研究者詳細 - 坂元　亨宇

論文 - 坂元　亨宇

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Ubiquitin-mediated proteasomal degradation of ABC transporters: a new aspect of genetic polymorphisms and clinical impacts

H. Nakagawa, Y. Toyoda, K. Wakabayashi-Nakao, H. Tamaki, M. Osumi and T. Ishikawa

J Pharm Sci 100 （ 9 ） 3602 - 19 2011年09月

ISSN 1520-6017

The interindividual variation in the rate of drug metabolism and disposition has been known for many years. Pharmacogenomics dealing with heredity and response to drugs is a part of science that attempts to explain variability of drug responses and to search for the genetic basis of such variations or differences. Genetic polymorphisms of drug metabolizing enzymes and drug transporters have been found to play a significant role in the patients' responses to medication. Accumulating evidence demonstrates that certain nonsynonymous polymorphisms have great impacts on the protein stability and degradation, as well as the function of drug metabolizing enzymes and transporters. The aim of this review article is to address a new aspect of protein quality control in the endoplasmic reticulum and to present examples regarding the impact of nonsynonymous single-nucleotide polymorphisms on the protein stability of thiopurine S-methyltransferase as well as ATP-binding cassette (ABC) transporters including ABCC4, cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), ABCC11, and ABCG2. Furthermore, we will discuss the molecular mechanisms underlying posttranslational modifications (intramolecular and intermolecular disulfide bond formation and N-linked glycosylation) and ubiquitin-mediated proteasomal degradation of ABCG2, one of the major drug transporter proteins in humans.

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Nrk, an X-linked protein kinase in the germinal center kinase family, is required for placental development and fetoplacental induction of labor

K. Denda, K. Nakao-Wakabayashi, N. Okamoto, N. Kitamura, J. Y. Ryu, Y. Tagawa, T. Ichisaka, S. Yamanaka and M. Komada

J Biol Chem 286 （ 33 ） 28802 - 10 2011年08月

ISSN 1083-351X

The complete mechanism of labor induction in eutherian mammals remains unclear. Although important roles for the fetus and placenta in triggering labor have been proposed, no gene has been shown to be required in the fetus/placenta for labor induction. Here we show that Nrk, an X-linked gene encoding a Ser/Thr kinase of the germinal center kinase family, is essential in the fetus/placenta for labor in mice. Nrk was specifically expressed in the spongiotrophoblast layer, a fetus-derived region of the placenta, and Nrk disruption caused dysregulated overgrowth of the layer. Due to preferential inactivation of the paternally derived X chromosome in placenta, Nrk heterozygous mutant placentas exhibited a similar defect to that in Nrk-null tissues when the wild-type allele was paternally derived. However, the phenotype was weaker than in Nrk-null placentas due to leaky Nrk expression from the inactivated X chromosome. Crossing of Nrk-null females to wild-type and Nrk-null males, as well as uterine transfer of Nrk-null fetuses to wild-type females, revealed that pregnant mice exhibit a severe defect in delivery when all fetuses/placentas are Nrk-null. In addition, Nrk was not expressed in female reproductive tissues such as the uterus and ovary, as well as the fetal amnion and yolk sac, in pregnant mice. Progesterone and estrogen levels in the maternal circulation and placenta, which control the timing of labor, were unaffected upon Nrk disruption. We thus provide evidence for a novel labor-inducing fetoplacental signal that depends on the X chromosome and possibly arises from the placenta.

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Identification by differential tissue proteome analysis of talin-1 as a novel molecular marker of progression of hepatocellular carcinoma

Kanamori, H., Kawakami, T., Effendi, K., Yamazaki, K., Mori, T., Ebinuma, H., Masugi, Y., Du, W., Nagasaka, K., Ogiwara, A., Kyono, Y., Tanabe, M., Saito, H., Hibi, T. and Sakamoto, M.

Oncology 80 （ 5-6 ） 406 - 15 2011年

ISSN 1423-0232

OBJECTIVE: Hepatocellular carcinoma (HCC) is characterized by a multistage process of tumor progression. This study addressed its molecular features to identify novel protein candidates involved in HCC progression. METHODS: Using liquid chromatography-tandem mass spectrometry, proteomes of 4 early HCCs and 4 non-HCC tissues derived from 2 cases of liver transplant surgery were compared with respect to the separation profiles of their tryptic peptides. Immunohistochemistry was performed on 106 HCC nodules to confirm the results of the proteomic analysis. RESULTS: Statistical analysis of the profiles selected the peptide peaks differentiating HCC from non-HCC. A database search of the tandem mass spectrometry data from those peptide peaks identified 61 proteins, including a cytoskeletal protein, talin-1, as upregulated in HCC. Talin-1 expression levels in HCC nodules were significantly associated with the dedifferentiation of HCC (p = 0.001). A follow-up survey of the examined clinical cases revealed a correlation between talin-1 upregulation and a shorter time to recurrence after resection (p = 0.039), which may be related to the higher rate of portal vein invasion in HCCs with talin-1 up-regulation (p = 0.029). CONCLUSIONS: Proteomic analysis led to identification of talin-1 as a promising HCC marker. Talin-1 upregulation is associated with HCC progression and may serve as a prognostic marker.

