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Kazuyuki Sugahara - One of the best experts on this subject based on the ideXlab platform.
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the tumor suppressor ext like gene extl2 encodes an alpha1 4 n acetylhexosaminyltransferase that transfers n acetylgalactosamine and n acetylglucosamine to the common glycosaminoglycan protein linkage region the key enzyme for the chain initiation of heparan sulfate
Journal of Biological Chemistry, 1999Co-Authors: Hiroshi Kitagawa, Hiromi Shimakawa, Kazuyuki SugaharaAbstract:Abstract We previously demonstrated a unique α-N-acetylgalactosaminyltransferase that transferredN-acetylgalactosamine (GalNAc) to the tetrasaccharide-serine, GlcAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser (GlcA represents glucuronic acid), derived from the common glycosaminoglycan-protein linkage region, through an α1,4-linkage. In this study, we purified the enzyme from the serum-free culture medium of a human Sarcoma Cell Line. Peptide sequence analysis of the purified enzyme revealed 100% identity to the multiple exostoses-like geneEXTL2/EXTR2, a member of the hereditary multiple exostoses (EXT) gene family of tumor suppressors. The expression of a soluble recombinant form of the protein produced an active enzyme, which transferred α-GalNAc from UDP-[3H]GalNAc to various acceptor substrates including GlcAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser. Interestingly, the enzyme also catalyzed the transfer ofN-acetylglucosamine (GlcNAc) from UDP-[3H]GlcNAc to GlcAβ1–3Galβ1-O-naphthalenemethanol, which was the acceptor substrate for the previously described GlcNAc transferase I involved in the biosynthetic initiation of heparan sulfate. The GlcNAc transferase reaction product was sensitive to the action of heparitinase I, establishing the identity of the enzyme to be α1,4-GlcNAc transferase. These results altogether indicate thatEXTL2/EXTR2 encodes the α1,4-N-acetylhexosaminyltransferase that transfers GalNAc/GlcNAc to the tetrasaccharide representing the common glycosaminoglycan-protein linkage region and that is most likely the critical enzyme that determines and initiates the heparin/heparan sulfate synthesis, separating it from the chondroitin sulfate/dermatan sulfate synthesis.
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the tumor suppressor ext like gene extl2 encodes an α1 4 n acetylhexosaminyltransferase that transfersn acetylgalactosamine and n acetylglucosamine to the common glycosaminoglycan protein linkage region the key enzyme for the chain initiation of heparan sulfate
Journal of Biological Chemistry, 1999Co-Authors: Hiroshi Kitagawa, Hiromi Shimakawa, Kazuyuki SugaharaAbstract:Abstract We previously demonstrated a unique α-N-acetylgalactosaminyltransferase that transferredN-acetylgalactosamine (GalNAc) to the tetrasaccharide-serine, GlcAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser (GlcA represents glucuronic acid), derived from the common glycosaminoglycan-protein linkage region, through an α1,4-linkage. In this study, we purified the enzyme from the serum-free culture medium of a human Sarcoma Cell Line. Peptide sequence analysis of the purified enzyme revealed 100% identity to the multiple exostoses-like geneEXTL2/EXTR2, a member of the hereditary multiple exostoses (EXT) gene family of tumor suppressors. The expression of a soluble recombinant form of the protein produced an active enzyme, which transferred α-GalNAc from UDP-[3H]GalNAc to various acceptor substrates including GlcAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser. Interestingly, the enzyme also catalyzed the transfer ofN-acetylglucosamine (GlcNAc) from UDP-[3H]GlcNAc to GlcAβ1–3Galβ1-O-naphthalenemethanol, which was the acceptor substrate for the previously described GlcNAc transferase I involved in the biosynthetic initiation of heparan sulfate. The GlcNAc transferase reaction product was sensitive to the action of heparitinase I, establishing the identity of the enzyme to be α1,4-GlcNAc transferase. These results altogether indicate thatEXTL2/EXTR2 encodes the α1,4-N-acetylhexosaminyltransferase that transfers GalNAc/GlcNAc to the tetrasaccharide representing the common glycosaminoglycan-protein linkage region and that is most likely the critical enzyme that determines and initiates the heparin/heparan sulfate synthesis, separating it from the chondroitin sulfate/dermatan sulfate synthesis.
Hiroshi Sonobe - One of the best experts on this subject based on the ideXlab platform.
