Faculty Profiles - SAKAGUCHI Masakiyo

MISC - SAKAGUCHI Masakiyo

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Introduction of an N-terminal peptide of S100C/A11 into human cells induces apoptotic cell death

E Makino, M Sakaguchi, K Iwatsuki, N Huh

JOURNAL OF MOLECULAR MEDICINE-JMM 82 ( 9 ) 612 - 620 2004.9

Language：English Publisher：SPRINGER

S100 proteins belong to the EF-hand Ca2+- binding protein family and are involved in the regulation of a variety of cellular processes. Individual S 100 proteins are expressed in cell- and tissue-specific manners, and functional deterioration of S100 proteins leads to a number of human diseases, including cancer. We previously demonstrated that S100C/A11 was translocated to nuclei and inhibited DNA synthesis in human keratinocytes when exposed to high Ca2+. In the present study we examined the effects of synthetic partial peptides of S100C/ A11 on human carcinoma cell lines. Only an N-terminal peptide with 19 amino acid residues (MAK19) showed cytotoxicity to the cell lines in dose- and time-dependent manners when introduced into cells by flanking the HIV-TAT protein transduction domain (TAT-MAK19). Pulse field electrophoresis revealed that DNA of the treated cells was partially degradated. Annexin V, a marker of cellular apoptosis, was detected in the cells treated with TAT-MAK19 by immunostaining and flow cytometry. The induction of apoptotic cell death was apparently independent of p53, p21(WAF1/CIP1), and caspase activity, but treatment with TAT-MAK19 resulted in partial translocation of apoptosis-inducing factor (AIF) from the cytoplasm to nuclei. These results indicate that MAK19 induces apoptosis in human cell lines and may therefore lead to the establishment of a new molecular target for the treatment of human cancer.

DOI： 10.1007/s00109-004-0560-1

Involvement of interferon regulatory factor 1 and S100C/A11 in growth inhibition by transforming growth factor beta 1 in human hepatocellular carcinoma cells

M Miyazaki, M Sakaguchi, Akiyama, I, Y Sakaguchi, S Nagamori, NH Huh

CANCER RESEARCH 64 ( 12 ) 4155 - 4161 2004.6

Language：English Publisher：AMER ASSOC CANCER RESEARCH

Growth inhibition by transforming growth factor (TGF)-beta1 has been attributed to the induction of cyclin-dependent kinase inhibitors, among which p21/Waf1 plays a major role in many biological contexts. In the present study, two new intracellular mediators for the induction of p21/Waf1 by TGF-beta1 were identified in a human hepatocellular carcinoma cell line (JHH-5) expressing mutant-type p53. After addition of TGF-beta1 to JHH-5 cells, a marked increase of the p21/Waf1 expression preceded the inhibition of DNA synthesis. Expression of IFN regulatory factor (IRF)-1, a known transacting factor for p21/Waf1 promoter, was elevated just before or in parallel with the increase of p21/Waf1. Transduction of antisense IRF-1 inhibited the increase in p21/Waf1 in JHH-5 cells treated with TGF-beta1 and partially released the cells from the growth arrest by TGF-beta1. Expression of S100C/A11, a member of the Ca2+-binding S100 protein family, also markedly increased after addition of TGF-beta1. S100C/A11 protein was translocated to and accumulated in nuclei of TGF-beta1-treated JHH-5 cells, where p21/Waf1 was concomitantly accumulated. When a recombinant S100C/A11 protein was introduced into nuclei of JHH-5 cells, DNA synthesis was markedly inhibited in a dose-dependent manner in the absence of TGF-beta1. Prior transfection of p21/Waf1-targeted small interfering RNA efficiently blocked decrease of DNA synthesis in JHH-5 cells caused by TAT-S100C/A11 or TGF-beta1 and markedly inhibited expression of p21/Waf1 protein in the cells. These results indicate that IRF-1 and S100C/A11 mediate growth inhibition by TGF-beta1 via induction of p21/Waf1.

