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

To investigate the behavior of nuclear proteins in apoptotic cells, we examined the changes in nucleolin and proteins of the nucleolar organizing region during apoptosis in human osteoblastic cell lines, Saos-2 and MG63. Apoptosis was induced by treatment of these cells with okadaic acid. Proteins prepared from apoptotic cells were subjected to Western blot analysis and a modified Western blot method using silver nitrate. The anti-nucleolin antibody recognized the 110-kDa band and the staining intensity of this band decreased in the proteins prepared from the okadaic acid-treated apoptotic cells. The additional band of an 80-kDa was also detected in the proteins prepared from the apoptotic cells. Two major silver nitrate-stained bands, 110-kDa and 37-kDa, were detected among the proteins obtained from control cells. Like the Western blot analysis, the intensity of the 110-kDa silver nitrate-staining band decreased; an 80-kDa band appeared and its staining intensity increased in the lysate from the okadaic acid-treated cells. The signal intensity of the 37-kDa protein did not change in the sample from the apoptotic cells. In a cell-free apoptotic system, the 80-kDa protein was also detected and the amount of the 110-kDa protein decreased in the extract of Saos-2 cell nuclei incubated with apoptotic cytosol. The change in nucleolin in Saos-2 cells induced to undergo apoptosis was examined by an immunocytochemical procedure using the anti-nucleolin antibody and Hoechst 33342. Nucleolin was visible as dots in nucleoli in the control cells; however, it was not detected in the cells undergoing apoptosis. The dual-exposure view of Hoechst 33342 and anti-nucleolin staining cells confirmed that nucleolin had disappeared from the apoptotic nuclei of Saos-2. (C) 2002 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S0006-291X(02)02942-X

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Okadaic acid stimulates the receptor activator of nuclear factor-kappa B ligand (RANKL) in mouse osteoblastic cells. Reviewed

Yoshida K, Okamura H, Morimoto H, Nagata T, Haneji T

Biomed. Res. 14 ( 2 ) 126 - 132 2003

Suppression of Egr-1 expression in human oral squamous carcinoma cells by okadaic acid Reviewed

H Okamura, H Morimoto, M Fujita, F Nasu, E Sasaki, T Haneji

ORAL ONCOLOGY 38 ( 8 ) 779 - 784 2002.12

Language：English Publishing type：Research paper (scientific journal) Publisher：PERGAMON-ELSEVIER SCIENCE LTD

We examined the expression of early growth response-1 (Egr-1) gene in human oral squamous carcinoma cell lines SCCKN and SCC-25 cells and human osteoblastic cell lines Saos-2 and MG63 cells treated with okadaic acid, a potent inhibitor of protein phosphatases type I and type 2A. Western blot analysis revealed that Egr-1 was strongly expressed in SCCKN cells and that okadaic acid decreased the expression of Egr-1 protein in these cells. However, Egr-1 was expressed at lower levels in SCC-25, Saos-2, and MG63 cells and transiently increased with the okadaic acid treatment. Suppression of Egr-1 protein expression in okadaic acid-treated SCCKN cells stemmed from the suppression of the Egr-1 mRNA level, as determined by the RT-RCR method. Formaldehyde-fixed and alcohol-permeabilized cultured SCCKN cells were reacted with the anti-Egr-1 antibody using immunohistochemical methods. Intense fluorescence was observed in the nuclei of the control SCCKN cells interacted with anti-Egr-1 antibody. However, only a weak reaction was observed in the nuclei in SCCKN cells treated with okadaic acid. A gel mobility shift assay showed that treatment of SCCKN cells with okadaic acid suppressed Egr-1 binding to the DIG-labeled Egr-1 consensus oligonucleotide probe. The present results indicate that the alteration of phosphorylation states in SCCKN cells regulates Egr-1 binding to its consensus sequence and its expression at the transcriptional level. (C) 2002 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S1368-8375(02)00039-8

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Suppression of Egr-1 expression in human oral squamous carcinoma cells by okadaic acid Reviewed

