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Biomedical subjects

Tomitaka Nakayama

Publications and source records attributed to Tomitaka Nakayama.

At least 19 recordsLinked to original sources
PubMed2006

In vitro transformation of mesenchymal stem cells by oncogenic H-rasVal12.

Tissue stem cells may serve as progenitors for malignant tumors derived from the same tissue. Here, we report the establishment of immortalized human mesenchymal stem cells (ihMSC) and tested the feasibility of using ihMSC as presarcomatous cells. Immortalization was achieved by introducing the genes for human telomerase reverse transcriptase and Bmi1. ihMSC retained the potential for multi-directional differentiation of the original MSC. To transform ihMSC, we introduced an oncogenic H-ras(Val12) gene, and established the cell line ihMSC-ras. ihMSC-ras had the phenotype of fully transformed cells and retained adipogenic and chondrogenic, but not osteogenic, potential. Interestingly, ihMSC-ras demonstrated morphological features of autophagy, and inhibition of the ERK pathway suppressed the production of autophagosomes, indicating that ras/ERK signaling is responsible for the induction of autophagy. Thus ihMSC will serve as a material with which to analyze the tumorigenic and differentiation-modifying effects of candidate oncogenes involved in the development of sarcomas.

Cell Culture Techniques↗
PubMed2006

Expression of claudin7 is tightly associated with epithelial structures in synovial sarcomas and regulated by an Ets family transcription factor, ELF3.

Synovial sarcoma, a soft tissue sarcoma that develops in adults, is pathologically subclassified into monophasic spindle synovial sarcoma and biphasic synovial sarcoma with epithelial components. The molecular mechanism building the epithelial components in biphasic synovial sarcoma is totally unknown. Here we investigated claudins, critical molecules in the tight junction, in biphasic synovial sarcoma. Expression profiles of 21 claudins in 17 synovial sarcoma tumor samples, including 9 biphasic tumors, identified claudin4, claudin7, and claudin10 as biphasic tumor-related claudins, and immunohistochemical analyses demonstrated the localization of these claudins in the epithelial component in biphasic tumors, with claudin7 the most closely associated with the epithelial component. The mRNA expression and protein localization of claudin7 coincided with those of the ELF3, an epithelia-specific member of the Ets family of transcription factors. Luciferase reporter assays demonstrated that the presence of the Ets-binding site at -150 in the promoter region of the claudin7 gene was critical for the transcriptional activity, and gel shift and chromatin immunoprecipitation assays confirmed the binding of ELF3 to the Ets site at -150. Inhibition of ELF3 expression by small interfering RNA simultaneously down-regulated the mRNA expression of the claudin7 gene, and the introduction of ELF3 expression in claudin7-negative cell lines induced mRNA expression of the claudin7 gene. Therefore, the induction of claudin7 expression by ELF3 appears critical to the formation of the epithelial structures in biphasic synovial sarcoma.

Base Sequence↗
PubMed2006

A variant of the SYT-SSX2 fusion gene in a case of synovial sarcoma.

Synovial sarcoma is a malignant soft tissue tumor harboring a tumor-specific fusion gene, SYT-SSX, of which exon 10 of the SYT gene is fused to exon 6 of the SSX gene is the common form. Here we report a case of synovial sarcoma with a novel form of the SYT-SSX2 fusion transcript, in which 75 bases were inserted at the common fusion junction. Computer analyses revealed that 15 bases were from intron 10 of the SYT gene, and 10 from the end of intron 4, and 50 from exon 5 of the SSX2 gene. Precise analyses of genomic breakpoints in SYT and SSX2 loci revealed that the reciprocal translocation creating the fusion gene was associated with a large deletion in both loci. The structure of SYT-SSX2 suggests that the fusion transcript in this case was created using a cryptic splicing acceptor site 15 bases upstream of the genomic fusion point, incorporating intronic sequences in mature mRNA. Reexamination of two variant SYT-SSX2 genes reported previously revealed that unknown sequences inserted at the common junction points were derived from intron sequences, as in the present case.

Amino Acid Sequence↗
PubMed2006

Regeneration of osteonecrosis of canine scapho-lunate using bone marrow stromal cells: possible therapeutic approach for Kienböck disease.

