Faculty Profiles - SAKAGUCHI Masakiyo

MISC - SAKAGUCHI Masakiyo

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Involvement of deterioration in S100C/A11-mediated pathway in resistance of human squamous cancer cell lines to TGF beta-induced growth suppression

Hiroyuki Sonegawa, Takamasa Nukui, Dai-Wei Li, Mikiro Takaishi, Masakiyo Sakaguchi, Nam-Ho Hub

JOURNAL OF MOLECULAR MEDICINE-JMM 85 ( 7 ) 753 - 762 2007.7

Language：English Publisher：SPRINGER

Recently, we demonstrated that S100C/A11 comprises an essential pathway for growth suppression by TGF beta in normal human keratinocytes. Nuclear transfer of S 100C/A11 was a hallmark of the activation of the process. In the present study, we examined the possible deterioration in the pathway in human squamous cancer cell lines, focusing on intracellular localization of S100C/A11 and its functional partners Smad3 and Smad4. All four human squamous cancer cell lines examined (A431, BSCC-93, DJM-1, and HSC-5) were resistant to growth suppression by TGF beta. In BSCC-93, DJM-1, and HSC-5 cells exposed to TGF beta, S100C/A11 was not transferred to the nuclei, and p21(WAF1) was not induced. Overexpression of nucleus-targeted S100C/A11 partially recovered induction of p21 (WAF1) and p15(INK4B) and growth suppression by TGF beta 1 in these cells. These results indicate that the deterioration in the S100C/A11-mediated pathway conferred upon the cancer cell lines resistance to TGF beta. In A431 cells, S100C/A11, Smad3, and Smad4 were simultaneously transferred to the nuclei, and p2l(WAF1) was induced upon exposure to TGF beta. We provide evidence to indicate that refractoriness of A431 cells to TGF beta was probably because the amount of p2l(WAF1) induced by TGF beta was insufficient to counteract cyclin A, which is highly overexpressed in A431 cells. Thus, the newly found S100C/A11-mediated pathway is at least partly involved in conferring upon human squamous cell cancers resistant to TGF beta-induced growth suppression, which is considered to play a critical role for the initiation and progression of many human cancers.

DOI： 10.1007/s00109-007-0180-7

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S100A8/A9, a key mediator for positive feedback growth stimulation and inflammation of normal human keratinocytes

R. Ehama, T. Nukui, C. Katagiri, M. Sakaguchi, H. Sonegawa, N. Huh, T. Hibino

JOURNAL OF INVESTIGATIVE DERMATOLOGY 127 S98 - S98 2007.4

Involvement of deterioration in S100C/A11-mediated pathway in resistance of human squamous cancer cell lines to TGF beta-induced growth suppression

SONEGAWA HIROYUKI, SAKAGUCHI MASAKIYO, HUH NAM-HO

26 ( 1 ) 63 - 63 2007.3

S100A8/A9, a Key Mediator for Positive Feedback Growth Stimulation of Normal Human Keratinocytes

EHMA RITSUKO, NUKUI TAKAMASA, SAKAGUCHI MASAKIYO, SONEGAWA HIROYUKI, KATAGIRI CHIKA, HIBINO TOSHIHIKO, HUH NAM-HO

26 ( 1 ) 73 - 73 2007.3

Autocrine growth-stimulation of normal human keratinocytes by secreted S100C/A11

SAKAGUCHI MASAKIYO, SONEGAWA HIROYUKI, HUH NUM-HO

26 ( 1 ) 74 - 74 2007.3

REIC/Dkk-3 as a potential gene therapeutic agent against human testicular cancer

TANIMOTO RYUTA, SAKAGUCHI MASAKIYO, FERNANDO ABARZUA, SONEGAWA HIROYUKI, NASU YASUTOMO, KUMON HIROMI, HUH NUM-HO

26 ( 1 ) 88 - 88 2007.3

REIC/Dkk-3 as a potential gene therapeutic agent against human testicular cancer

Ryuta Tanimoto, Fernando Abarzua, Masakiyo Sakaguchi, Mikiro Takaishi, Yasutomo Nasu, Hiromi Kumon, Nam-Ho Huh

