Papers - IKEDA Ryuji
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Ushiyama M., Ikeda R., Nitta T., Tazitsu Y., Miyawaki A., Yamaguchi T., Shimodouzono Y., Ushinohama K., Matsui R., Sugihara K., Nakamura N., Yamada K.
Oral Therapeutics and Pharmacology 27 ( 3 ) 143 - 150 2008.12
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Oral Therapeutics and Pharmacology
In our hospital, hospital preparations of Azunol Saline Gargle (AS, saline solution containing 0.006% sodium gualenate hydrate), and Azunol Lidocaine Saline Gargle (ALS, AS with lidocaine) are used as a treatment for oral cancer with oral mucositis. However, little is known about the stability and microbiological safety of AS and ALS. In this study, the stability and microbiological safety of AS and ALS were assessed as the pH and the percent of sodium gualenate hydrate remaining in both preparations after exposure to various light and temperature conditions and the colony formation, respectively. As a result, we found that under fluorescent lamp lighting, AS and ALS were stable for 7 days at 4°C compared with 25°C or room temperature. Furthermore, by light shielding, they were stable for at least 14 days at 4°C. Bacterial contamination of AS was prevented by preserving at 4°C for 14 days. We have demonstrated the stability and microbiological safety of AS and ALS and established an appropriate preservation method. This study provides useful information regarding the management of oral mucositis in oral cancer patients.
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Copper transport systems are involved in multidrug resistance and drug transport
Furukawa T., Komatsu M., Ikeda R., Tsujikawa K., Akiyama S.
Current Medicinal Chemistry 15 ( 30 ) 3268 - 3278 2008.12
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Current Medicinal Chemistry
Copper is an essential trace element and several copper containing proteins are indispensable for such processes as oxidative respiration, neural development and collagen remodeling. Copper metabolism is precisely regulated by several transporters and chaperone proteins. Copper Transport Protein 1 (CTR1) selectively uptakes copper into cells. Subsequently three chaperone proteins, HAH1 (human atx1 homologue 1), Cox17p and CCS (copper chaperone for superoxide dismutase) transport copper to the Golgi apparatus, mitochondria and copper/zinc superoxide dismutase respectively. Defects in the copper transporters ATP7A and ATP7B are responsible for Menkes disease and Wilson's disease respectively. These proteins transport copper via HAH1 to the Golgi apparatus to deliver copper to cuproenzymes. They also prevent cellular damage from an excess accumulation of copper by mediating the efflux of copper from the cell. There is increasing evidence that copper transport mechanisms may play a role in drug resistance. We, and others, found that ATP7A and ATP7B are involved in drug resistance against the anti-tumor drug cis-diamminedichloroplatinum (II) (CDDP). A relationship between the expression of ATP7A or ATP7B in tumors and CDDP resistance is supported by clinical studies. In addition, the copper uptake transporter CTR1 has also been reported to play a role in CDDP sensitivity. Furthermore, we have recently found that the effect of ATP7A on drug resistance is not limited to CDDP. Using an ex vivo drug sensitivity assay, the histoculture drug response assay (HDRA), the expression of ATP7A in human surgically resected colon cancer cells correlated with sensitivity to 7-ethyl-10-hydroxy-camptothecin (SN-38). ATP7Aoverexpressing cells are resistant to many anticancer drugs including SN-38, 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11), vincristine, paclitaxel, etoposide, doxorubicin (Dox), and mitoxantron. The mechanism by which ATP7A and copper metabolism modulate drug transport appears to involve modulation of drug cellular localization via modulation of the vesicle transport system. In ATP7A overexpressing cells, Dox accumulates in the Golgi apparatus. In contrast, in the parental cells, Dox is localized in the nuclei, where the target molecules of Dox, topoisomerase II and DNA, are found. Disruption of the intracellular vesicle transport system with monensin, a Na+/H+ ionophore, induced the relocalization of Dox from the Golgi apparatus to the nuclei in the ATP7A overexpressing cells. These data suggested that ATP7A-related drug transport is dependent on the vesicle transport system. Thus copper transport systems play important roles in drug transport as well as in copper metabolism. Components of copper metabolism are therefore likely to include target molecules for the modulation of drug potency of not only anti-cancer agents but also of other drugs. © 2008 Bentham Science Publishers Ltd.
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Zhao H., Ooyama A., Yamamoto M., Ikeda R., Haraguchi M., Tabata S., Furukawa T., Che X., Iwashita K., Oka T., Fukushima M., Nakagawa M., Ono M., Kuwano M., Akiyama S.