DOI： 10.1158/0008-5472.CAN-03-2750

Establishment of an immortalized human-liver endothelial cell line with SV40T and hTERT

T Matsumura, M Takesue, KA Westerman, T Okitsu, M Sakaguchi, T Fukazawa, T Totsugawa, H Noguchi, S Yamamoto, DB Stolz, N Tanaka, P Leboulch, N Kobayashi

TRANSPLANTATION 77 ( 9 ) 1357 - 1365 2004.5

Language：English Publisher：LIPPINCOTT WILLIAMS & WILKINS

Background and Aims. Liver endothelial cells (LECs) perform an essential role in important pathophysiologic functions in the liver. Establishment of a human LEC line facilitates advances in LEC research. Here, we present immortalization of human LECs using retroviral gene transfer of simian virus 40 large T antigen (SV40T) and human telomerase reverse transcriptase (hTERT). We also demonstrate excision of SV40T and hTERT with TAT-mediated Cre/loxP recombination and subsequent cell sorting.
Methods. First, human LECs were transduced with a retroviral vector somatostatin receptor (SSR)#69 expressing SV40T and hygromycin-resistance genes flanked by a pair of loxA recombination targets. Then, cells were retrovirally superinfected with SSR#197 encoding hTERT and green fluorescent protein (GFP) cDNAs that were intervened by two loxBs. One SV40T- and hTERT-immortalized LEC clone, TMNK-1, was established and analyzed for its biologic characteristics.
Results. The cells were hygromycin-resistant and uniformly positive for GFP expression. TMNK-1 expressed EC markers, including factor VIII, vascular endothelial growth factor receptors (flt-1, KDR/Flk-1), and CD34, showed uptake of Di-I-acetylated-low-density lipoprotein and angiogenic potential in Matrigel assays. After lipopolysaccharide treatment, TMNK-1 produced tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 and exhibited increased expression of intracellular adhesive molecule-1, vascular cellular adhesive molecule-1, and VE-cadherin. After treatment with TAT-Cre recombinase fusion protein, approximately 60% of TMNK-1 was negative for GFP expression, and subsequent cell sorting of this population for GFP allowed for collection of the reverted form of TMNK-1.
Conclusions. This study demonstrates the utility and efficiency of the reversible immortalization procedure to expand primary human LECs for basic studies.

DOI： 10.1097/01.TP.0000124286.82961.7E

Differential expression of S100C in thyroid lesions

C Torres-Cabala, A Panizo-Santos, HC Krutzsch, H Barazi, M Namba, M Sakaguchi, DD Roberts, MJ Merino

INTERNATIONAL JOURNAL OF SURGICAL PATHOLOGY 12 ( 2 ) 107 - 115 2004.4

Language：English Publisher：WESTMINSTER PUBL INC

Identification of new potential markers that may help in the diagnosis of benign and malignant thyroid lesions is needed. By comparative 2-dimensional gel electrophoresis of microdissected cells from tumors and normal thyroid tissue, we identified a new protein, S100C, which is highly expressed in papillary carcinomas. In order to validate this finding, we investigated the immunohistochemical expression and the potential role in diagnosis of these markets in 94 specimens representing the spectrum of malignant and benign thyroid lesions. Normal thyroid tissue was evaluated in 57 specimens. Galectin-3, a marker reported as specific for malignant lesions, was also evaluated in the same lesions. S100C protein was expressed in the nuclei of normal tissue, hyperplastic nodules, and follicular adenomas and carcinomas. Papillary carcinomas showed a strong, but cytoplasmic, pattern of staining. Galectin-3 immunostaining was strongly positive in papillary carcinomas, and negative in benign lesions, confirming its value in differential diagnosis. These findings Suggest that immunohistochemical Staining of S100C could be helpful in the pathological study of thyroid lesions, especially in cases in which follicular variants of papillary carcinoma and follicular carcinoma are considered in the differential diagnosis.

DOI： 10.1177/106689690401200203

Growth Regulation of Normal Human Keratinocytes : PKCa Mediates TGFb-induced Growth Inhibition via Phosphorylation of S100C/A11

SAKAGUCHI Masakiyo, MIYAZAKI Masahiro, SONEGAWA Hiroyuki, KASHIWAGI Mariko, OHBA Motoi, KUROKI Toshio, NAMBA Masayoshi, HUH Namho

23 ( 1 ) 38 - 38 2004.3

Decreased expression of REIC/Dkk-3 in human renal clear cell carcinoma

K Kurose, M Sakaguchi, Y Nasu, S Ebara, H Kaku, R Kariyama, Y Arao, M Miyazaki, T Tsushima, M Namba, H Kumon, NH Huh