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 19 ( 3 ) 363 - 368 2007.3

Language：English Publisher：PROFESSOR D A SPANDIDOS

Human testicular cancer is very sensitive to chemotherapy and radiation therapy and is regarded as a curable cancer. The cancer prevails in the young reproductive generation and testicular dysfunction is often observed as a side effect, remaining a serious challenge. In the present study, we examined the potential utility of REIC/Dkk-3-based gene therapy against human testicular cancer. Expression of REIC/Dkk-3 was reduced in all of the human seminoma and non-seminomatous germ cell tumor tissues. Overexpression of REIC/Dkk-3 using an adenovirus vector (Ad-REIC) induced apoptosis in a testicular germ cell cancer cell line NCCIT but not in normal human fibroblasts. c-Jun terminal kinase (JNK) was activated by Ad-REIC and the induction of apoptosis was abrogated by a JNK inhibitor. A single intratumoral injection of Ad-REIC markedly inhibited the tumorigenic growth of NCCIT cells in nude mice. These results indicate that Ad-REIC may lead to developing less insulting and non-genotoxic therapeutic measures against human testicular cancer.

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Denatured and reversibly cationized p53 readily enters cells and simultaneously folds to the functional protein in the cells

H Murata, M Sakaguchi, J Futami, M Kitazoe, T Maeda, H Doura, M Kosaka, H Tada, M Seno, N Huh, H Yamada

BIOCHEMISTRY 45 ( 19 ) 6124 - 6132 2006.5

Language：English Publisher：AMER CHEMICAL SOC

Cationization is a powerful strategy for internalizing a protein into living cells. On the other hand, a reversibly cationized denatured protein through disulfide bonds is not only soluble in water but also able to fold to the native conformation in vitro. When these advantages in cationization were combined, we developed a novel method to deliver a denatured protein into cells and simultaneously let it fold to express its function within cells. This "in-cell folding" method enhances the utility of recombinant proteins expressed in Escherichia coli as inclusion bodies; that is, the recombinant proteins in inclusion bodies are solubilized by reversible cationization through cysteine residues by disulfide bonds with aminopropyl methanethiosulfonate or pyridyldithiopropionylpolyethylenimine and then incubated with cells without an in vitro folding procedure. As a model protein, we investigated human tumor-suppressor p53. Treatment of p53-null Saos-2 cells with reversibly cationized p53 revealed that all events examined as indications of the activation of p53 in cells, such as reduction of disulfide bonds followed by tetramer formation, localization into the nucleus, induction of p53 target genes, and induction of apoptosis of cells, occurred. These results suggest that reversible cationization of a denatured protein through cysteine residues is an alternative method for delivery of a functional protein into cells. This method would be very useful when a native folded protein is not readily available.

DOI： 10.1021/bi052642a

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Rescue of 90%-hepatectomized rats with acute liver failure by intrasplenic transplantation of hepatocytes derived from rat bone marrow cells

MIYAZAKI MASAHIRO, HARDJO MARHAEN, MEDINA REINHOLD, MASAKA TAKURO, NAILA MAHMUT, TOMIYAMA KOJI, SAKAGUCHI MASAKIYO, TAKAHASHI MIKIRO, HUH NAM-HO

25 ( 1 ) 75 - 75 2006.3

In vitro trans-differentiation of bone marrow derived hepatocyte-like cells into insulin producing cells

MASAKA TAKURO, MIYAZAKI MASAHIRO, HARDJO MARHAEN, MEDINA REINHOLD, SAKAGUCHI MASAKIYO, TAKAISHI MIKIRO, HUH NAM-HO

25 ( 1 ) 77 - 77 2006.3

HNF4 転写因子による肝機能制御機構の解明

阪口政清

ファルマシア 2006

Adenovirus-mediated overexpression of REIC/Dkk-3 selectively induces apoptosis in human prostate cancer cells through activation of c-Jun-NH2-kinase

