Takeharu Nakamata

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.

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.

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.

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.

The effect of walking speed on gait kinematics and kinetics after endoprosthetic knee replacement following bone tumor resection

Gait function is one of the most important components of functional outcome evaluation in patients with a tumor around the knee. In addition to walking at a preferred speed, the patients might be sometimes required to walk fast in daily life (e.g., schooling and working) because the major types of bone tumors often occur in adolescence and young adults. Therefore, recovering the ability to walk fast would increase the quality of life of these patients. To clarify which parts of the lower limb are exerted while walking fast, we investigated the kinematic and kinetic changes during fast walking in patients who underwent endoprosthetic knee replacement after bone tumor resection. Laboratory-based gait analysis was performed on eight patients who had undergone endoprosthetic knee replacement following resection of a tumor around the knee. Patients walked at a preferred and faster speed, and the gait parameters were compared between the two walking speeds for each leg. To increase walking speed, patients tended to rely on the bilateral hip, ankle, and contralateral knee to generate additional power. Kinetic analysis showed that involved-side vertical body support was not significantly increased during late stance to increase walking speed, which was associated with a small increase in ankle plantarflexion moment and concentric power. These results suggest to patients after knee reconstruction how to effectively increase their walking speed or redistribute the mechanical load on the muscles and joints to prevent excessive stress on the lower limbs.

Perforation of enteric duplication during chemotherapy for osteosarcoma

A 9‐year‐old boy undergoing chemotherapy for conventional osteosarcoma complained of severe abdominal pain associated with rebound tenderness and muscular defense. Abdominal computed tomography indicated intraperitoneal free air. On surgical investigation, a diverticulum‐like lesion, perforated at the base, was found on the sidewall of the ileum. The anatomic location of the lesion was indicative of enteric duplication. Although the frequency of complications is very rare, perforations of the digestive tract should be considered in patients suffering severe abdominal pain while receiving chemotherapy.