Cancer Letters 270 ( 1 ) 156 - 163 2008.10
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Cancer Letters
5-FU is commonly used for treatment of various solid tumors including colon carcinoma. We have previously demonstrated that Egr-1 induced by 5-FU enhanced TSP-1 expression in human colon cancer KM12C cells. In this study, a Genechip analysis of KM12C cells treated with 5-FU revealed down-regulation of 924 genes and up-regulation of 460 genes. The decreased expression of c-Myc mRNA and phosphorylated c-Myc were detected and confirmed by RT-PCR and immunoblotting. Since 5-FU induced the expression of TSP-1, we examined the effect of c-Myc on the TSP-1 promoter. Deletion of the TSP-1 promoter region in which binding sites for c-Myc reside had no effect on the TSP-1 promoter activity induced by 5-FU. Meanwhile, 5-FU dose-dependently decreased the expression of miR-17-92 cluster. These findings suggest that 5-FU decreased the expression of c-Myc and consequently miR-17-92 cluster and increased the expression of TSP-1 mRNA. © 2008 Elsevier Ireland Ltd. All rights reserved.
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Ikeda R., Iwashita K., Sumizawa T., Beppu S., Tabata S., Tajitsu Y., Shimamoto Y., Yoshida K., Furukawa T., Che X., Yamaguchi T., Ushiyama M., Miyawaki A., Takeda Y., Yamamoto M., Zhao H., Shibayama Y., Yamada K., Akiyama S.
Experimental Cell Research 314 ( 16 ) 3017 - 3026 2008.10
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Experimental Cell Research
The major vault protein (MVP) is the major constituent of the vault particle, the largest ribonuclear protein complex described to date and is identical to lung resistance-related protein (LRP). Although MVP is also expressed in several normal tissues, little is known about its physiological role. MVP played a protective role against some xenobiotics and other stresses. We thus investigated the effect of osmotic stress on MVP expression by treating human colon cancer SW620 cells with sucrose or NaCl. The expression level of both MVP protein and MVP mRNA was increased by the osmostress. Sucrose or sodium chloride could also enhance MVP promoter activity. Inhibition of p38 MAPK in SW620 cells by SB203580 inhibited the expression of MVP under hyperosmotic stress. These findings suggested that osmotic stress up-regulated the MVP expression through p38 MAPK pathway. Down-regulation of MVP expression by MVP interfering RNA (RNAi) in SW620 cells increased the sensitivity of the cells to hyperosmotic stress and enhanced apoptosis. Furthermore, MVP RNAi prevented the osmotic stress-induced, time-dependent increase in phosphorylated Akt. These findings suggest that the PI3K/Akt pathway might be implicated in the cytoprotective effect of MVP. Our data demonstrate that exposure of cells to hyperosmotic stress induces MVP that might play an important role in the protection of the cells from the adverse effects of osmotic stress. © 2008 Elsevier Inc. All rights reserved.
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Molecular basis for the induction of an angiogenesis inhibitor, thrombospondin-1, by 5-fluorouracil
Zhao H., Ooyama A., Yamamoto M., Ikeda R., Haraguchi M., Tabata S., Furukawa T., Che X., Zhang S., Oka T., Fukushima M., Nakagawa M., Ono M., Kuwano M., Akiyama S.
Cancer Research 68 ( 17 ) 7035 - 7041 2008.9
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Cancer Research
5-Fluorouracil (5-FU) is one of the most commonly used anticancer drugs in chemotherapy against various solid tumors. 5-FU dose-dependently increased the expression levels of intrinsic antiangiogenic factor thrombospondin-1 (TSP-1) in human colon carcinoma KM12C cells and human breast cancer MCF7 cells. We investigated the molecular basis for the induction of TSP-1 by 5-FU in KM12C cells. Promoter assays showed that the region with the Egr-1 binding site is critical for the induction of TSP-1 promoter activity by 5-FU. The binding of Egr-1 to the TSP-1 promoter was increased in KM12C cells treated with 5-FU. Immunofluorescence staining revealed that 5-FU significantly increased the level of Egr-1 in the nuclei of KM12C cells. The suppression of Egr-1 expression by small interfering RNA decreased the expression level of TSP-1. Furthermore, 5-FU induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and heat shock protein 27 (HSP27). Blockade of the p38 MAPK pathway by SB203580 remarkably inhibited the phosphorylation of HSP27 induced by 5-FU and decreased the induction of Egr-1 and TSP-1 by 5-FU in KM12C cells. These findings suggest that the p38 MAPK pathway plays a crucial role in the induction of Egr-1 by 5-FU and that induced Egr-1 augments TSP-1 promoter activity, with the subsequent production of TSP-1 mRNA and protein. ©2008 American Association for Cancer Research.