JOURNAL OF UROLOGY 171 ( 3 ) 1314 - 1318 2004.3

Language：English Publisher：LIPPINCOTT WILLIAMS & WILKINS

Purpose: We examined the expression of REIC/Dkk-3, a possible candidate for a tumor suppressor gene, in human renal clear cell carcinoma (RCCC) cell lines and sporadic RCCC surgical specimens.
Materials and Methods: Human RCCC cell lines (Caki-1, Caki-2, ACHN and KPK-1) and several control cell lines were used to examine the expression of REIC/Dkk-3 mRNA and characterize a newly raised antibody specific for REIC/Dkk-3 protein. Pairs of cancerous and adjacent noncancerous tissues were obtained from 20 patients with RCCC. Of them 17 and 7 cases were analyzed by real-time quantitative reverse transcriptase-polymerase chain reaction, and by Western blot analysis and/or immunohistochemical analysis, respectively.
Results: The decreased expression of REIC/Dkk-3 mRNA and protein in human RCCC cell lines, and the specificity of the new antibody were confirmed. In a real-time quantitative reverse transcriptase-polymerase chain reaction study using 17 pairs of RCCC and adjacent normal tissues REIC/Dkk-3 mRNA levels were significantly decreased in carcinoma tissues (by 25% to approximately 95% in 15 pairs). Western blot analysis and immunohistochemistry revealed a significant decrease in REIC/Dkk-3 protein levels in 6 of the 7 and 13 of the 14 RCCC cases analyzed, respectively.
Conclusions: The decrease in REIC/Dkk-3 mRNA and protein levels was observed irrespective of tumor grade and stage, indicating the involvement of REIC/Dkk-3 in an initial step of malignant conversion. Consequently REIC/Dkk-3 could be a new molecular target for therapeutic measures against RCCC.

DOI： 10.1097/01.ju.0000101047.64379.d4

Establishment of a highly differentiated immortalized human cholangiocyte cell line with SV40T and hTERT

M Maruyama, N Kobayashi, KA Westerman, M Sakaguchi, JE Allain, T Totsugawa, T Okitsu, T Fukazawa, A Weber, DB Stolz, P Leboulch, N Tanaka

TRANSPLANTATION 77 ( 3 ) 446 - 451 2004.2

Language：English Publisher：LIPPINCOTT WILLIAMS & WILKINS

Background. Cholangiocytes perform an essential role in important pathophysiologic functions in the liver. Establishment of a human cholangiocyte line facilitates advances in cholangiocyte research and clinical applications for cell therapies. Here, we describe the immortalization of human cholangiocytes using serial transfection of simian virus 40 large T (SV40T) followed by human telomerase reverse transcriptase (hTERT).
Methods. SV40T-transduced human liver OUMS-21 cells were superinfected with a retroviral vector SSR#197 encoding hTERT and green fluorescent protein (GFP) cDNAs. Resulting cell lines were evaluated for gene expression, functional cholangiogenic characteristics in vitro and in vivo, and response to lipopolysaccharide (LPS).
Results. One of the SV40T- and hTERT-immortalized cholangiocyte clones, MMNK-1, was established. MMNK-1 expressed cholangiocyte markers, including cytokeratin (CK)-7 and -19 and exhibited cholangiogenic tubule formation in a Matrigel assay. When transplanted into the immunodeficient mice, MMNK-1 cells developed bile duct-like structures in the spleen. After LPS treatment, MMNK-1 cells produced interleukin-6 and failed to form well-developed tubular structures in Matrigel.
Conclusion. We have established an immortalized cholangiocyte cell line, MMNK-1, using SV40T and hTERT transduction.