We evaluated the ability of canine bone marrow stromal cells (cBMSCs) to regenerate bone in a cavity of the scapholunate created by curretage and freeze-thawing with liquid nitrogen (LN). Autologous BMSCs were harvested from the iliac crest and expanded in vitro. Their potential to differentiate into osteo-, chondro-, and adipogenic lineages was confirmed using a standard differentiation induction assay. LN-treated scapholunates showed no regeneration of bone tissue when the cavity was left alone, demonstrating severe collapse and deformity as observed in human Kienböck disease. A combination of beta-tri-calcium phosphate and a vascularized bone graft with autologous fibroblasts failed to regenerate bone in the LN-treated cavity. When the same procedure was performed using BMSCs, however, LN-treated scapholunates showed no collapse and deformity, and the cavity was completely filled with normal cancerous bone within 4 weeks. These results suggested the potential of using BMSCs to treat Kienböck disease.

Adipogenesis↗
PubMed2005

Disruption of fibroblast growth factor signal pathway inhibits the growth of synovial sarcomas: potential application of signal inhibitors to molecular target therapy.

PURPOSE: Synovial sarcoma is a soft tissue sarcoma, the growth regulatory mechanisms of which are unknown. We investigated the involvement of fibroblast growth factor (FGF) signals in synovial sarcoma and evaluated the therapeutic effect of inhibiting the FGF signal. EXPERIMENTAL DESIGN: The expression of 22 FGF and 4 FGF receptor (FGFR) genes in 18 primary tumors and five cell lines of synovial sarcoma were analyzed by reverse transcription-PCR. Effects of recombinant FGF2, FGF8, and FGF18 for the activation of mitogen-activated protein kinase (MAPK) and the growth of synovial sarcoma cell lines were analyzed. Growth inhibitory effects of FGFR inhibitors on synovial sarcoma cell lines were investigated in vitro and in vivo. RESULTS: Synovial sarcoma cell lines expressed multiple FGF genes especially those expressed in neural tissues, among which FGF8 showed growth stimulatory effects in all synovial sarcoma cell lines. FGF signals in synovial sarcoma induced the phosphorylation of extracellular signal-regulated kinase (ERK1/2) and p38MAPK but not c-Jun NH2-terminal kinase. Disruption of the FGF signaling pathway in synovial sarcoma by specific inhibitors of FGFR caused cell cycle arrest leading to significant growth inhibition both in vitro and in vivo. Growth inhibition by the FGFR inhibitor was associated with a down-regulation of phosphorylated ERK1/2 but not p38MAPK, and an ERK kinase inhibitor also showed growth inhibitory effects for synovial sarcoma, indicating that the growth stimulatory effect of FGF was transmitted through the ERK1/2. CONCLUSIONS: FGF signals have an important role in the growth of synovial sarcoma, and inhibitory molecules will be of potential use for molecular target therapy in synovial sarcoma.

Animals↗
PubMed2004

PGE2 signal through EP2 promotes the growth of articular chondrocytes.

UNLABELLED: EP2 was identified as the major PGE2 receptor expressed in articular cartilage. An EP2 agonist increased intracellular cAMP in articular chondrocytes, stimulating DNA synthesis in both monolayer and 3D cultures. Hence, the EP2 agonist may be a potent therapeutic agent for degenerative cartilage diseases. INTRODUCTION: Prostaglandin E2 (PGE2) exhibits pleiotropic effects in various types of tissue through four types of receptors, EP1-4. We examined the expression of EPs and effects of agonists for each EP on articular chondrocytes. MATERIALS AND METHODS: The expression of each EP in articular chondrocytes was examined by immunohistochemistry and RT-PCR. A chondrocyte cell line, MMA2, was established from articular cartilage of p53(-/-) mice and used to analyze the effects of agonists for each EP. A search for molecules downstream of the PGE2 signal through the EP2 agonist was made by cDNA microarray analysis. The growth-promoting effect of the EP2 agonist on chondrocytes surrounded by cartilage matrix was examined in an organ culture of rat femora. RESULTS AND CONCLUSION: EP2 was identified as the major EP expressed in articular cartilage. Treatment of MMA2 cells with specific agonists for each EP showed that only the EP2 agonist significantly increased intracellular cAMP levels in a dose-dependent manner. Gene expression profiling of MMA2 revealed a set of genes upregulated by the EP2 agonist, including several growth-promoting and apoptosis-protecting genes such as the cyclin D1, fibronectin, integrin alpha5, AP2alpha, and 14-3-3gamma genes. The upregulation of these genes by the EP2 agonist was confirmed in human articular chondrocytes by quantitative mRNA analysis. On treatment with the EP2 agonist, human articular chondrocytes showed an increase in the incorporation of 5-bromo-2-deoxyuracil (BrdU), and the organ culture of rat femora showed an increase of proliferating cell nuclear antigen (PCNA) staining in articular chondrocytes surrounded by cartilage matrix, suggesting growth-promoting effects of the PGE2 signal through EP2 in articular cartilage. These results suggested that the PGE2 signal through EP2 enhances the growth of articular chondrocytes, and the EP2 agonist is a candidate for a new therapeutic compound for the treatment of degenerative cartilage diseases.