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analysis of transforming activity of human synovial Sarcoma associated chimeric protein syt ssx1 bound to chromatin remodeling factor hbrm hsnf2α
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Makoto Nagai, Hiroshi Sonobe, Masumi Tsuda, Akio Minami, Shinya Tanaka, Shuichi Endo, Hiroyuki Kato, Hiroaki Hiraga, Hiroshi Nishihara, Hirofumi SawaAbstract:Human synovial Sarcoma has been shown to exclusively harbor the chromosomal translocation t(X;18) that produces the chimeric gene SYT-SSX. However, the role of SYT-SSX in Cellular transformation remains unclear. In this study, we have established 3Y1 rat fibroblast Cell Lines that constitutively express SYT, SSX1, and SYT-SSX1 and found that SYT-SSX1 promoted growth rate in culture, anchorage-independent growth in soft agar, and tumor formation in nude mice. Deletion of the N-terminal 181 amino acids of SYT-SSX1 caused loss of its transforming activity. Furthermore, association of SYT-SSX1 with the chromatin remodeling factor hBRM/hSNF2α, which regulates transcription, was demonstrated in both SYT-SSX1-expressing 3Y1 Cells and in the human synovial Sarcoma Cell Line HS-SY-II. The binding region between the two molecules was shown to reside within the N-terminal 181 amino acids stretch (aa 1–181) of SYT-SSX1 and 50 amino acids (aa 156–205) of hBRM/hSNF2α and we found that the overexpression of this binding region of hBRM/hSNF2α significantly suppressed the anchorage-independent growth of SYT-SSX1-expressing 3Y1 Cells. To analyze the transcriptional regulation by SYT-SSX1, we established conditional expression system of SYT-SSX1 and examined the gene expression profiles. The down-regulation of potential tumor suppressor DCC was observed among 1,176 genes analyzed by microarray analysis, and semi-quantitative reverse transcription–PCR confirmed this finding. These data clearly demonstrate transforming activity of human oncogene SYT-SSX1 and also involvement of chromatin remodeling factor hBRM/hSNF2α in human cancer.
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a new human synovial Sarcoma Cell Line hs sy 3 with a truncated form of hybrid syt ssx1 gene
International Journal of Cancer, 1999Co-Authors: Hiroshi Sonobe, Mutsuo Furihata, J Iwata, Yuji Ohtsuki, Tamotsu Takeuchi, Shengben Liag, Takahiro Taguchi, Kazunari Yuri, Kiichi Shimizu, Joseph R TestaAbstract:Recent cytogenetical and molecular studies have indicated that synovial Sarcoma harbors a t(X;18)(p11.2;q11.2) translocation, resulting in the formation of a hybrid SYT/SSX (SSX1 or SSX2) gene. We newly established a human Cell Line, HS-SY-3, from a synovial Sarcoma. HS-SY-3 Cells were shown to harbor the pathognomonic t(X;18)(p11.2;q11.2) translocation by chromosome analysis but not to exhibit the classical hybrid SYT/SSX transcripts induced by this translocation, using RT-PCR. To determine the reason for this discrepancy, we analyzed cDNA from HS-SY-3 Cells, as well as the original Sarcoma tissue by the rapid amplification of cDNA 3′ end assay, and found that the chimaeric cDNA was 240 bp shorter than the previously established SYT/SSX1 cDNA due to truncation of the 3′ side of SSX1. The HS-SY-3 Cells should be useful for future functional studies of the SYT/SSX chimeric gene. Int. J. Cancer 82:459–464, 1999. © 1999 Wiley-Liss, Inc.
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further characterization of the human clear Cell Sarcoma Cell Line hs mm demonstrating a specific t 12 22 q13 q12 translocation and hybrid ews atf 1 transcript
The Journal of Pathology, 1999Co-Authors: Hiroshi Sonobe, Mutsuo Furihata, Tamotsu Takeuchi, Takahiro Taguchi, Kiichi Shimizu, Jun Iwata, Yuji OhtsukiAbstract:Only a small number of clear Cell Sarcoma (CCS) Cell Lines have been reported, including the Cell Line HS-MM. In the present study, this Cell Line, maintained for more than 4 years since establishment, was further characterized by cytogenetic studies, fluorescence in situ hybridization (FISH) analysis, and reverse transcriptase-polymerase chain reaction (RT-PCR). HS-MM Cells both in vitro and in vivo exhibited pseudodiploid karyotypes with the specific t(12;22)(q13;q12) translocation. The translocation between chromosomes 12 and 22 was confirmed by FISH analysis and the hybrid EWS/ATF-1 transcript induced by this translocation was detected by RT-PCR. The HS-MM Cell Line will be useful for further studies of CCS.