DOI： 10.1097/01.TP.0000110292.73873.25

Transplantation of reversibly immortalized insulin-secreting human hepatocytes controls diabetes in pancreatectomized pigs

T Okitsu, N Kobayashi, HS Jun, S Shin, SJ Kim, J Han, H Kwon, M Sakaguchi, T Totsugawa, M Kohara, KA Westerman, N Tanaka, P Leboulch, JW Yoon

DIABETES 53 ( 1 ) 105 - 112 2004.1

Language：English Publisher：AMER DIABETES ASSOC

Type 1 diabetes results from the destruction of insulin-producing pancreatic beta-cells by a beta-cell-specific autoimmune process. Although converting other cell types into insulin-producing cells may compensate for the loss of the beta-cell mass while evading beta-cell-specific T-cell responses, proof-of-principle of this approach in large animal models is lacking. This investigation was initiated to determine whether an insulin-producing human hepatocyte line can control diabetes when transplanted into totally pancreatectomized diabetic pigs. We established a reversibly immortalized human hepatocyte line, YOCK-13, by transferring a human telomerase reverse transcriptase cDNA and a drug-inducible Cre recombinase cassette, followed by cDNA for a modified insulin under the control of the L-type pyruvate kinase (L-PK) promoter. YOCK-13 cells produced small amounts of modified insulin and no detectable endogenous L-PK at low glucose concentrations, whereas they produced large amounts of both modified insulin and L-PK in response to high glucose concentrations. Xenotransplantation of YOCK-13 cells via the portal vein into immunosuppressed, totally pancreatectomized pigs decreased hyperglycemia and prolonged survival without adverse effects such as portal thrombosis, liver necrosis, pulmonary embolism, and tumor development. We suggest that this reversibly immortalized, insulin-secreting human hepatocyte line may overcome the shortage of donor pancreata for islet transplantation into patients with type 1 diabetes.

DOI： 10.2337/diabetes.53.1.105

Propagation of adult rat bone marrow-derived hepatocyte-like cells by serial passages in vitro

M Miyazaki, T Masaka, Akiyama, I, E Nakashima, M Sakaguchi, NH Huh

CELL TRANSPLANTATION 13 ( 4 ) 385 - 391 2004

Language：English Publisher：COGNIZANT COMMUNICATION CORP

Previously, we found that hepatocyte growth factor receptor (c-Met)-and alpha-fetoprotein (AFP)-expressing cells were present in adult rat bone marrow, and that these cells also expressed hematopoietic stem cell markers, such as CD34, Thy-1, and c-Kit. When bone marrow cells were cultured in a hepatocyte growth medium (HGM) with HGF and EGF, colonies composed of polygonal cells resembling mature hepatocytes appeared by 2 weeks and grew very slowly because of overgrowth of stromal cells. At days 34-41, 2-mm(2) sheets of hepatocyte-like cells were cut out of their colonies by scratching with an injection needle under observation with a phase contrast microscope, transferred into wells of 24-well plates, and cultured in the HGM medium in the presence or absence of HGF and EGF. When cells reached confluence, cells were detached with trypsin and EDTA and transferred step by step into bigger culture vessels. Thus, hepatocyte-like cells were expanded 1000-fold during less than 4 months. These cells were immunocytochemically stained for albumin and also for AFP and the hematopoietic stem cell markers described above, showing characteristics of oval cells. By RT-PCR, we detected mRNAs of tryptophan-2,3-dioxygenase and tyrosine aminotransferase, markers of hepatocytes at a terminal differentiation stage. The present culture system may be useful for supply of hepatocyte resources for cell transplantation therapy.

NMR STUDIES OF THE N-TERMINAL SEGMENT OF S100C/A11 PROTEIN

T. Kouno, M. Mizuguchi, M. Sakaguchi, E. Makino, N. H. Huh, K. Kawano

JOURNAL OF PEPTIDE SCIENCE 10 273 - 273 2004

S100C/A11 is a key mediator of Ca2+-induced growth inhibition of human epidermal keratinocytes

M Sakaguchi, M Miyazaki, M Takaishi, Y Sakaguchi, E Makino, N Kataoka, H Yamada, M Namba, NH Huh

JOURNAL OF CELL BIOLOGY 163 ( 4 ) 825 - 835 2003.11

Language：English Publisher：ROCKEFELLER UNIV PRESS

An increase in extracellular Ca2+ induces growth arrest and differentiation of human keratinocytes in culture. We examined possible involvement of S100C/A11 in this growth regulation. On exposure of the cells to high Ca2+, S100C/A11 was specifically phosphorylated at (10)Thr and (94)Ser. Phosphorylation facilitated the binding of S100C/A11 to nucleolin, resulting in nuclear translocation of S100C/A11. In nuclei, S100C/A11 liberated Sp1/3 from nucleolin. The resulting free Sp1/3 transcriptionally activated p21(CIP1/WAF1), a representative negative regulator of cell growth. Introduction of anti-S100C/A11 antibody into the cells largely abolished the growth inhibition induced by Ca2+ and the induction of p21(CIP1/WAF1). In the human epidermis, S100C/A11 was detected in nuclei of differentiating cells in the suprabasal layers, but not in nuclei of proliferating cells in the basal layer. These results indicate that S100C/A11 is a key mediator of the Ca2+-induced growth inhibition of human keratinocytes in culture, and that it may be possibly involved in the growth regulation in vivo as well.