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Thymidine phosphorylase inhibits the expression of proapoptotic protein BNIP3
Ikeda R., Tajitsu Y., Iwashita K., Che X., Yoshida K., Ushiyama M., Furukawa T., Komatsu M., Yamaguchi T., Shibayama Y., Yamamoto M., Zhao H., Arima J., Takeda Y., Akiyama S., Yamada K.
Biochemical and Biophysical Research Communications 370 ( 2 ) 220 - 224 2008.5
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Biochemical and Biophysical Research Communications
An angiogenic factor, thymidine phosphorylase (TP), confers resistance to apoptosis induced by hypoxia. We investigated the molecular basis for the suppressive effect of TP on hypoxia-induced apoptosis using Jurkat cells transfected with TP cDNA, Jurkat/TP, and a mock transfectant, Jurkat/CV. TP and 2-deoxy-d-ribose, a degradation product of thymidine generated by TP enzymatic activity, suppressed hypoxia-induced apoptosis. They also inhibited the upregulation of hypoxia-inducible factor (HIF) 1α and the proapoptotic factor, BNIP3, and caspase 3 activation induced by hypoxia. Introduction of siRNA against BNIP3 in Jurkat cells decreased the proportion of apoptotic cells under hypoxic condition. These findings suggest that the suppression of BNIP3 expression by TP prevents, at least in part, hypoxia-induced apoptosis. Expression levels of TP are elevated in many malignant solid tumors and thus 2-deoxy-d-ribose generated by TP in these tumors might play an important role in tumor progression by preventing hypoxia-induced apoptosis. © 2008 Elsevier Inc. All rights reserved.
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Saigo K., Yoshida K., Ikeda R., Sakamoto Y., Murakami Y., Urashima T., Asano T., Kenmochi T., Inoue I.
Human Mutation 29 ( 5 ) 703 - 708 2008.5
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Human Mutation
Integration of hepatitis B virus (HBV) DNA into host DNA is detected in about 90% of HBV-related hepatocellular carcinoma (HCC), but the preferential sites of the viral integration etiologically relevant to oncogenesis have been controversial. By using an adaptor-ligation/suppression-PCR, we identified four integrations into the myeloid/lymphoid or mixed-lineage leukemia 4 (MLL4) gene from 10 HCC patients with positive HBV surface antigen (HBsAg). Determination of the cellular-virus DNA junction demonstrated that various lengths of the virus were integrated within 300 bp of intron 3 flanked by the Alu element of MLL4. Chimeric hepatitis B virus X gene (HBx)/MLL4 transcripts and the HBx fusion proteins were detected. DNA microarray revealed that HBx/MLL4 fusion proteins suppressed unique genes in HepG2 cells. Finally, chromosomal translocations of intron 3 of MLL4 to the specific region of chromosome 17p11.2 in 22 out of 32 HCC patients were observed, showing that the intron 3 region of MLL4 gene would be a target of translocation breakpoint. In conclusion, the present data suggest that the translocation breakpoint of MLL4 gene is one of the preferential targets for HBV DNA integration into the MLL4 gene and the HBV DNA integration may be involved in liver oncogenesis. © 2008 Wiley-Liss, Inc.
DOI： 10.1002/humu.20701
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Yokomakura N., Natsugoe S., Okumura H., Ikeda R., Uchikado Y., Mataki Y., Takatori H., Matsumoto M., Owaki T., Ishigami S., Aikou T.