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Expression of Keratin 19 Is Related to High Recurrence of Hepatocellular Carcinoma after Radiofrequency Ablation

Tsuchiya, K., Komuta, M., Yasui, Y., Tamaki, N., Hosokawa, T., Ueda, K., Kuzuya, T., Itakura, J., Nakanishi, H., Takahashi, Y., Kurosaki, M., Asahina, Y., Enomoto, N., Sakamoto, M. and Izumi, N.

Oncology 80 （ 3-4 ） 278 - 288 2011年

ISSN 0030-2414

Objective: Keratin (K) 19 positivity has been reported to be a useful predictive marker for recurrence in patients with hepatocellular carcinoma (HCC) who have undergone hepatic resection. We investigated the clinical usefulness of K19 positivity in patients who had received curative radiofrequency ablation (RFA). Methods: We retrospectively evaluated the clinicopathological features, including imaging and K19 expression, in 246 patients with HCC who were within the Milan criteria and had received curative RFA. Using a two-step insertion method, tumor biopsies were obtained just prior to RFA and were evaluated histologically. Results: Tumor seeding due to liver biopsy and RFA was not observed. Ten patients (4.1%) had K19-positive HCC. Imaging findings were similar between K19-positive and -negative HCC (p = 0.187). Nine out of 10 patients (90%) who had K19-positive HCC had recurrence of HCC after RFA, and intrahepatic recurrences were observed within 12 months in 6 out of 10 (60.0%). K19 positivity was a significant risk factor for recurrence (p < 0.0001) and early recurrence (<1 year after RFA; p = 0.012). K19 expression (p = 0.016) was an independent risk factor for tumor status exceeding the Milan criteria after RFA. Conclusion: Expression of K19 is related to high recurrence of HCC after curative RFA. Copyright (C) 2011 S. Karger AG, Basel

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Reduced transforming growth factor-beta receptor II expression in hepatocellular carcinoma correlates with intrahepatic metastasis

Mamiya, T., Yamazaki, K., Masugi, Y., Mori, T., Effendi, K., Du, W. L., Hibi, T., Tanabe, M., Ueda, M., Takayama, T. and Sakamoto, M.

Laboratory Investigation 90 （ 9 ） 1339 - 1345 2010年09月

ISSN 0023-6837

Hepatocellular carcinoma (HCC) occurs mainly in the liver associated with chronic hepatitis and hepatic cirrhosis as a result of prolonged viral infection. Transforming growth factor-beta (TGF-beta) induces the fibrosis in hepatic cirrhosis, although it is also an inhibitor of hepatocyte proliferation. To understand the role of TGF-beta signaling in HCC progression, we analyzed gene expression in HCC cells in relation to TGF-beta signaling using a two-way clustering algorithm. By the analysis, five HCC cell lines were classified into two groups according to their metastatic capacity. TGF-beta receptor II (TGFBR2) was downregulated in metastatic cells, which did not show a response to TGF-beta. Immunohistochemistry demonstrated clear membrane distribution of TGFBR2 in noncancerous hepatocytes, whereas reduced TGFBR2 expression was observed in 34 of 136 HCCs. In clinical cases, reduced TGFBR2 expression correlated with larger tumor size (P<0.001), poor differentiation (P<0.001), portal vein invasion (P = 0.002), intrahepatic metastasis (IM) (P<0.001), and shorter recurrence-free survival (P 0.022). In conclusion, reduced TGFBR2 expression was associated with aggressive features of HCC such as IM, and may represent an immunohistochemical biomarker to detect aggressive HCC. Laboratory Investigation (2010) 90, 1339-1345; doi:10.1038/labinvest.2010.105; published online 7 June 2010

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Molecular diagnosis of multistage hepatocarcinogenesis

Sakamoto, M., Effendi, K. and Masugi, Y.