DOI： 10.1083/jcb.200304017

Dynamic alteration of human beta-defensin 2 localization from cytoplasm to intercellular space in psoriatic skin

WK Huh, T Oono, Y Shirafuji, H Akiyama, J Arata, M Sakaguchi, NH Huh, K Iwatsuki

JOURNAL OF MOLECULAR MEDICINE-JMM 80 ( 10 ) 678 - 684 2002.10

Language：English Publisher：SPRINGER-VERLAG

Defensins are cationic antimicrobial peptides with a broad spectrum. Recently human P-defensin 2 (hBD-2) has been isolated from psoriatic skin; however, its exact localization and fate have not been fully under,stood. We studied the distribution pattern of hBD-2 in skin tissues of psoriasis and other inflammatory skin diseases. In the upper spinous and granular layer of psoriasis vulgaris hBD-2 was present in the cytoplasm. In the horny layer the positive signals were in a basket-weave pattern, indicating possible accumulation of hBD-2 in the intercellular space. The similar pattern of hBD-2 distribution was observed in the lesions of nummular eczema and atopic dermatitis. hBD-2 was not detected in the section of normal elbow and knee skin. When isolated psoriatic scales were stained. hBD-2 was detected in a wrapping paper-like distribution pattern surrounding the corneocytes. In horny layer of psoriatic skin hBD-2 was closely associated or c localized with elafin, which is known to be in extracellular space, as demonstrated by double staining. Western blot analysis using cultured human keratinocytes detected hBD-2 with an expected size in the conditioned medium and in the cell lysates when stimulated with 5% FCS or IL-alpha. These results indicate that hBD-2 was synthesized and remained in cytoplasm in the upper spinous and granular layer, and then secreted into intercellular space in the horny layer. This dynamic change in hBD-2 distribution in epidermis is certainly relevant to function as an innate host defense mechanism against invading microorganisms.

DOI： 10.1007/s00109-002-0373-z

新規タンパク質リン酸化酵素BRPKの解析

阪口政清, 洪梅, 片岡健, 宮崎正博, 許南浩

日本癌学会総会記事 61回 81 - 81 2002.10

Improvement in the differentiated hepatic phenotype of immortalized human hepatocytes by adenovirus mediated p21 gene transfer

N Kobayashi, M Sakaguchi, T Okitsu, T Totsugawa, M Maruyama, T Matsumura, T Watanabe, H Noguchi, Y Kosaka, T Fujiwara, N Tanaka

ASAIO JOURNAL 48 ( 4 ) 355 - 359 2002.7

Language：English Publisher：LIPPINCOTT WILLIAMS & WILKINS

The p21 molecule, a potent cyclin dependent kinase inhibitor, regulates the transition from the G1 phase to the S phase of the cell cycle and is involved in terminal cellular differentiation. The overexpression of p21 has been shown to induce differentiation in various cell lines. We have made an effort to establish a reliable human hepatocyte cell line as a source of hepatic function in bioartificial liver (BAL) therapy. In this work, we investigated the effect of p21 on the differential phenotype of simian virus 40 large T antigen (SV40Tag) immortalized human hepatocytic NKNT-3 cells. A recombinant adenoviral vector expressing a p21 gene under control of the cytomegalovirus (CMV) promoter (Ad-p21) was used to efficiently transfer genes into NKNT-3 cells. The morphologic alterations, the cell cycle progression, and the expression of p-450 associated enzymes (CYPs) were carefully examined in NKNT-3 cells that had been infected with Ad-p21. Adenovirus mediated gene delivery of p21 was efficiently achieved in NKNT-3 cells without affecting cellular structure. After Ad-p21 infection, NKNT-3 cells were GO/G1 arrested in cell cycle analysis. NKNT-3 cells that had been infected with Ad-p21 showed differentiated hepatic phenotypes in morphology and improvement in protein expression of CYP 3A4 and CYP 2C9. In the present work, we demonstrate that the exogenous expression of p21 enhances the differential phenotype of immortalized hepatocytic NKNT-3 cells.