Adult↗
PubMed2004

Mutation analysis of the RECQL4 gene in sporadic osteosarcomas.

Osteosarcoma (OS) is the most prevalent malignant tumor among cases of Rothmund-Thomson syndrome (RTS) with germline mutations of the RECQL4 gene, a member of the RecQ helicase family. We investigated the involvement of the RECQL4 gene in the development of OS unrelated to RTS. RECQL4 mRNA was detected in 9 of 9 OS cell lines by Northern blotting and 26 of 26 OS tumors by RT-PCR. Direct sequencing of the entire coding region along with flanking splice junctions and 13 small (< 100 bp) introns in 71 OS tumors revealed 2 sites with a single-base change causing an amino acid change (G1814A for R355Q and C2474T for P441S) and one site with a 6 bp inframe deletion (4837-42delTGCACC for CT857-8del). Identical genotypes were found in corresponding normal tissues in all cases, and the frequency of each allele was not significantly different between OS and control populations. Our data indicate that the RECQL4 gene is not a frequent target for somatic mutations in sporadic OS unrelated to RTS.

Adenosine Triphosphatases↗
PubMed2004

Methylation in the core-promoter region of the chondromodulin-I gene determines the cell-specific expression by regulating the binding of transcriptional activator Sp3.

Transcriptional regulation of cell- and stage-specific genes is a crucial process in the development of mesenchymal tissues. Here we have investigated the regulatory mechanism of the expression of the chondromodulin-I (ChM-I) gene, one of the chondrocyte-specific genes, in osteogenic cells using osteosarcoma (OS) cells as a model. Methylation-specific sequence analyses revealed that the extent of methylation in the core-promoter region of the ChM-I gene was correlated inversely with the expression of the ChM-I gene in OS primary tumors and cell lines. 5-Aza-deoxycytidine treatment induced the expression of the ChM-I gene in ChM-I-negative OS cell lines, and the induction of expression was associated tightly with the demethylation of cytosine at -52 (C(-52)) in the middle of an Sp1/3 binding site to which the Sp3, but not Sp1, bound. The replacement of C(-52) with methyl-cytosine or thymine abrogated Sp3 binding and also the transcription activity of the genomic fragment including C(-52). The inhibition of Sp3 expression by small interfering RNA reduced the expression of the ChM-I gene in ChM-I-positive normal chondrocytes, indicating Sp3 as a physiological transcriptional activator of the ChM-I gene. These results suggest that the methylation status of the core-promoter region is one of the mechanisms to determine the cell-specific expression of the ChM-I gene through the regulation of the binding of Sp3.

5-Methylcytosine↗
PubMed2004

Expression of the chondromodulin-I gene in chondrosarcomas.

We investigated the expression of the Chondromodulin-I (ChM-I) gene, a putative tumor suppressor gene in cartilaginous tumors, by quantitative RT-PCR in 15 chondrosarcomas (CSs). Eight CSs expressed the ChM-I gene at the level higher than those in articular cartilage (positive cases), whereas the expression of the ChM-I gene in the remaining seven CSs was lower than those in articular cartilage (negative cases). All of five peripheral CS were positive, and the ChM-I positive tumors shared expression profiles of cartilage-related genes with articular cartilage cells. On the other hand, all of four central CSs without extramedullary lesions were negative, and the ChM-I negative tumors expressed the parathyroid hormone-related peptide gene at the lower level and the COL10A1 genes at the higher level than articular cartilage cells. Neither the histological grade nor the rate of recurrence showed clear association with the level of ChM-I gene expression. These results suggested that the expression of ChM-I gene in CS has no direct role in tumorigenesis but rather reflects the site of tumor development and therefore precursor of tumor cells.