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establishment and characterization of a new human synovial Sarcoma Cell Line hs sy ii
Laboratory Investigation, 1992Co-Authors: Hiroshi Sonobe, Yuiko Manabe, Mutsuo Furihata, J Iwata, T Oka, Yuji Ohtsuki, H Mizobuchi, H Yamamoto, O Kumano, Syuiti AbeAbstract:BACKGROUND The Line of differentiation in synovial Sarcoma still remains controversial. Thus far, only a few human synovial Sarcoma Cell Lines have been described. However, their morphologic characteristics have not been fully established. EXPERIMENTAL DESIGN We established a new synovial Sarcoma Cell Line (HS-SY-II) from pleural effusion with lung metastasis in a typical example of the monophasic spindle Cell type. The HS-SY-II Cells, in vitro and in vivo, were examined by light microscopy, immunohistochemistry, electron microscopy, and cytogenetics. RESULTS The HS-SY-II Cells showed a hypertriploid karyotype with complex chromosome abnormalities including pathognomonic t(X;18)(p11;q11), and have been stably maintained for more than 40 months in vitro, showing rather small spindle or polygonal shape without conspicuous pleomorphism. Histologic features of initially and serially transplanted tumors in nude mice were essentially the same as those of the original Sarcoma, corresponding to the monophasic spindle Cell variant with a prominent palisading pattern and calcified foci in parts. The HS-SY-II Cells in vitro and in vivo similarly expressed vimentin and cytokeratin by immunohistochemistry, and also exhibit the same ultrastructural features such as irregularly shaped nuclei with prominent nucleoli, many paranuclearly running intermediate filaments, and filopodia-like processes. CONCLUSIONS This HS-SY-II Cell Line retaining the distinct morphological characteristics as the monophasic spindle Cell type of synovial Sarcoma therefore will be extremely useful for various pathomorphologic investigations on synovial Sarcoma.
Lor R Randall - One of the best experts on this subject based on the ideXlab platform.
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doxorubicin induces Cell senescence preferentially over apoptosis in the fu sy 1 synovial Sarcoma Cell Line
Journal of Orthopaedic Research, 2006Co-Authors: David E Joyner, Jeffrey D Bastar, Lor R RandallAbstract:Surgical resection coupled with adjuvant radiotherapy and/or doxorubicin based chemotherapy are the mainstays of synovial Sarcoma (SS) treatment. Although effective as a SS adjuvant, the proposed mechanism of action of doxorubicin remains controversial. Current opinion supports DNA damage-induced apoptosis. This in vitro study used cDNA gene expression profiling to investigate whether apoptosis, alone or in combination with Cell senescence, is induced by doxorubicin in SS Cells. Cell cultures of the FU-SY-1 SS, the pleomorphic SW982 Sarcoma, and a primary dermal fibroblast (NHDF), were exposed to 500 nM doxorubicin, and then processed for cDNA microarray analysis. The one class response option of SAM (Significance Analysis of Microarrays) was used to test for significant overexpression of 15 apoptosis-related genes and nine senescence-related genes. Drug-induced Cell senescence was quantified by measuring beta-galactosidase activity. None of 15 apoptosis-related genes and only two of nine senescence-related genes were identified by SAM as significantly overexpressed in doxorubicin-treated cultures. Drug-induced senescence as reflected by beta-galactosidase activity was significantly increased (p < 0.05) only in FU-SY-1 SS cultures. Apoptosis does not appear to be a major determinant of doxorubicin-induced mortality in FU-SY-1 SS or NHDF cultures, but may impact SW982 Cells via the overexpression of BAX relative to Bcl-2. Doxorubicin-induced Cell senescence was prominent in FU-SY-1 SS cultures, but negligible in SW982 and NHDF cultures. Likely, both apoptosis and Cell senescence contribute to doxorubicin-induced Cell death in this synovial Sarcoma Cell Line.