Oncology Reports 18 ( 3 ) 561 - 567 2007.9
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Oncology Reports
Chemoradiation therapy (CRT), a combination of X-ray irradiation and anticancer agents as a radiosensitizer, has been found to be an effective treatment for esophageal cancer and has been linked to p53 genetics. The p53 gene family regulates cell-cycle arrest, apoptosis and DNA damage repair. A recently identified ribonucleotide reductase, p53R2, is directly regulated by p53 in the supply of nucleotides for repairing damaged DNA. In the present study, we investigated the improvement in radiosensitivity of human esophageal squamous cell carcinoma (ESCC) cell lines using p53R2 small interfering RNA (siRNA). p53R2 expression in ESCC cells (TE-8) with or without transfection of p53R2 siRNA was examined by Western blot analysis and reverse transcription-polymerase chain reaction (RT-PCR). The radiosensitivity of TE-8 cells was also measured by cell survival assay. In addition, we investigated the relationship between the expression of p53R2 mRNA in the biopsy specimens of untreated primary tumors and the efficacy of CRT, using RT-PCR. The expression of p53R2 was amplified after X-ray irradiation (14 Gy) and diminished after X-ray irradiation following the transfection of p53R2 siRNA in TE-8 cells. The radiosensitivity of the TE-8 cells significantly improved following the transfection of p53R2 siRNA. In the clinical study, a significantly lower p53R2 mRNA expression was detected in the effective response cases. We demonstrated that p53R2 is associated with the radiosensitivity of ESCC cell lines, and that p53R2 expression is reduced after X-ray irradiation following the transfection of p53R2 siRNA. This protocol could potentially improve the efficacy of radiation therapy.
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Gene expression changes during the chondrogenic differentiation of human mesenchymal stem cells
Ikeda R., Tsukaara S., Yoshida K., Inoue I.
Journal of Biological Sciences 7 ( 5 ) 729 - 736 2007.7
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Biological Sciences
To gain more insight into the molecular mechanisms of chondrogenic differentiation induced by transforming growth factor (TGF)-β1 and insulin-like growth factor (IGF)-I and insulin, we performed cDNA microarray experiments during the chondrogenic differentiation of human mesenchymal stem cells (hMSCs), which provide an excellent in vitro model system for chondrogenesis. Our repeated cDNA microarray analyses identified the up regulation of 23 transcripts and the down regulation of 25 transcripts after 14 days of chondrogenic induction. We found that many of the up regulated and down regulated genes belonged to overlapping gene categories; specifically, 44 and 40% of the up regulated and down regulated genes were associated with the extracellular matrix and metabolic pathways, respectively. The expression of the identified genes was confirmed by RT-PCR. These analyses suggest that the transcriptional control induced by TGF-β1, IGF-I and insulin signaling during the chondrogenic differentiation of hMSCs is mainly targeted to genes belonging to specialized gene categories. © 2007 Asian Network for Scientific Information.
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Ushiyama M., Ikeda R., Sugawara H., Yoshida M., Mori K., Kangawa K., Inoue K., Yamada K., Miyata A.
Molecular Pharmacology 72 ( 1 ) 103 - 111 2007.7
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Molecular Pharmacology
Pituitary adenylate cyclase-activating polypeptide (PACAP), a pleiotropic neuropeptide, performs a variety of physiological functions. The PACAP-specific receptor PAC1 has several variants that result mainly from alternative splicing in the mRNA regions encoding the first extracellular (EC1) domain and the third intracellular cytoplasmic (IC3) loop. The effects on down-stream signaling produced by combinations of alternative splicing events in the EC1 domain and IC3 loop have not yet been clarified. In this study, we have used semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to examine the tissue distributions of four PAC1 isoforms in mice. We then established cell lines constitutively expressing each of the PAC1 isoforms and characterized the binding properties of each isoform to PACAP-38, vasoactive intestinal polypeptide (VIP), and the PAC1-specific agonist maxadilan, as well as the resulting effects on two major intracellular signaling pathways: cAMP production and changes in the intracellular calcium concentration. The results demonstrate that the variants of the IC3 loop affect the binding affinity of the ligands for the receptor, whereas the variants of the EC1 domain primarily affect the intracellular signaling downstream of PAC1. Accordingly, this study indicates that the combination of alternative splicing events in the EC1 domain and the IC3 loop create a variety of PAC1 isoforms, which in turn may contribute to the functional pleiotropism of PACAP. This study not only contributes to the understanding of the multiple functions of PACAP but also helps to elucidate the relationship between the structures and functions of G-protein-coupled receptors. Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics.
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Molecular basis for the inhibition of anticancer agents-induced apoptosis by thymidine phosphorylase
Ikeda R.