F Abarzua, M Sakaguchi, M Takaishi, Y Nasu, K Kurose, S Ebara, M Miyazaki, M Namba, H Kumon, N Huh

CANCER RESEARCH 65 ( 21 ) 9617 - 9622 2005.11

Language：English Publisher：AMER ASSOC CANCER RESEARCH

Alteration in genes which takes place during malignant conversion and progression could be potential targets for gene therapy. We previously identified REIC/Dkk-3 as a gene whose expression is reduced in many human cancers. Here, we showed that expression of REIC/Dkk-3 was consistently reduced in human prostate cancer tissues in a stage-dependent manner. Forced expression of REIC/Dkk-3 induced apoptosis in human prostate cancer cell lines lacking endogenous REIC/Dkk-3 expression but not in REIC/Dkk3-proficient normal prostate epithelial and stromal cells. The apoptosis involved c-jun-NH2-kinase activation, mitochondrial translocation of Bax, and reduction of Bcl-2. A single injection of an adenovirus vector carrying REIC/Dkk-3 showed a dramatic antitumor effect on a xenotransplanted human prostate cancer. Thus, REIC/Dkk-3 could be a novel target for gene-based therapy of prostate cancer.

DOI： 10.1158/0008-5472.CAN-05-0829

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Bifurcated converging pathways for high Ca2+- and TGF beta-induced inhibition of growth of normal human keratinocytes

M Sakaguchi, H Sonegawa, T Nukui, Y Sakaguchi, M Miyazaki, M Namba, N Huh

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 102 ( 39 ) 13921 - 13926 2005.9

Language：English Publisher：NATL ACAD SCIENCES

Growth suppression of normal human keratinocytes by high Ca2+ or TGF beta was shown to be mediated by p2l(WAF1/C1P1) and Sp1 [Pardali, K., et al. (2000) J. Biol. Chem. 275, 29244-29256; Santini, M. P., Talora, C., Seki, T., Bolgan, L. & Dotto, G. P. (2001) Proc. Nat. Acad Sci. USA 98, 9575-9580; Al-Daraji, W. I., Grant, K. R., Ryan, K., Saxton, A., & Reynolds, N. J. (2002) J. Invest. Dermato/. 118, 779-788]. We previously demonstrated that S100C/A11 is a key mediator for growth inhibition of normal human epidermal keratinocytes (NHK) triggered by high Ca2+ or TGF beta [Sakaguchi, M., et al. (2003) J. Cell Biol. 163, 825-835; Sakaguchi, M., et al. (2004) 164, 979-984]. On exposure of NHK cells to either agent, S100C/ All is transferred to nuclei, where it induces p21(WAF1/CIP1) through activation of Sp1/Sp3. In the present study, we found that high Ca2+ activated NFAT1 through calcineurin-dependent dephosphorylation. In growing NHK cells, Krueppel-like factor (KLF)16, a member of the Sp/KLF family, bound to the p21(WAF1/CIP1) promoter and, thereby, inhibited the transcription of p21(WAF1/CIP1). Sp1 complexed with NFAT1 in high Ca2+ -treated cells or with Smad3 in TGF beta 1-treated cells, but not Sp1 alone, replaced KLF16 from the p21(WAF1/CIP1) promoter and transcriptionally activated the p21(WAF1/CIP1) gene. Thus, high Ca2+ and TGF beta 1 have a common S1OOC/A11-mediated pathway in addition to a unique pathway (NFAT1-mediated pathway for high Ca2+ and Smad-mediated pathway for TGF beta 1) for exhibiting a growth inhibitory effect on NHK cells, and both pathways were shown to be indispensable for growth inhibition.