DOI： 10.1097/00002480-200207000-00005

Reversible immortalization of human hepatocytes using the Cre/Loxp system for cell therapies

T Totsugawa, N Kobayashi, T Okitsu, H Noguchi, T Watanabe, T Matsumura, M Maruyama, M Hikida, A Ohmori, M Sakaguchi, KA Westerman, P Leboulch, N Tanaka

GASTROENTEROLOGY 123 ( 1 ) 54 - 54 2002.7

Controlled expansion of human endothelial cell Populations by cre-loxP-based reversible immortalization

H Noguchi, N Kobayashi, KA Westerman, M Sakaguchi, T Okitsu, T Totsugawa, T Watanabe, T Matsumura, T Fujiwara, T Ueda, M Miyazaki, N Tanaka, P Leboulch

HUMAN GENE THERAPY 13 ( 2 ) 321 - 334 2002.1

Language：English Publisher：MARY ANN LIEBERT INC PUBL

Endothelial cells (ECs) play multiple physiological functions and are central to many pathological processes. Various biological studies as well as cell and gene therapy applications would benefit substantially from a procedure that would result in the expansion in culture of large numbers of highly differentiated human ECs. Here, we report the amplification in vitro of human EC populations, which occurred during the first phase of reversible immortalization resulting from the retroviral transfer of an oncogene that was subsequently excised by Cre-loxP-mediated site-specific recombination. Human umbilical vein endothelial cells (HUVECs) and human liver sinusoidal endothelial cells (HLSECs) were transduced with a retroviral vector that expresses the simian virus 40 large T (SV40T) gene flanked by positive and negative selectable markers and a pair of loxP recombination targets. Transduced HUVECs and HLSECs yielded clones with greatly extended life spans, referred to as HNNT-1 and HNNT-2 cells, respectively. HNNT-1 and HNNT-2 cells showed morphological characteristics of ECs and were maintained in culture up to population doubling level (PDL) 80 for HNNT-1 and PDL 65 for HNNT-2 cells. HNNT-1 and HNNT-2 cells were not tumorigenic when transplanted into severe combined immunodeficiency mice and were sensitive to ganciclovir as well as G418. Both cell clones expressed EC markers, which include factor VIII, VEGF receptors (Flt-1 and KDR/Flk-1), and CD34, and endocytosed acetylated low-density lipoproteins. Formation of capillary-like structures in a Matrigel assay was observed with HNNT-1 and HNNT-2 cells until at least PDL 50. Complete elimination of the transferred SV40T gene was achieved in virtually 100% of HNNT-1 and HNNT-2 cells after infection with a recombinant adenovirus expressing the Cre recombinase fused to a nuclear localization signal and subsequent selection with G418. Reverted cells maintained their differentiated EC phenotype. This study extends the utility of the reversible immortalization procedure and provides a means to expand primary human ECs of various sources for basic studies and possible cell and gene therapies.

DOI： 10.1089/10430340252769833

レンチウイルスベクターシステム樹立に伴う安全配慮.

都津川敏範, 小林直哉, 丸山昌伸, 小坂芳和, 興津輝, 荒田尚, 阪口政清, 藤原俊義, 倉林譲, 田中紀章

Organ Biology 9,4,357-364 2002

Lentiviral transfer of the LacZ gene into human endothelial cells and human bone marrow mesenchymal stem cells

T Totsugawa, N Kobayashi, T Okitsu, H Noguchi, T Watanabe, T Matsumura, M Maruyama, T Fujiwara, M Sakaguchi, N Tanaka