Yakugaku Zasshi 127 ( 7 ) 1097 - 1102 2007.7
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Yakugaku Zasshi
An angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-D-ribose, a degradation product of thymidine generated by TP enzymatic activity, partially prevented hypoxia-induced apoptosis. TP was expressed at higher levels in tumor tissues compared to the adjacent non-neoplastic tissues in a variety of human carcinomas. High expression of TP is associated with an unfavorable prognosis. To investigate the effect of TP on cisplatin-induced apoptosis, human leukemia Jurkat cells were transfected with wild-type or mutant (L148R) TP cDNA. Jurkat cells transfected with TP cDNA (Jurkat/TP) and mutant TP cDNA (Jurkat/TPMu) expressed high levels of TP, while Jurkat/CV cells which were transfected with a control vector did not express TP. A high TP enzyme activity was detected in Jurkat/TP cells, but not in Jurkat/CV and Jurkat/TPMu cells. Sensitivities to cisplatin of these cells were determined by MTT assay. IC50 values for cisplatin of Jurkat/CV, Jurkat/TP, and Jurkat/TPMu cells were 4.50, 14.08, 13.40 μM, respectively. Jurkat/TP and Jurkat/TPMu cells were about three times more resistant to cisplatin than Jurkat/CV cells. TP inhibited activation of caspase 3, 9 and mitochondrial cytochrome c release induced by cisplatin. These findings suggest a mechanism by which TP confers the resistance to cisplatin-induced apoptosis. Moreover, mutant TP that has no enzymatic activity also suppressed the cisplatin-induced apoptosis. These suggest that TP molecules have cytoprotective functions against cytotoxic agents. © 2007 The Pharmaceutical Society of Japan.
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Effect of pre-treatment with St John's Wort on nephrotoxicity of cisplatin in rats
Shibayama Y., Kawachi A., Onimaru S., Tokunaga J., Ikeda R., Nishida K., Kuchiiwa S., Nakagawa S., Takamura N., Motoya T., Takeda Y., Yamada K.
Life Sciences 81 ( 2 ) 103 - 108 2007.6
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Life Sciences
A herbal health care supplement, St John's Wort (SJW, Hypericum perforatum) has become widely used in the treatment of depression, and is known to interact with therapeutic drugs. Here we report a preventive effect of SJW on cisplatin nephrotoxicity in rats. Rats were given SJW (400 mg/kg/day, p.o.) for 10 consecutive days, and were injected with cisplatin (5 mg/kg, i.v.) on the day after the final SJW treatment. Cisplatin treatment increased the serum creatinine level, which is an index of nephrotoxicity, to 1.51 ± 0.22 mg/dl (mean ± SE) from 0.28 ± 0.05 mg/dl (control) on day 5 after the cisplatin injection. This increase fell significantly to 0.86 ± 0.13 mg/dl by pre-treatment with SJW. Cisplatin-induced histological abnormality of the kidney was blocked by pre-treatment with SJW. When SJW was administered for 10 days, the amounts of renal metallothionein (MT) and hepatic multidrug resistance protein 2 (Mrp2) were increased to 164.8 ± 13.0% and 220.8 ± 39.3% (mean ± SE) of controls, respectively. GSH levels in the kidney and liver were not changed. Total and free cisplatin concentration in serum was not influenced by SJW treatment. In conclusion, the results suggest that pre-treatment with SJW may diminish cisplatin nephrotoxicity. © 2007 Elsevier Inc. All rights reserved.
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Komatsu M., Furukawa T., Ikeda R., Takumi S., Nong Q., Aoyama K., Akiyama S., Keppler D., Takeuchi T.
Toxicological Sciences 97 ( 2 ) 407 - 416 2007.6
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Toxicological Sciences
The serine/threonine protein phosphatase (PP) 2A inhibitor, microcystin-LR, selectively induces liver damage and promotes hepatocarcinogenesis. It is thought that microcystin-LR affects hepatocellular viability mainly through inhibition of PP2A, partially through PP1, and, in addition, by generation of reactive oxygen species (ROS). However, the molecular basis of the selective liver damage and the balance between cell death and survival remained unclear. We analyzed the cytotoxicity of low doses of microcystin-LR using HEK293 cells stably expressing the human hepatocyte uptake transporters, organic anion transporting polypeptide (OATP)1B1 (HEK293-OATP1B1 cells) and OATP1B3 (HEK293-OATP1B3 cells). HEK293-OATP1B1 (IC50 6.6nM) and HEK293-OATP1B3 cells (IC50 6.5nM) were equally very sensitive to microcystin-LR. In contrast, control-vector-transfected (HEK293-CV) cells were resistant to microcystin-LR. Using HEK293-OATP1B3 cells, the cytotoxicity was attenuated by substrates and inhibitors of OATP1B3, including bromosulfophthalein, rifampicin, and cyclosporin A. Microcystin-LR was transported into HEK293-OATP1B3 cells with 1.2mM Km value, and its uptake was inhibited by above substances. Accumulation of microcystin-LR in the HEK293-OATP1B1 and HEK293-OATP1B3 cells was increased in a dose-dependent manner but not in HEK293-CV cells. Cellular serine/threonine PP activity of HEK293-OATP1B3 cells was decreased by microcystin-LR but not in HEK293-CV cells. Apoptotic changes were observed after incubation of the HEK293-OATP1B3 cells with microcystin-LR. We found by FACS analysis that microcystin-LR induced apoptosis but not necrosis in HEK293-OATP1B3 cells. Microcystin-LR activated several mitogen-activated protein kinases (MAPKs) including ERK1/2, JNK, and p38 through inhibition of PP2A. In addition, the cytotoxicity of microcystin-LR was attenuated by the inhibitors of MAPK pathways, including U0126, SP600125, and SB203580. The ROS scavenger N-acetyl-L-cysteine partially attenuated the cytotoxicity of microcystin-LR. Thus, the present study demonstrates that microcystin-LR induces apoptosis through activation of multiple MAPK pathways subsequent to its selective uptake via OATP1B1 and OATP1B3 and followed by inhibition of PP2A, in addition to the ROS generation which might contribute to apoptosis. © The Author 2007. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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Owatari S., Akune S., Komatsu M., Ikeda R., Firth S., Che X., Yamamoto M., Tsujikawa K., Kitazono M., Ishizawa T., Takeuchi T., Aikou T., Mercer J., Akiyama S., Furukawa T.