DOI： 10.1073/pnas.0500630102

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Protein transduction assisted by polyethylenimine-cationized carrier proteins

M Kitazoe, H Murata, J Futami, T Maeda, M Sakaguchi, M Miyazaki, M Kosaka, H Tada, M Seno, N Huh, M Namba, M Nishikawa, Y Maeda, H Yamada

JOURNAL OF BIOCHEMISTRY 137 ( 6 ) 693 - 701 2005.6

Language：English Publisher：JAPANESE BIOCHEMICAL SOC

Previously, we have reported that cationized-proteins covalently modified with polyethylenimine (PEI) (direct PEI-cationization) efficiently enter cells and function in the cytosol [Futami et al. (2005) J. Biosci. Bioeng. 99,95-1031. However, it may be more convenient if a protein could be delivered into cells just by mixing the protein with a PEI-cationized carrier protein having a specific affinity (indirect PEI-cationization). Thus, we prepared PEI-cationized avidin (PEI-avidin), streptavidin (PEI-streptavidin), and protein G (PEI-protein G), and examined whether they could deliver biotinylated proteins and antibodies into living cells. PEI-avidin (and/or PEI-streptavidin) carried biotinylated GFPs into various mammalian cells very efficiently. A GFP variant containing a nuclear localization signal was found to arrive even in the nucleus. The addition of a biotinylated RNase A derivative mixed with PEI-streptavidin to a culture medium of 3T3-SV-40 cells resulted in remarkable cell growth inhibition, suggesting that the biotinylated RNase A derivative entered cells and digested intracellular RNA molecules. Furthermore, the addition of a fluorescein-labeled antiS100C (beta-actin binding protein) antibody mixed with PEI-protein G to human fibroblasts resulted in the appearance of a fluorescence image of actin-like filamentous structures in the cells. These results indicate that indirect PEI-cationization using non-covalent interaction is as effective as the direct PEI-cationization for the transduction of proteins into living cells and for expression of their functions in the cytosol. Thus, PEI-cationized proteins having a specific affinity for certain molecules such as PEI-streptavidin, PEI-avidin and PEI-protein G are concluded to be widely applicable protein transduction carrier molecules.

DOI： 10.1093/jb/mvi081

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Intracellular delivery of proteins into mammalian living cells by polyethylenimine-cationization

J Futami, M Kitazoe, T Maeda, E Nukui, M Sakaguchi, J Kosaka, M Miyazaki, M Kosaka, H Tada, M Seno, Y Sasaki, NH Huh, M Namba, H Yamada

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 99 ( 2 ) 95 - 103 2005.2

Language：English Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN

In the post-genomic era, there is pressing need for development of protein manipulation methodology to analyze functions of proteins in living cells. For this purpose, techniques to deliver functional proteins into living cells are currently being evaluated as alternative approaches to the introduction of transcriptionally active DNA. Here, we describe a novel method for efficient protein transduction into living cells in which a protein is simply cationized with polyethylenimine (PEI) by limited chemical conjugation. PEI-cationized proteins appear to adhere to the cell surface by ionic charge interaction and then internalize into cells in a receptor- and transporter-independent fashion. Since PEI is an organic macromolecule with a high cationic-charge density, limited coupling with PEI results in endowment of sufficient cationic charge to proteins without causing serious decline in their fundamental functions. A number of PEI-cationized proteins, such as ribonuclease (RNase), green fluorescent protein (GFP) and immunoglobulin (IgG), efficiently entered cells and functioned in the cytosol. Our results suggest that protein cationization techniques using PEI will be useful for the development of protein transduction technology.

DOI： 10.1263/jbb.99.095

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Targeted disruption of transcriptional regulatory function of p53 by a novel efficient method for introducing a decoy oligonucleotide into nuclei