CELL TRANSPLANTATION 11 ( 5 ) 481 - 488 2002

Language：English Publisher：COGNIZANT COMMUNICATION CORP

Because one of the attractive characteristics of human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors is that it can infect even nondividing cells, a lentivirus-mediated gene delivery system is currently being paid a great deal of attention as an innovative tool for gene transfer into target cells. The purpose of the work was to investigate the efficacy of lentiviral transfer of the LacZ gene into human umbilical vein endothelial cells (HUVECs) and human bone marrow mesenchymal stem cells (HMSCs) in vitro. For the present study, a vesicular stomatitis virus G-protein (VSV-G)-pseudotyped lentiviral vector encoding the E. coli LacZ gene tagged with nuclear localization signal (NLS) was generated in 293T cells by means of the three-plasmid system. The resulting lentiviral vector, LtV-NLS/LacZ, was allowed to infect HUVECs and HMSCs. Approximately 70% of HUVECs were positive for LacZ expression and 50% of HMSCs showed LacZ activity. There was no significant difference in transduction efficacy between early and late-passage phases in both cells. LtV-NLS/LacZ-transduced HUVECs showed gene expression of endothelial markers including CD34 and flt-1 and KDR/flk-1 of vascular endothelial growth factor (VEGF) receptors and had angiogenic potential as efficiently as primarily cultured HUVECs in a Matrigel assay. These findings provide evidence that lentiviral vectors are efficient tools for gene transfer and expression in human endothelial cells and stem cells that could be useful for tissue engineering.

Transduction of immortalized human hepatocytes with p21 to enhance differentiated phenotypes

T Kunieda, N Kobayashi, M Sakaguchi, T Okitsu, T Totsugawa, T Watanabe, T Matsumura, M Maruyama, H Noguchi, M Takesue, N Shibata, K Ohmoto, T Fujiwara, S Yamamoto, N Tanaka

CELL TRANSPLANTATION 11 ( 5 ) 421 - 428 2002

Language：English Publisher：COGNIZANT COMMUNICATION CORP

We previously constructed an immortal human hepatocyte line NKNT-3 with a simian virus 40 T antigen (SV40T) to develop cell-based biological therapies. p21 is a molecule that regulates the transition from the G, phase to the S phase of the cell cycle. Investigators have demonstrated that overexpression of p21 induces differentiation in various cell lines. In the current study we examined the effect of p21 on differentiated phenotypes of SV40T-immortalized NKNT-3 cells. A replication-deficient adenovirus vector expressing a human wild-type p21 cDNA under the control of the CMV promoter (Ad5CMVp21) and a human wild-type p21 protein fused to the protein transduction domain from the human immunodeficiency virus (HIV) TAT protein (TAT/p21) were utilized to achieve efficient delivery of p21 into NKNT-3 cells. Morphological alterations, cell cycle progression, and expression of albumin and p-450 associated enzymes (CYPs) 3A4 and 2C9 were evaluated in NKNT-3 cells treated with Ad5CMVp21 and TAT/p21. Efficient adenovirus-based p21 transfer and TAT-mediated p21 protein delivery were confirmed in NKNT-3 cells in an immunofluorescence study and Western blotting analysis. Transduction of NKNT-3 cells with p21 predominantly arrested the cell cycle at the G, checkpoint, resulting in differentiated hepatic phenotypes in morphology and improvement in protein expression of albumin, CYP 3A4, and CYP C29. We here show that exogenous expression of p21 augments cellular differentiation in immortalized human NKNT-3 cells.

Reactivation of liver-specific gene expression in an immortalized human hepatocyte cell line by introduction of the human HNF4 alpha 2 gene

Y Inoue, M Miyazaki, T Tsuji, M Sakaguchi, K Fukaya, NH Huh, M Namba

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 8 ( 5 ) 481 - 487 2001.11

Language：English Publisher：PROFESSOR D A SPANDIDOS

An immortalized human hepatocyte cell line (OUMS-29) was established from fetal liver by transfection with the SV-40 large T antigen gene that has certain liver-specific functions such as albumin production and enzyme activities of CYP1A1, 1A2, and 2E1. To make OUMS-29 cells express other liver-specific functions, the human hepatocyte nuclear factor 4 alpha2 (HNF4 alpha2) gene was introduced into the cells, because this gene was found to be markedly down-regulated. The transduced HNF4 alpha2 was overexpressed in the nuclei of the transfected cells, and its DNA-binding activity was also detected. The liver-specific genes such as apolipoprotein AI, CII, CIII, blood coagulation factor X, alpha1-antitrypsin, and HNF1 alpha were up-regulated. Thus, this cell line is expected to be a useful tool for studying the differentiated human hepatocyte, functions.

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