Taisuke Hosaka

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.

The Id2 gene is a novel target of transcriptional activation by EWS-ETS fusion proteins in Ewing family tumors

We report here that the Id2 (inhibitor of DNA binding 2) gene is a novel target of transcriptional activation by EWS–FLI1 and EWS–ERG, two fusion proteins that characterize Ewing family tumors (EFTs). To identify downstream targets of these EWS–ETS fusion proteins, we introduced EWS–ETS fusion constructs into a human fibrosarcoma cell line by retroviral transduction. cDNA microarray analysis revealed that Id2 expression was up-regulated by introducing the EWS–ETS fusion gene but not by the normal full-length ETS gene. An Id2 promoter-luciferase reporter assay showed that transactivation by EWS–ETS involves the minimal Id2 promoter and may function in cooperation with c-Myc within the full-length regulatory region. A chromatin immunoprecipitation assay revealed direct interaction between the Id2 promoter and EWS-FLI1 fusion protein in vivo. Significantly higher expression of Id2 and c-Myc was observed in all of the six EFT cell lines examined compared to six other sarcoma cell lines. Moreover, high levels of Id2 expression were also observed in five of the six primary tumors examined. Id2 is generally thought to affect the balance between cell differentiation and proliferation in development and is highly expressed in several cancer types. Considering these previous studies, our data suggest that the oncogenic effect of EWS–ETS may be mediated in part by up-regulating Id2 expression.

Characteristics of genomic breakpoints in TLS-CHOP translocations in liposarcomas suggest the involvement of Translin and topoisomerase II in the process of translocation

Fusion of TLS/FUS and CHOP gene by reciprocal translocation t(12;16)(q32;q16) is a common genetic event found in myxoid and round-cell liposarcomas. Characterization of this genetic event was performed by three methods, Southern blot, RT – PCR, and genomic long-distance PCR in nine myxoid and three round-cell liposarcomas. All but one tumors showed genetic alternations indicating the fusion of TLS/FUS and CHOP gene. Two novel types of fusion transcripts were found, of which one lacked exon 2 sequence of CHOP gene, and the other lacked 3′ half of exon 5 of TLS gene. The latter case was caused by a cryptic splicing site which was created by the genomic fusion. Detailed analyses genomic fusion points revealed several sequence characteristics surrounding the fusion points. Homology analyses of breakpoint sequences with known sequence motifs possibly involve in the process of translocation uncovered Translin binding sequences at both of TLS/FUS and CHOP breakpoints in two cases. Translocations were always associated with other genetic alterations, such as deletions, duplications, or insertions. Short direct repeats were almost always found at both ends of deleted or duplicated fragments some of which had apparently been created by joining of sequences that flank the rearrangement. Finally, consensus topoisomerase II cleavage sites were found at breakpoints in all cases analysed, suggesting a role of this enzyme in creating staggered ends at the breakpoint. These data suggested that sequence characteristics may play an important role to recruit several factors such as Translin and topoisomerase II in the process of chromosomal translation in liposarcomas.

High incidence of SV40-like sequences detection in tumour and peripheral blood cells of Japanese osteosarcoma patients

Recent studies have revealed the evidence for the significance of SV40 genome in human malignancies. In this paper, the presence of SV40-like sequences was investigated in 54 Japanese osteosarcomas in which mutations of the retinoblastoma (Rb), p53, MDM2, and CDK4 genes had been already analysed. Using polymerase chain reaction and Southern hybridization, SV40-like sequences were detected in 25 cases (46.3%). In most cases, only a part of SV40 genome was detected, and the regulatory region containing enhancer sequences was most frequently found (21/54, 38.9%). There was no apparent relationship between the presence of SV40-like sequences and tumour suppressor genes mutations in each tumour. The SV40-like sequences were also detected in peripheral blood cells of substantial proportion of the patients (43.3%), whereas the incidence was much lower (4.7%) in normal healthy controls. This difference is statistically highly significant (P< 0.0001), suggesting that the presence of SV40-like sequences, even if only a part, may play some roles to predispose individuals to osteosarcoma.