Cancer Research 67 ( 10 ) 4860 - 4868 2007.5
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Cancer Research
We and others have shown that the copper transporters ATP7A and ATP7B play a role in cellular resistance to cis-diaminedichloroplatinum (II) (CDDP). In this study, we found that ATP7A transfection of Chinese hamster ovary cells (CHO-K1) and fibroblasts isolated from Menkes disease patients enhanced resistance not only to CDDP but also to various anticancer drugs, such as vincristine, paclitaxel, 7-ethyl-10-hydroxy-camptothecin (SN-38), etoposide, doxorubicin, mitoxantron, and 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11). ATP7A preferentially localized doxorubicin fluorescence to the Golgi apparatus in contrast to the more intense nuclear staining of doxorubicin in the parental cells. Brefeldin A partially and monensin completely altered the distribution of doxorubicin to the nuclei in the ATP7A-expressing cells. ATP7A expression also enhanced the efflux rates of doxorubicin and SN-38 from cells and increased the uptake of SN-38 in membrane vesicles. These findings strongly suggested that ATP7A confers multidrug resistance to the cells by compartmentalizing drugs in the Golgi apparatus and by enhancing efflux of these drugs, and the trans-Golgi network has an important role of ATP7A-related drug resistance. ATP7A was expressed in 8 of 34 (23.5%) clinical colon cancer specimens but not in the adjacent normal epithelium. Using the histoculture drug response assay that is useful for the prediction of drug sensitivity of clinical cancers, ATP7A-expressing colon cancer cells were significantly more resistant to SN-38 than ATP7A-negative cells. Thus, ATP7A confers resistance to various anticancer agents on cancer cells and might be a good index of drug resistance in clinical colon cancers. ©2007 American Association for Cancer Research.
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Ikeda R., Yoshida K., Inoue I.
Journal of Cellular Biochemistry 101 ( 1 ) 147 - 154 2007.5
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Cellular Biochemistry
Bone morphogenetic protein 2 (BMP2) is a key factor in the regulation of osteoblastic differentiation; however, its downstream mediators are not fully understood. Previously, we identified and characterized transcription factor promyelocytic leukemia zinc finger protein (PLZF), composed of an N-terminal BTB/POZ and C-terminal zinc finger motifs, as an upstream factor of CBFA1 (Runx2/core-binding factor 1). PLZF was induced in an osteoblastic differentiation medium, but was not induced by BMP2. Here, we report the identification of transcription factor fanconi anemia zinc finger protein (FAZF), which is closely related to PLZF. FAZF was induced by BMP2 in human mesenchymal stem cells (hMSCs). In addition to the full-length FAZF, we also identified alternatively spliced mRNAs in which the C-terminal zinc finger motifs were deleted (designated BTB/POZ-only FAZF). Both the full-length and BTB/POZ-only FAZF mRNAs were equally expressed in BMP2-treated hMSCs. The full-length FAZF was exclusively detected in the nucleus, whereas the BTB/POZ-only FAZF protein was localized in the cytoplasm of the transfected cells. The full-length FAZF, but not the BTB/ POZ-only FAZF, increased the expression of osteoblastic differentiation markers, including CBFA1, collagen 1A1, osteocalcin, and alkaline phosphatase in C2C12 cells. In conclusion, both FAZF and PLZF differentially participate in the regulation of osteoblastic differentiation via the BMP2 and CBFA1 signaling pathways, respectively. © 2006 Wiley-Liss, Inc.