Aggrecans↗
PubMed2003

FR901228 induces tumor regression associated with induction of Fas ligand and activation of Fas signaling in human osteosarcoma cells.

We investigated the antitumor effects of FR901228, a HDAC inhibitor, on human osteosarcoma cells, in vitro and in vivo to explore its possible utility in the treatment of pediatric bone cancers. FR901228 caused marked growth inhibition with a 50% inhibitory concentration of 1.2-7.3 nM and induction of apoptosis in all eight osteosarcoma cell lines tested. These effects of FR901228 were also observed in vivo xenograft models on BALB/c nude mice, and treatment with 5.6 mg/kg/day resulting in a >70% reduction in the mean final tumor volume compared with the mean initial tumor volume. TUNEL assays demonstrated extensive apoptosis in tumor sections of mice treated with FR901228. Induction of apoptosis was preceded by increased expression of Fas ligand (FasL) mRNA, resulting in expression of membrane-bound FasL, which was followed by sequential activation of caspase-8 and -3. The level of apoptosis induction was reduced using a neutralizing anti-FasL antibody and overexpression of either the dominant-negative FADD or the viral FLICE inhibitory protein. Furthermore, treatment with a suboptimal dose of FR901228 greatly sensitized osteosarcoma cells to agonistic anti-Fas antibody-mediated apoptosis. These findings suggest that FR901228 is a highly promising antitumor agent against osteosarcoma, inducing apoptosis by the activation of the Fas/FasL system.

Animals↗
PubMed2003

In vitro demonstration of cell-to-cell interaction in growth plate cartilage using chondrocytes established from p53-/- mice.

Three clonal cell lines (MMR14, MMR17, and MMR32) were established from the costal cartilage derived from p53-/- mice. Expression profiles of cartilage-related molecules in MMR14 and MMR17 were compatible with those in cells of the hypertrophic zone. Prolonged in vitro culture induced the expression of calcification-related genes in both cell lines, but calcified nodules were observed only in MMR14. The expression profile of cartilage-related molecules in MMR32 was compatible with that of cells in the perichondrium, with high expression levels of decorin, bone morphogenetic protein-3, and parathyroid hormone-related peptide (PTHrP). When MMR14 was co-cultured with an equal amount of MMR32 without direct contact, the nodule formation was completely inhibited, whereas no such inhibition was observed when MMR14 was co-cultured with MMR17, indicating that soluble factors produced by MMR32 were responsible for the inhibition. Blocking the effects of PTHrP by either antagonizing peptide or neutralizing antibody against PTHrP failed to rescue the inhibitory effects of MMR32, and no increase of the cyclic adenosine monophosphate production in MMR14 was observed when co-cultured with MMR32, suggesting that soluble factors other than PTHrP produced by MMR32 were responsible for the inhibition of terminal differentiation of hypertrophic chondrocytes. This report is the first to show cell-to-cell interaction in the growth plate using cell lines, which will be useful material to investigate the regulatory mechanism of chondrocyte differentiation.

Animals↗
PubMed2002

Mutation analyses of the NFAT1 gene in chondrosarcomas and enchondromas.

Mice lacking nuclear factor of activated T cell 1 (NFAT1) showed an abnormal proliferation of chondrocytes in articular cartilage and formed an extraosseous cartilaginous mass resembling a neoplastic lesion, suggesting that the NFAT1 gene is a tumor suppressor gene in cartilaginous neoplasms. Here we performed mutation analyses of the NFAT1 gene in human cartilaginous tumors including 30 chondrosarcomas and 15 enchondromas. Reverse transcription-polymerase chain reaction (PCR) analysis revealed the expression of the NFAT1 gene in 15/15 chondrosarcomas and 12/13 enchondromas. To find subtle alterations, the genomic structure of the NFAT1 gene was determined using human genome draft sequences, and a mutation analysis was performed using the exon-by-exon PCR-single-strand conformation polymorphism method. Two heterozygous missense mutations, A1557T (His446Leu) and C2859T (Pro880Leu), were found in eight tumor samples, but the same mutation was also present in the constitutional cells of corresponding patients. The incidence of the mutant alleles in the patient and control groups showed no significant difference, suggesting that these mutations are rare single nucleotide polymorphisms unrelated with tumorigenesis. These results suggest that the NFAT1 gene is not likely to be a tumor suppressor gene in human cartilaginous tumors.