Jpn J Clin Oncol 40 （ 9 ） 891 - 6 2010年09月

ISSN 1465-3621

Human hepatocellular carcinoma is recognized as a good model for multistage carcinogenesis, as the malignant steps from chronic liver disease through to advanced human hepatocellular carcinoma are relatively clear. We address the activation of different molecular pathways during hepatocarcinogenesis that is especially useful in the diagnosis of pathological multistage human hepatocellular carcinoma. In chronic liver disease, the gene-expression signature as well as the degree of liver fibrosis could help us to predict the development of human hepatocellular carcinoma or survival outcome after treatment for human hepatocellular carcinoma. Several genes, such as HSP70, CAP2 and GPC3, have been identified as potential biomarkers for early human hepatocellular carcinoma. Classical oncogenes or tumor suppressor genes, such as beta-catenin and p53, are mutated during the progression from early to advanced human hepatocellular carcinoma. Also, the presence of hepatoblastic feature like CK19 in advanced human hepatocellular carcinoma can be used as a predictor of aggressive human hepatocellular carcinoma. Although many advances have been made in the diagnosis of multistage hepatocarcinogenesis, we still need further useful markers to more precisely evaluate each step of hepatocarcinogenesis for better treatment choices, and that will promote future molecular-targeted therapy.

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Bmi-1 gene is upregulated in early-stage hepatocellular carcinoma and correlates with ATP-binding cassette transporter B1 expression

Effendi, K., Mori, T., Komuta, M., Masugi, Y., Du, W. L. and Sakamoto, M.

Cancer Science 101 （ 3 ） 666 - 672 2010年03月

ISSN 1347-9032

Overexpression of "stemness gene'' Bmi-1 has been identified in some solid tumors. We investigated Bmi-1 expression in hepatocellular carcinoma (HCC) and ATP-binding cassette transporter B1 (ABCB1) as a new potential target for Bmi-1. Bmi-1 was highly expressed in HCC cell lines and the most well differentiated cell line, KIM-1, showed the highest expression. Immunohistochemical, immunocytochemical, and immunoelectron microscopic analysis showed the Bmi-1 protein as having a high intensity of small dots within the nucleus which reflected concentrated sites of Bmi-1 repressive activity. Clear "dot-pattern'' staining was observed in 24 of 37 (65%) well differentiated HCC (including 13 of 21 early nodules [62%]), in 32 of 71 (45%) moderately differentiated HCC, and 7 of 14 (50%) poorly differentiated HCC. A similar expression was not observed in non-cancerous background regions. High Bmi-1 expression was observed in the early and well differentiated HCC. Furthermore, overexpression and suppression of Bmi-1 was followed by a respective increase and decrease in ABCB1 expression. As with Bmi-1, high ABCB1 expression was also observed in the early and well differentiated HCC. A strong correlation between ABCB1 and Bmi-1 mRNA expression was seen in HCC cell lines and clinical samples (Pearson's correlation coefficient 0.95 and 0.90, respectively). The Bmi-1 gene is upregulated in HCC, and in particular is highly expressed in early and well differentiated HCC. The fact that this expression correlated with that of ABCB1 suggests a new regulation target for Bmi-1, and gives new insight into early hepatocarcinogenesis mechanisms and potential targets for future HCC treatment. (Cancer Sci 2010; 101: 666-672)

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Production of cells with targeted integration of gene variants of human ABC transporter for stable and regulated expression using the Flp recombinase system

K. Wakabayashi-Nakao, A. Tamura, S. Koshiba, Y. Toyoda, H. Nakagawa and T. Ishikawa

Methods Mol Biol 648 139 - 59 2010年

ISSN 1940-6029

The vector-mediated introduction of cDNA into mammalian cells by calcium phosphate co-precipitation or permeation with lipofectamine is widely used for the integration of cDNA into genomic DNA. Such integration, however, of cDNA occurs randomly at unpredictable sites in the host's chromosomal DNA, and the number of integrated recombinant DNAs is not controllable. To overcome this problem, we developed the Flp-In method to integrate one single copy of cDNA encoding the human ABC transporter ABCG2 into FRT-tagged genomic DNA. Examination of more than 20 metaphase spreads for both fluorescence in situ hybridization (FISH) mapping and multicolor-FISH analysis revealed that ABCG2 cDNA was incorporated into the telomeric region of the short arm on one of chromosomes 12 in Flp-In-293 cells. By using those cells, we investigated the effect of genetic polymorphisms and post-translational modifications of human ABC transporter ABCG2 on the protein expression and degradation. On the basis of our experience, it has been concluded that the Flp recombinase system provides a useful tool to quantitatively analyze the protein stability and endoplasmic reticulum (ER)-associated degradation of proteins like the ABC transporter. This system is also applicable for similar studies of the biogenesis of other proteins using the secretory pathway as well as proteins with other cellular localizations.