DOI： 10.1002/jcb.21165
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Hayashi R., Goto Y., Ikeda R., Yokoyama K., Yoshida K.
Journal of Biological Chemistry 281 ( 47 ) 35633 - 35648 2006.11
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Biological Chemistry
The TRIP-Br1/p34 SEI-1 family proteins participate in cell cycle progression by coactivating E2F1- or p53-dependent transcriptional activation. Here, we report the identification of human CDCA4 (also know as SEI-3/Hepp) as a novel target gene of transcription factor E2F and as a repressor of E2F-dependent transcriptional activation. Analysis of CDCA4 promoter constructs showed that an E2F-responsive sequence in the vicinity of the transcription initiation site is necessary for the E2F1-4-induced activation of CDCA4 gene transcription. Chromatin immunoprecipitation analysis demonstrated that E2F1 and E2F4 bound to an E2F-responsive sequence of the human CDCA4 gene. Like TRIP-Br1/p34 SEI-1 and TRIP-Br2 (SEI-2), the transactivation domain of CDCA4 was mapped within C-terminal acidic region 175-241. The transactivation function of the CDCA4 protein was inhibited by E2F1-4 and DP2, but not by E2F5-8. Inhibition of CDCA4 transactivation activity by E2F1 partially interfered with retinoblastoma protein overexpression. Conversely, CDCA4 suppressed E2F1-3-induced reporter activity. CDCA4 (but not acidic region-deleted CDCA4) suppressed E2F1-regulated gene promoter activity. These findings suggest that the CDCA4 protein functions as a suppressor at the E2F-responsive promoter. Small interfering RNA-mediated knockdown of CDCA4 expression in cancer cells resulted in up-regulation of cell growth rates and DNA synthesis. The CDCA4 protein was detected in several human cells and was induced as cells entered the G 1/S phase of the cell cycle. Taken together, our results suggest that CDCA4 participates in the regulation of cell proliferation, mainly through the E2F/retinoblastoma protein pathway. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.
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Shibayama Y., Ushinohama K., Ikeda R., Yoshikawa Y., Motoya T., Takeda Y., Yamada K.
Cancer Science 97 ( 11 ) 1260 - 1266 2006.11
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Cancer Science
The ATP binding cassette (ABC) transporters, multidrug resistance protein 2 (Mrp2; Abcc2) and breast cancer resistance protein (Bcrp; Abcg2), and organic anion transporters (Oats) mediate excretion of methotrexate (MTX) and many other drugs. However, it is not known whether MTX treatment leads to any changes in the expression of these transporters. We examined the effect of MTX treatment on expression of Mrp2, Bcrp and Oats in rats. MTX was single injected intraperitoneally at doses of 10, 50 and 150 mg/kg, and then Western blot analysis was performed. The levels of Mrp2, Oat1 and Oat2 on day 1 after the treatment showed no significant change. Four days after injection of 150 mg/kg MTX, the Mrp2 levels in the liver and ileum, but not in the kidney, were markedly down-regulated to 20 ± 3.6% and 8.9 ± 3.8% (mean ± SEM) of controls, respectively. Renal Oat1 and Oat3 were also down-regulated to 56.4 ± 4.3% (Oat1) and 54.3 ± 5.5% (Oat3) of controls. These effects of MTX were almost recovered by leucovorin which rescues normal cells from MTX toxicity. MTX treatment also decreased mRNA levels of constitutive androstane receptor (CAR) and pregnane X receptor (PXR) to 65.5 ± 17.9% and 59.6 ± 14.5% of controls in the liver, respectively. MTX treatment has no apparent effect on expression levels of Bcrp, cytochrome P450 2B6 and 3A1. In conclusion, these data indicate that MTX treatment down-regulates expression levels of Mrp2, Oat1 and Oat3, and its effects are recovered by leucovorin. © 2006 Japanese Cancer Association.
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Tsukahara S., Ikeda R., Goto S., Yoshida K., Mitsumori R., Sakamoto Y., Tajima A., Yokoyama T., Toh S., Furukawa K., Inoue I.