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g3139 antisense oligonucleotide directed against antiapoptotic bcl 2 enhances doxorubicin cytotoxicity in the fu sy 1 synovial Sarcoma Cell Line
Journal of Orthopaedic Research, 2006Co-Authors: David E Joyner, Jeffrey D Bastar, Karen H Albritton, Lor R RandallAbstract:Synovial Sarcoma (SS) is a highly aggressive, periarticular soft tissue Sarcoma that causes death in more than half of affected children, adolescents, and young adults. Five- and 10-year survival rates are as low as 36 and 20%, respectively. Bcl-2, a negative regulator of apoptosis, is overexpressed in up to 90% of SS. Increased Bcl-2 expression not only leads to the development of cancer, but also to resistance of many anticancer chemotherapeutic agents. We hypothesized reducing Bcl-2 expression in SS should enhance doxorubicin cytotoxicity. Cell cultures representing two human Sarcomas (FU-SY-1 SS and the pleomorphic SW982) and a primary human dermal fibroblast comparator (NHDF) were exposed in vitro to doxorubicin, or to doxorubicin preceded by Bcl-2 (G3139) antisense oligonucleotides, and assayed for Cell survival, apoptosis, and modulations in Bcl-2 and Bcl-xL mRNA and protein content. SW982 Sarcoma Cells proved most susceptible to doxorubicin, while NHDF mesenchymal Cells were least sensitive to doxorubicin. Treatment of FU-SY-1 SS with G3139 reduced Bcl-2 mRNA and protein levels, which enhanced doxorubicin-induced Cell killing. There was a concurrent reduction in Bcl-xL mRNA following G3139 application in FU-SY-1 and NHDF cultures, but not in SW982. G3139 anti-Bcl-2 intervention sensitized the FU-SY-1 SS to doxorubicin, due to increased apoptosis. G3139 intervention was ineffective in the two non-SS Cell Lines.
Syuiti Abe - One of the best experts on this subject based on the ideXlab platform.
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establishment of a new continuous clear Cell Sarcoma Cell Line morphological and cytogenetic characterization and detection of chimaeric ews atf 1 transcripts
Virchows Archiv, 1997Co-Authors: Hiroaki Hiraga, Syuiti Abe, Takayuki Nojima, Katsushige Yamashiro, Shinya Yamawaki, Kiyoshi Kaneda, Kazuo NagashimaAbstract:Clear Cell Sarcoma (CCS), a rare tumour of deep soft tissues, often has a t(12; 22) (q13; q12) translocation that induces the formation of a hybrid EWS/ATF-1 gene. To investigate these alterations further, we established a new continuous Cell Line directly from a CCS taken from a 9-year-old girl. The cultures were characterized with respect to morphological, ultrastructural, immunohistochemical and karyotypical features and were tested by reverse transcription PCR (RT-PCR) for chimaeric EWS/ATF-1 transcripts. The continuous Cell Line, designated KAO, is tumorigenic in nude mice, and the resultant tumours resemble the primary CCS. The tumour Cells and the cultured Cells have melanosomes in their cytoplasm and are immunoreactive with the melanoma-specific antibody HMB45, but do not express S-100 protein. The cultured CCS Cells have the t(12; 22)(q13; q12) translocation and express the hybrid EWS/ATF-1 gene. No transcripts of the hybrid gene were detected in a malignant cutaneous melanoma tested simultaneously. Although CCS and malignant melanoma are morphologically related, the present results suggest that their geneses differ at the chromosome and molecular levels. They also indicate that chromosome analysis and detection of fusion EWS/ATF-1 transcripts may be useful adjuvant tools for the diagnosis of CCS.
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establishment and characterization of a new human synovial Sarcoma Cell Line hs sy ii
Laboratory Investigation, 1992Co-Authors: Hiroshi Sonobe, Yuiko Manabe, Mutsuo Furihata, J Iwata, T Oka, Yuji Ohtsuki, H Mizobuchi, H Yamamoto, O Kumano, Syuiti AbeAbstract:BACKGROUND The Line of differentiation in synovial Sarcoma still remains controversial. Thus far, only a few human synovial Sarcoma Cell Lines have been described. However, their morphologic characteristics have not been fully established. EXPERIMENTAL DESIGN We established a new synovial Sarcoma Cell Line (HS-SY-II) from pleural effusion with lung metastasis in a typical example of the monophasic spindle Cell type. The HS-SY-II Cells, in vitro and in vivo, were examined by light microscopy, immunohistochemistry, electron microscopy, and cytogenetics. RESULTS The HS-SY-II Cells showed a hypertriploid karyotype with complex chromosome abnormalities including pathognomonic t(X;18)(p11;q11), and have been stably maintained for more than 40 months in vitro, showing rather small spindle or polygonal shape without conspicuous pleomorphism. Histologic features of initially and serially transplanted tumors in nude mice were essentially the same as those of the original Sarcoma, corresponding to the monophasic spindle Cell variant with a prominent palisading pattern and calcified foci in parts. The HS-SY-II Cells in vitro and in vivo similarly expressed vimentin and cytokeratin by immunohistochemistry, and also exhibit the same ultrastructural features such as irregularly shaped nuclei with prominent nucleoli, many paranuclearly running intermediate filaments, and filopodia-like processes. CONCLUSIONS This HS-SY-II Cell Line retaining the distinct morphological characteristics as the monophasic spindle Cell type of synovial Sarcoma therefore will be extremely useful for various pathomorphologic investigations on synovial Sarcoma.