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Specific expression of hepatocyte nuclear factor-1βin the ovarian clear cell adenocarcinoma and its application to cytogical diagnosis;

坂元亨宇

Cancer science. 98 （ 3 ） 387-391 2007年03月

研究論文（学術雑誌）, 単著

Efficient immortalization of primary human cells by p16INK4a-specific short hairpin RNA or Bmi-1,combined with introduction of hTERT;

坂元亨宇

Cancer science. 98 （ 2 ） 147-154 2007年02月

研究論文（学術雑誌）, 単著

cancer stem cells「細胞の不死化・腫瘍化と多分化能の維持」

森　泰昌、坂元　亨宇

病理と臨床 25 （ 4 ） 2007年

研究論文（学術雑誌）, 共著

Gene therapy of liver tumors with liver-specific nanoparticles;

坂元亨宇

Cancer Gene Therapy 14 74-81 2007年

研究論文（学術雑誌）, 共著

Genome-wide array-based comparative genomic hybridization anlysis of pancreatic adenocarcinoma:Identification of genetic indicators that predict patient outcome:

坂元亨宇

Cancer science 98 （ 3 ） 392-400 2007年

研究論文（学術雑誌）, 単著

Granulomatous arteritis in cutaneous lesions of churg-strauss syndrome;

坂元亨宇

J cutaneous pathology. 134 （ 4 ） 330-337 2007年

研究論文（学術雑誌）, 単著

「6　肝」第2部　各臓器、疾患で用いられる抗体とその応用

柴田　理恵　坂元　亨宇

　病理と臨床「臨時増刊号」 25 2007年

研究論文（学術雑誌）, 単著

Overexpression of cyclase-associated protein 2 in multistage hepatocarcinogenesis

Shibata, R., Mori, T., Du, W. L., Chuma, M., Gotoh, M., Shimazu, M., Ueda, M., Hirohashi, S. and Sakamoto, M.

Clinical Cancer Research 12 （ 18 ） 5363 - 5368 2006年09月

ISSN 1078-0432

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Overexpression of Cyclase-Associated Protein 2 in Multistage Hepatocarcinogenesis

坂元亨宇

Clinical Cancer Research 12 （ 18 ） 5363-5368 2006年09月

研究論文（学術雑誌）, 共著

CT Evaluation of the progression of hypoattenuating nodular lesions in virus-related chronic liver disease

Takayasu, K., Muramatsu, Y., Mizuguchi, Y., Okusaka, T., Shimada, K., Takayama, T. and Sakamoto, M.

AJR Am J Roentgenol 187 （ 2 ） 454 - 63 2006年08月

ISSN 1546-3141

OBJECTIVE: The purpose of this study was to clarify the natural outcomes of hypoattenuating nodular lesions in patients with virus-related chronic liver disease depicted on dynamic CT. MATERIALS AND METHODS: Sixty lesions (mean size, 1.3 cm) exhibiting hypoattenuation or isoattenuation in the arterial and delayed phases of dynamic CT were retrospectively evaluated with additional CT (mean, six examinations) for a mean period of 838 days. The primary end point was emergence of hyperattenuating areas within hypoattenuating lesions, a phenomenon called attenuation conversion. Cumulative attenuation conversion rates suggesting rates of malignant transformation were calculated with the Kaplan-Meier method, and factors affecting attenuation conversion rate were analyzed with the Cox proportional hazard model. RESULTS: Thirty-six (60%) of 60 hypoattenuating lesions developed to hyperattenuating lesions, 21 were unchanged, and three disappeared spontaneously. The 36 lesions that became hyperattenuating were divided into two subgroups according to lesion enhancement pattern: hyper-in-hypoattenuating (n = 25) and entirely hyperattenuating (n = 11). The cumulative attenuation conversion rates for the 60 hypoattenuating lesions were 15.8%, 44.3%, and 58.7% at 1, 2, and 3 years. The hyper-in-hypoattenuating lesions showed more rapid progression to entirely enhanced lesions. Positive results for hepatitis C viral antibody (p = 0.028) and initial lesion size (p = 0.007) showed a positive correlation with attenuation conversion rate. CONCLUSION: Hypoattenuating hepatic nodular lesions in chronic liver disease depicted on dynamic CT have high malignant potential and should be followed with special attention to conversion from hypoattenuation to hyperattenuation to determine the optimal timing of treatment.

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Establishment of perineural invasion models and analysis of gene expression revealed an invariant chain (CD74) as a possible molecule involved in perineural invasion in pancreatic cancer.

森泰昌

Clin Cancer Res. 12 （ 8 ） 2419-26. 2006年05月

研究論文（学術雑誌）, 共著

Establishment of perineural invasion models anda analysis of gene ezpression revealed an invariant chain(CD74)as a possible molecule involved in perineural invasion in pancreatic cancer:

坂元亨宇

clin cancer res. 12 （ 8 ） 2006年

研究論文（学術雑誌）, 共著

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