Adolescent↗
PubMed2002

Genome-wide analysis of gene expression in synovial sarcomas using a cDNA microarray.

Among a histologically heterogeneous group of soft tissue sarcomas, synovial sarcoma (SS) is regarded as a "miscellaneous" entity of uncertain origin. Although recent molecular analysis has disclosed involvement of a specific chromosomal translocation in the pathogenesis of SS, its genetic features remain largely unclear. In the work reported here we examined genome-wide gene expression profiles of 13 SS cases and 34 other spindle-cell sarcoma cases by cDNA microarray consisting of 23,040 genes. A hierarchical clustering analysis grouped SS and malignant peripheral nerve sheath tumor into the same category, and these two types of tumor shared expression patterns of numerous genes relating to neural differentiation. Several genes were up-regulated in almost all SS cases, and the presumed functions of known genes among them were related to migration or differentiation of neural crest cells, suggesting the possibility of neuroectodermal origin of SS. Moreover, we identified a set of genes that divided SS cases into two putative subclasses, a feature that may shed light on novel biological aspects of SS in addition to those having to do with epithelial differentiation. These data have provided clues for understanding the origin and tumorigenesis of SS.

Cluster Analysis↗
PubMed2002

Morphological and biological heterogeneity of three tumorigenic cell lines derived from a single p53-/- osteoblast-like cell line, MMC2.

Osteosarcoma is a malignant tumor with heterogeneous features both in histological and biological aspects. We have established three tumorigenic cell lines, MMOS1, MMOS2, and MMOS3, from three independent tumors that developed in nude mice after the inoculation of MMC2, an osteoblast-like cell line derived from p53-/- mice. Expression patterns of the osteoblast-related genes showed a marked difference between MMOS2 and the other two cell lines, and were correlated well with the features of the original tumors, ranging from an osteoblastic osteosarcoma (MMOS2) to tumors with scarce or no osteoid formation (MMOS1 and MMOS3). The properties of malignant cells also varied in the three cell lines. MMOS1, which was the most serum-dependent in vitro, developed markedly larger tumors in vivo than the other two cell lines. MMOS3 showed the fastest growth in low-serum conditions and produced the largest number of colonies in soft agar, but did not develop lung metastases, whereas MMOS1 and MMOS2 developed lung metastases with a frequency of 30 and 50%. These data suggest that the biological activities in vivo do not necessarily reflect those in vitro. Because the three tumorigenic cell lines share MMC2 as a common precursor, our data showed an example that the heterogeneity of osteosarcoma was created by genetic alterations that took place during the transformation process of each tumor.

Alkaline Phosphatase↗
PubMed2002

Clonal heterogeneity in differentiation potential of immortalized human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are bone marrow stroma-derived cells, which can differentiate into several types of mesenchymal tissues. Although regarded as tissue-specific stem cells, human MSCs (hMSCs) have a low proliferative ability with a finite life span, which is a hurdle to further analysis of their biology. Here we attempted to establish immortalized hMSCs by retrovirus-mediated gene transfer. The gain in telomerase activity obtained on expression of human telomerase reverse transcriptase (hTERT) was found not to be enough to make the cell line immortal. A combination of hTERT with human papillomavirus E6 and E7 successfully immortalized hMSCs without affecting the potential for adipogenic, osteogenic, and chondrogenic differentiation. From the parental immortalized hMSC, 100 single-cell derived clones were established, of which the differentiation properties varied considerably, including tri-, bi-, and uni-directional clones, suggesting that hMSCs are constituted by a group of cells with different differentiation potential. These cell lines, being the first established immortalized clonal cell lines of hMSCs, could provide insights into the mechanisms regulating the early steps of differentiation from undifferentiated MSCs into a specific lineage.

Animals↗