Hiroshi Kitagawa - One of the best experts on this subject based on the ideXlab platform.
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the tumor suppressor ext like gene extl2 encodes an alpha1 4 n acetylhexosaminyltransferase that transfers n acetylgalactosamine and n acetylglucosamine to the common glycosaminoglycan protein linkage region the key enzyme for the chain initiation of heparan sulfate
Journal of Biological Chemistry, 1999Co-Authors: Hiroshi Kitagawa, Hiromi Shimakawa, Kazuyuki SugaharaAbstract:Abstract We previously demonstrated a unique α-N-acetylgalactosaminyltransferase that transferredN-acetylgalactosamine (GalNAc) to the tetrasaccharide-serine, GlcAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser (GlcA represents glucuronic acid), derived from the common glycosaminoglycan-protein linkage region, through an α1,4-linkage. In this study, we purified the enzyme from the serum-free culture medium of a human Sarcoma Cell Line. Peptide sequence analysis of the purified enzyme revealed 100% identity to the multiple exostoses-like geneEXTL2/EXTR2, a member of the hereditary multiple exostoses (EXT) gene family of tumor suppressors. The expression of a soluble recombinant form of the protein produced an active enzyme, which transferred α-GalNAc from UDP-[3H]GalNAc to various acceptor substrates including GlcAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser. Interestingly, the enzyme also catalyzed the transfer ofN-acetylglucosamine (GlcNAc) from UDP-[3H]GlcNAc to GlcAβ1–3Galβ1-O-naphthalenemethanol, which was the acceptor substrate for the previously described GlcNAc transferase I involved in the biosynthetic initiation of heparan sulfate. The GlcNAc transferase reaction product was sensitive to the action of heparitinase I, establishing the identity of the enzyme to be α1,4-GlcNAc transferase. These results altogether indicate thatEXTL2/EXTR2 encodes the α1,4-N-acetylhexosaminyltransferase that transfers GalNAc/GlcNAc to the tetrasaccharide representing the common glycosaminoglycan-protein linkage region and that is most likely the critical enzyme that determines and initiates the heparin/heparan sulfate synthesis, separating it from the chondroitin sulfate/dermatan sulfate synthesis.
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the tumor suppressor ext like gene extl2 encodes an α1 4 n acetylhexosaminyltransferase that transfersn acetylgalactosamine and n acetylglucosamine to the common glycosaminoglycan protein linkage region the key enzyme for the chain initiation of heparan sulfate
Journal of Biological Chemistry, 1999Co-Authors: Hiroshi Kitagawa, Hiromi Shimakawa, Kazuyuki SugaharaAbstract:Abstract We previously demonstrated a unique α-N-acetylgalactosaminyltransferase that transferredN-acetylgalactosamine (GalNAc) to the tetrasaccharide-serine, GlcAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser (GlcA represents glucuronic acid), derived from the common glycosaminoglycan-protein linkage region, through an α1,4-linkage. In this study, we purified the enzyme from the serum-free culture medium of a human Sarcoma Cell Line. Peptide sequence analysis of the purified enzyme revealed 100% identity to the multiple exostoses-like geneEXTL2/EXTR2, a member of the hereditary multiple exostoses (EXT) gene family of tumor suppressors. The expression of a soluble recombinant form of the protein produced an active enzyme, which transferred α-GalNAc from UDP-[3H]GalNAc to various acceptor substrates including GlcAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser. Interestingly, the enzyme also catalyzed the transfer ofN-acetylglucosamine (GlcNAc) from UDP-[3H]GlcNAc to GlcAβ1–3Galβ1-O-naphthalenemethanol, which was the acceptor substrate for the previously described GlcNAc transferase I involved in the biosynthetic initiation of heparan sulfate. The GlcNAc transferase reaction product was sensitive to the action of heparitinase I, establishing the identity of the enzyme to be α1,4-GlcNAc transferase. These results altogether indicate thatEXTL2/EXTR2 encodes the α1,4-N-acetylhexosaminyltransferase that transfers GalNAc/GlcNAc to the tetrasaccharide representing the common glycosaminoglycan-protein linkage region and that is most likely the critical enzyme that determines and initiates the heparin/heparan sulfate synthesis, separating it from the chondroitin sulfate/dermatan sulfate synthesis.