A Novel Type of EWS-CHOP Fusion Gene in Two Cases of Myxoid Liposarcoma

Fusion genes consisting of TLS/FUS and CHOP or EWS and CHOP are characteristic markers for myxoid/round cell liposarcomas (MLS/RCLS). Several different structures of the fusion genes were reported in the case of the TLS/FUS-CHOP form, whereas only one type of structure has so far been found for the EWS-CHOP form, which consisted of exons 1 to 7 of the EWS and exons 2 to 4 of the CHOP gene. Here we describe a novel type of EWS-CHOP fusion gene in two cases of MLS/RCLS, which were found in a consecutive analysis of 21 cases. This fusion gene consisted of exons 1 to 10 of the EWS and exons 2 to 4 of the CHOP gene. The two cases with this fusion gene shared several clinical features, such as a large tumor mass, rapid and invasive growth, and local recurrence within 12 months after surgical resection. Histopathological findings also showed common features characterized by the diffuse proliferation of small spindle cells with a primitive mesenchymal appearance. The association of these clinical and histopathological features suggests a distinct biological property for this rare type of fusion product.

Translin binds to the sequences adjacent to the breakpoints of the TLS and CHOP genes in liposarcomas with translocation t(12;16)

Myxoid and round-cell liposarcomas share the translocation t(12;16)(q13;p11) creating the TLS-CHOP fusion gene as a common genetic alteration. We previously reported several unique characteristics of genomic sequences around the breakpoints in the TLS and CHOP loci, and among them was the presence of consensus recognition motifs of Translin, a protein that associates with chromosomal translocations of lymphoid neoplasms. We further extended our search for Translin binding motifs in sequences adjacent to breakpoints and investigated whether Translin binds to these sequences in vitro by mobility-shift assay. Computer-assisted search found sequences highly homologous (>70%) with Translin binding motifs adjacent to the breakpoints in 10 out of 11 liposarcomas with the TLS-CHOP fusion genes. All of 13 oligonucleotides corresponding to the putative binding sequences in these cases bind to Hela cell extract and also recombinant Translin protein, although the binding affinity of each motif showed considerable differences. The DNA-protein complex formation was inhibited by non-labeled competitor or anti-Translin antibody, suggesting the specificity of the complex formation. Considering the high incidence and specific binding property, the presence of Translin binding motif may be one of the important determinants for the location of breakpoints in the TLS and CHOP genes in liposarcomas.

Natural course of desmoid-type fibromatosis

BackgroundDesmoid-type fibromatosis is a locally aggressive tumor known to have high potential for local recurrence after resection, while it exhibits self-limiting behavior and shows growth arrest or spontaneous regression in many patients. Thus, its natural course is not well known, and the proper treatment has not yet been established.MethodsWe retrospectively reviewed clinical outcome and changes in tumor size in 11 consecutive patients with extremity and trunk desmoid-type fibromatoses, who were basically observed without any treatment after diagnosis.ResultsFor two patients in whom the tumors were initially incorrectly diagnosed as other tumors, surgical resection was performed. For another two patients, surgical resections were performed in the follow-up periods due to tumor enlargement or joint contracture. In all four patients who underwent surgery, tumors recurred shortly after resection and re-resection was not performed. During the follow-up periods with a median length of 56 months, ten tumors eventually stopped growing, and three of them regressed spontaneously. At the time of final follow-up, ten patients were alive with residual disease without severe morbidity. In one patient, the tumor enlarged to over 30 cm in diameter with a substantial functional deficit.ConclusionsSimple observation is a noninvasive and function-preserving treatment for desmoid-type fibromatosis.

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.

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.

TLS-CHOP target gene DOL54 expression in liposarcomas and malignant fibrous histiocytomas

Downstream of the gene for the liposarcoma‐associated fusion oncoprotein 54 (DOL54) is a target gene of the myxoid liposarcoma and round cell liposarcoma (M‐LPS/RC‐LPS) oncogene, TLS/FUS‐CHOP. The DOL54 gene product is closely associated with adipogenic differentiation. DOL54 overexpression resulted in tumorigenicity when Chinese Hamster Ovary (CHO) cells were injected subcutaneously into nude mice. The biological significance of DOL54 expression for human malignant soft tissue tumors, however, has not yet been investigated. We examined TLS‐CHOP and DOL54 expression in M‐LPS/RC‐LPS, well‐differentiated liposarcoma and malignant fibrous histiocytoma (MFH), a tumor whose cellular origin has not been determined. We observed DOL54 expression in 50% of M‐LPS/RC‐LPS cases (in which TLS‐CHOP was also expressed) and 33% of MFH cases, suggesting that a portion of MFH lesions may either derive from adipocytic precursor cells or have the potential to undergo adipogenic differentiation. In this manner, M‐LPS/RC‐LPS and MFH lesions may share tumorigenic characteristics, resulting from the unscheduled expression of DOL54.