Biochemical Journal 398 ( 3 ) 595 - 603 2006.9
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Biochemical Journal
To better understand the molecular pathogenesis of OPLL (ossification of the posterior longitudinal ligament) of the spine, an ectopic bone formation disease, we performed cDNA microarray analysis on cultured ligament cells from OPLL patients. We found that TSG-6 (tumour necrosis factor α-stimulated gene-6) is down-regulated during osteoblastic differentiation. Adenovirus vector-mediated overexpression of TSG-6 inhibited osteoblastic differentiation of human mesenchymal stem cells induced by BMP (bone morphogenetic protein)-2 or OS (osteogenic differentiation medium). TSG-6 suppressed phosphorylation and nuclear accumulation of Smad 1/5 induced by BMP-2, probably by inhibiting binding of the ligand to the receptor, since interaction between TSG-6 and BMP-2 was observed in vitro. TSG-6 has two functional domains, a Link domain (a hyaluronan binding domain) and a CUB domain implicated in protein interaction. The inhibitory effect on osteoblastic differentiation was completely lost with exogenously added Link domain-truncated TSG-6, while partial inhibition was retained by the CUB domain-truncated protein. In addition, the inhibitory action of TSG-6 and the in vitro interaction of TSG-6 with BMP-2 were abolished by the addition of hyaluronan. Thus, TSG-6, identified as a down-regulated gene during osteoblastic differentiation, suppresses osteoblastic differentiation induced by both BMP-2 and OS and is a plausible target for therapeutic intervention in OPLL. © 2006 Biochemical Society.
DOI： 10.1042/BJ20060027
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Ikeda R., Yoshida K., Ushiyama M., Yamaguchi T., Iwashita K., Futagawa T., Shibayama Y., Oiso S., Takeda Y., Kariyazono H., Furukawa T., Nakamura K., Akiyama S., Inoue I., Yamada K.
Biological and Pharmaceutical Bulletin 29 ( 9 ) 1815 - 1819 2006.9
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Biological and Pharmaceutical Bulletin
Myoblasts respond to growth factor deprivation either by diffentiation into multinucleated myotubes or by undergoing apoptosis. The induction of apoptosis and differentiation in myogenic lineage may use overlapping cellular mechanisms. Here we demonstrate that the expression of the small heat shock protein αB-crystallin as well as MyoD and myogenin is induced during myogenic differentiation in C2C12 cells, and these inductions occur at an early stage in the differentiation in vitro. To investigate the effect of αB-crystallin on myogenic differentiation and apoptosis, C2C12 cells were infected with adenovirus vector bearing full-length αB-crystallin cDNA. Overexpression of αB-crystallin in C2C12 cells suppressed differentiation-induced apoptosis and activation of caspase 3, and also decreased the expression of MyoD and myogenin during myogenic differentiation of C2C12 cells induced by the differentiation medium. Our findings suggest that stress such as growth factor deprivation plays an important role in triggering apoptosis associated with myogenic differentiation and αB-crystallin suppressed the differentiation, apoptosis and caspase 3 activity. © 2006 Pharmaceutical Society of Japan.
DOI： 10.1248/bpb.29.1815
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Che X., Zheng C., Owatari S., Mutoh M., Gotanda T., Jeung H., Furukawa T., Ikeda R., Yamamoto M., Haraguchi M., Arima N., Akiyama S.
Blood 107 ( 12 ) 4880 - 4887 2006.6
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Blood
Patients with acute- or lymphoma-type adult T-cell leukemia (ATL) have a poor outcome because of the intrinsic drug resistance to chemotherapy. Protection from apoptosis is a common feature involved in multidrug-resistance of ATL. IAP (inhibitor of apoptosis) family proteins inhibit apoptosis induced by a variety of stimuli. In this study, we investigated the expression of IAP family members (survivin, cIAP1, cIAP2, and XIAP) in the primary leukemic cells from patients with ATL. We found that survivin was overexpressed in ATL, especially in acute-type ATL. Sodium arsenite was shown to down-regulate the expression of survivin at both the protein and RNA levels in a time- and dose-dependent manner, thus inhibiting cell growth, inducing apoptosis, and enhancing the caspase-3 activity in ATL cells. Nuclear factor-κB (NF-κB) enhances the transcriptional activity of survivin. Sodium arsenite suppressed the constitutive NF-κB activation by preventing the IκB-α degradation and the nuclear translocation of NF-κB. These findings suggest that survivin is an important antiapoptotic molecule that confers drug resistance on ATL cells. Sodium arsenite was shown to down-regulate the expression of survivin through the NF-κB pathway, thus inhibiting cell growth and promoting apoptosis of ATL cells. © 2006 by The American Society of Hematology.