H. Okamura, H. Morimoto, M. Fujita, F. Nasu, E. Sasaki, T. Haneji

Oral Oncology 38 ( 8 ) 779 - 784 2002.12

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

We examined the expression of early growth response-1 (Egr-1) gene in human oral squamous carcinoma cell lines SCCKN and SCC-25 cells and human osteoblastic cell lines Saos-2 and MG63 cells treated with okadaic acid, a potent inhibitor of protein phosphatases type 1 and type 2A. Western blot analysis revealed that Egr-1 was strongly expressed in SCCKN cells and that okadaic acid decreased the expression of Egr-1 protein in these cells. However, Egr-1 was expressed at lower levels in SCC-25, Saos-2, and MG63 cells and transiently increased with the okadaic acid treatment. Suppression of Egr-1 protein expression in okadaic acid-treated SCCKN cells stemmed from the suppression of the Egr-1 mRNA level, as determined by the RT-RCR method. Formaldehyde-fixed and alcohol-permeabilized cultured SCCKN cells were reacted with the anti-Egr-1 antibody using immunohistochemical methods. Intense fluorescence was observed in the nuclei of the control SCCKN cells interacted with anti-Egr-1 antibody. However, only a weak reaction was observed in the nuclei in SCCKN cells treated with okadaic acid. A gel mobility shift assay showed that treatment of SCCKN cells with okadaic acid suppressed Egr-1 binding to the DIG-labeled Egr-1 consensus oligonucleotide probe. The present results indicate that the alteration of phosphorylation states in SCCKN cells regulates Egr-1 binding to its consensus sequence and its expression at the transcriptional level. © 2002 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S1368-8375(02)00039-8

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Interaction of protein phosphatase 1 delta with nucleolin in human osteoblastic cells Reviewed

H Morimoto, H Okamura, T Haneji

JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 50 ( 9 ) 1187 - 1193 2002.9

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

We examined the expression and cytolocalization of the protein phosphatase type 1 delta (PP1delta) isoform and nucleolin in human osteoblastic MG63 and Saos-2 cells. Cellular fractionation of MG63 cells was done and protein was prepared from each fraction. Anti-nucleolin antibody interacted with the 100- and 95-kD proteins present in the whole-cell lysate. The 100-kD protein was detected in nuclear and nucleolar fractions. The 95-kD protein was detected in cytosolic and nucleoplasmic fractions. PP1delta and nucleolin were co-localized in the nucleolus in MG63 and Saos-2 cells revealed by an immunofluorescence method. PP1delta and nucleolin were also co-immunoprecipitated with anti-nucleolin and anti-PP1delta antibodies. In the actinomycin D-treated cells, the subcellular localization of PP1delta and nucleolin was changed. Expression of PP1delta was upregulated with actinomycin D treatment. The level of 100-kD protein did not change in the actinomycin D-treated cells. However, the level of the 95-kD band increased with actinomycin D treatment. These results indicate that PP1delta was associated with nucleolin in the nucleolus of MG63 and Saos-2 cells and that nucleolin is a possible candidate substrate for PP1delta.

DOI： 10.1177/002215540205000905

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Peplomycin-induced apoptosis in oral squamous carcinoma cells depends on bleomycin sensitivity Reviewed

H Okamura, H Morimoto, T Haneji

ORAL ONCOLOGY 37 ( 4 ) 379 - 385 2001.6

Language：English Publishing type：Research paper (scientific journal) Publisher：PERGAMON-ELSEVIER SCIENCE LTD

Oral squamous carcinoma cell line SSCKN cells were shown to be highly sensitive to bleomycin, whereas SCCTF cells were minimally sensitive to this reagent. To determine whether the anticancer drug resistance to oral squamous carcinoma cells could be related to the degree of the drug-induced apoptosis, we examined the effects of peplomycin on induction of apoptosis in these cells. After reaching subconfluence, SCCKN and SCCTF cells were exposed to various concentrations of peplomycin. Peplomycin caused cytotoxicity in both SCCKN and SCCTF cells in a dose-dependent fashion with the maximal effect at concentrations of 1 and 10 muM, respectively, as determined by phase-contrast microscopy and WST-1 cell viability assay. By using the Hoechst 33342 staining, we observed marked nuclear condensation and fragmentation of chromatin in SCCKN cells treated with 1 muM peplomycin. How ever, SCCTF cells treated with 1 muM peplomycin showed neither nuclear condensation nor fragmentation. DNA ladder formation was also detected in both cell lines by treatment with peplomycin. The induced DNA ladder formation in SCCKN and SCCTF cells was dose-dependent, with the maximal effect at concentrations of 5 and 50 muM, respectively. Bleomycin also induced DNA ladder formation in SCCKN and SCCTF cells with different sensitivities. Mitomycin C induced DNA laddering in both SCCKN and SCCTF cells; however, the intensity of DNA ladder formation was almost the same in both cell lines. The present results indicate that peplomycin-induced apoptosis in oral squamous carcinoma cell lines depends on the sensitivity of these cells to bleomycin. (C) 2001 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S1368-8375(00)00101-9

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Alteration of argyrophilic nucleolar organizer region associated (Ag-NOR) proteins in apoptosis-induced human salivary gland cells and human oral squamous carcinoma cells Reviewed

Y Morimoto, S Kito, T Ohba, H Morimoto, H Okamura, T Haneji

JOURNAL OF ORAL PATHOLOGY & MEDICINE 30 ( 4 ) 193 - 199 2001.4

Language：English Publishing type：Research paper (scientific journal) Publisher：MUNKSGAARD INT PUBL LTD