M Sakaguchi, T Nukui, H Sonegawa, H Murata, J Futami, H Yamada, N Huh

NUCLEIC ACIDS RESEARCH 33 ( 9 ) 2005

Language：English Publisher：OXFORD UNIV PRESS

Decoy oligonucleotides have been used for functional sequestering of transcription factors. Efficient introduction into cells is a prerequisite for the oligonucleotides to exert their blocking function. Lipofection is the most widely used technique for that purpose because of its convenience and relatively high efficiency. However, the transduction efficiency of lipofection largely depends on cell types and experimental conditions and the introduced nucleotides are not specifically directed to nuclei where they exert their major function. In the present study, we designed a new system for transporting oligonucleotides into cell nuclei. The vehicle is composed of glutathione-S-transferase, 7 arginine residues, the DNA- binding domain of GAL4 and a nuclear localization signal, which are linked with flexible glycine stretches. The p53-responsive element linked to the GAL4 upstream activating sequence was efficiently transferred by the vehicle protein into nuclei of primary cultures of neuronal cells, embryonic stem cells and various human normal cells. Transcriptional activation of p21(WAF1/CIP1) and Bax by p53 on exposure to cisplatin was completely blocked by introducing the p53 decoy oligonucleotide. Thus, the system developed in the present study can be a convenient and powerful tool for specifically disrupting the function of DNA- binding proteins in culture.

DOI： 10.1093/nar/gni088

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siRNAによる遺伝子発現抑制法

阪口政清, 曽根川裕之, 温井孝昌, 許南浩

Surgery Frontier 2005

PKCa mediates TGFb-induced growth inhibition of human keratinocytes via phosphorylation of S100C/A11

M Sakaguchi, M Miyazaki, H Sonegawa, M Kashiwagi, M Ohba, T Kuroki, M Namba, N Huh

MOLECULAR BIOLOGY OF THE CELL 15 449A - 449A 2004.11

Increased expression of calcium-binding protein S100 in human uterine smooth muscle tumours

T Kanamori, K Takakura, M Mandai, M Kariya, K Fukuhara, M Sakaguchi, NH Huh, K Saito, T Sakurai, J Fujita, S Fujii

MOLECULAR HUMAN REPRODUCTION 10 ( 10 ) 735 - 742 2004.10

Language：English Publisher：OXFORD UNIV PRESS

S100 proteins belong to the EF-hand Ca2+ -binding protein family and regulate a variety of cellular processes via interaction with different target proteins. Several diseases, including cancer and melanoma, are related to the abnormal expression of S100 proteins, which are expressed in cell- and tissue-specific manners. We investigated the expression of S100 family members in human uterine smooth muscle tumours. Expression of six members of the S100 protein family: S100A1, A4, A6, A7, A10 and A11, was found in human uterine leiomyoma and myometrium tissue, but expression of other members was not detected by RT-PCR. Real-time PCR showed that S100A11 expression was significantly increased in leiomyoma compared with myometrium. Suppression of S100A11 by small interfering RNA (siRNA) led to apoptosis, and the overexpression of S100A11 inhibited apoptosis in human uterine smooth muscle tumour cells. These findings suggest that S100A11 has an anti-apoptotic function and is related to the process of growth of human uterine leiomyoma.

DOI： 10.1093/molehr/gah100

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Expression of CYP3A4 by an immortalized human hepatocyte line in a three-dimensional culture using a radial-flow bioreactor

Akiyama, I, K Tomiyama, M Sakaguchi, M Takaishi, M Mori, M Hosokawa, S Nagamori, N Shimizu, NH Huh, M Miyazaki

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 14 ( 4 ) 663 - 668 2004.10

Language：English Publisher：PROFESSOR D A SPANDIDOS

Cytochrome P450 (CYP) 3A is responsible for about 50% of drug metabolizing activity in the liver. The present study was undertaken to establish a CYP3A4-active model for in vitro analysis of human drug metabolism. The cells used were immortalized normal human fetal hepatocytes (OUMS-29) and its HNF4alpha-introduced subline (OUMS-29/H-11). The cells were cultivated under high-density three-dimensional conditions in a radial-flow bioreactor (RFB). The number of OUMS-29 cells increased 15-fold over 49 days and their apical surfaces were covered with abundant microvilli, a characteristic of hepatocytes in vivo. The amount of albumin secreted by OUMS-29 cells in the three-dimensional RFB culture was 6-fold higher than those in a monolayer culture. CYP3A4 protein and an intermediate metabolite of testosterone by CYP3A4 were detected in OUMS-29/H11 cells cultivated in RFB >29 days. These results indicate that the RFB; culture of OUMS-29/H-11 cells is useful for screening and developing new drugs.

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