The level of argyrophilic nucleolar organizer regions (AgNORs) and AgNOR-associated proteins (Ag-NOR proteins) varies with cell activity, including ribosomal biogenesis occurring in proliferating cells. Proteins associated with some AgNORs are detected by a specific silver staining. To investigate a possible relationship between apoptosis and the AgNORs or Ag-NOR proteins, we examined the changes of AgNORs and Ag-NOR proteins during apoptosis in a human salivary gland cell line, HSG cells, and a human oral squamous carcinoma cell line, SCC-25 cells. Apoptosis was induced by treatment of HSG and SCC-25 cells with okadaic acid. Proteins prepared from HSG and SCC-25 cells treated with varying concentrations of okadaic acid (OA) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by transferring to transfer membranes and staining for Ag-NOR proteins by modified Western blot analysis. Four major bands (110 kDa, 43 kDa, 39kDa, and 37 hDa) were detected in the proteins obtained from the control cells. The level of the 110-kDa protein decreased in the proteins prepared from OA-induced apoptotic cells; however, the reaction intensity of the other three bands was changed in apoptotic cells. An additional band of an 80-kDa Ag-NOR protein appeared and increased in the apoptotic cells. Cellular fractionation of HSG cells and SCC-25 cells was done with or without apoptotic induction. An 80-kDa Ag-NOR protein was detected in the nuclear fraction prepared from the apoptotic cells, while the 110-kDa protein decreased in the nuclear fraction of these cells. The 110-kDa Ag-NOR protein may be nucleolin (C23) as deduced from its AgNOR staining features, including molecular weight. The 80-kDa protein may be the cleavage product of the 110-kDa protein, in the cell-free apoptotic system, in which intact nuclei of HSG cells were incubated with the cytosol fraction of apoptotic HSG and SCC-25 cells, the 80-kDa Ag-NOR protein was detected in nuclei incubated with the cytosol fraction of apoptotic cells, while the level of the 110-kDa protein decreased. The changes of Ag-NOR proteins in nuclei prepared from SCC-25 cells incubated with cytosol fractions prepared from HSG and SCC-25 cells were identical to those of the HSG cells. The alternation of AgNORs in apoptosis-induced HSG cells was also examined using double staining with Hoechst 33342 and silver nitrate. Hoechst staining revealed typical apoptotic nuclei, which exhibited highly fluorescent condensed chromatin in OA-treated HSG cells. Silver grains representing AgNORs were not detected in the cells undergoing apoptosis. The dual-imposition view confirmed that AgNORs, which are visible as dots in nucleoli in the control cells, disappeared from the apoptotic nuclei of HSG cells. Our results indicate that the 110-kDa nucleolar Ag-NOR protein is associated with apoptosis and is cleaved during apoptosis.

DOI： 10.1034/j.1600-0714.2001.300401.x

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Upregulation of the expression of Fas antigen and Fas ligand in a human submandibular gland ductal cell line by okadaic acid Reviewed

Y Morimoto, H Morimoto, H Okamura, K Nomiyama, N Nakamuta, S Kobayashi, S Kito, T Ohba, T Haneji

ARCHIVES OF ORAL BIOLOGY 45 ( 8 ) 657 - 666 2000.8

Language：English Publishing type：Research paper (scientific journal) Publisher：PERGAMON-ELSEVIER SCIENCE LTD

Fas receptor is a member of a superfamily of receptors characterized by cysteine-rich motifs in the extracellular domain of the molecule. Binding of Fas ligand to the receptor leads to receptor activation and the induction of intracellular signals that result in apoptotic cell death. In the present study, the expression of mRNA and proteins of Fas receptor and Fas ligand were examined in human submandibular gland ductal (HSG) cells treated with okadaic acid by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analysis. Six hundred and eighty-two bp of the PCR product of Fas receptor mRNA was detected in HSG cells and a protein with an estimated molecular weight of 58,000 was expressed in HSG cells. Treatment of HSG cells with an agonistic anti-Fas monoclonal antibody resulted in death of HSG cells, indicating that the functional Fas receptor protein is expressed in HSG cells. Fas receptor protein expression stimulated by okadaic acid was elevated in a dose- and time-dependent manner, with maximal expression at 20 nM and 48 h treatment. Fas ligand mRNA was also detected constitutively in HSG cells by RT-PCR. Okadaic acid stimulated the expression of Fas ligand protein in HSG cells in a time-dependent manner, while the expression of the ligand was low in untreated HSG cells. The molecular weight of Fas ligand was estimated as 68,000. An antagonistic anti-Fas ligand monoclonal antibody prevented okadaic acid-induced death in HSG cells in a dose-dependent fashion as determined by WST-1 assay. The results indicate that the expression of Fas receptor and ligand is regulated by protein phosphatase(s) sensitive to okadaic acid and is involved in okadaic acid-induced apoptosis in HSG cells, The results also suggest that the Fas receptor-ligand system might regulate apoptosis in HSG cells. (C) 2000 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S0003-9969(00)00038-8

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Papers - OKAMURA Hirohiko