Toru Morishita

Overexpression of matrix metalloproteinase (MMP)-9 correlates with metastatic potency of spontaneous and 4-hydroxyaminoquinoline 1-oxide (4-HAQO)-induced transplantable osteosarcomas in rats

In the present experiment, the expression of matrix metalloproteinase (MMP)-2 and MMP-9, key proteins in the MMP family, and the tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2, antagonistic proteins against MMP-2 and MMP-9, respectively, were investigated by Northern blot analysis in rat transplantable osteosarcomas with high and low metastatic potencies. Two transplantable osteosarcomas, one induced with the carcinogen, 4-hydroxyaminoquinoline 1-oxide (4-HAQO) (COS, chemical carcinogen-induced osteosarcoma), and the other, a spontaneous lesion (SOS, spontaneous osteosarcoma), were repeatedly transplanted from lung nodules to generate lines with high metastatic potency, C-SLM (chemical carcinogen-induced osteosarcoma, selected lung metastatic lesions) and S-SLM (spontaneous osteosarcoma, selected lung metastatic lesions), respectively. MMP-9 was overexpressed in both S-SLM and C-SLM, and TIMP-2 in the case of S-SLM. Neither MMP-2 nor TIMP-1 was overexpressed in either of the transplantable osteosarcomas with high metastatic potentials. The active form MMP-9, studied by zymography, increased in S-SLM and C-SLM but not in SOS and COS. MMP-9 mRNA expression was highly correlated with the gelatinolytic activity of active form MMP-9 (r = 0.85, P < 0.0001) and with the activation ratio of MMP-9 (r = 0.83, P < 0.0001). However, the active form MMP-2 was not detectable in all cases. These results suggest that overexpression of MMP-9 mRNA is one of the essential factors in the acquisition of metastatic potential in rat transplantable osteosarcomas.

Bone marrow-derived mesenchymal cells can rescue osteogenic capacity of devitalized autologous bone

In clinical cases, many orthopaedists have been troubled with bone fragility, such as fractures after devitalization therapy for bone tumour, pathological fractures and metastatic tumours. The aim of this study was to determine whether loss of osteogenic capacity of devitalized autologous bones can be rescued using cultured bone marrow‐derived mesenchymal cells. A devitalized bone model was produced from rat femur by irradiation and three groups were prepared: intact bone, irradiated bone and irradiated bone combined with cultured mesenchymal cells. Each bone was transplanted subcutaneously into a syngeneic rat. At 2 or 4 weeks after transplantation, biochemical analyses [alkaline phosphatase (ALP) activity and osteocalcin mRNA expression] and histological measurement were performed. Moreover, we verified the origin of newly formed bone, using the sex‐determining region Y (sry) gene as a marker to distinguish between donor and recipient. In both intact bone and irradiated bone with mesenchymal cells, ALP activity and osteocalcin mRNA expression were detected and living osteoblasts together with newly formed bone were clearly seen histologically. Furthermore, analysis of the origin of de novo formed bone indicated that newly formed bone in irradiated bone with mesenchymal cells was derived from cultured bone marrow‐derived mesenchymal cells. These results proved that the osteogenic capacity of devitalized autologous bone can be rescued using tissue‐engineering techniques. This procedure should contribute to various clinical treatments, such as local metastatic tumours, pathological fracture after devitalization therapy and reconstruction after wide‐margin tumour resection. The benefits would be applicable to all types of devitalized bone. Copyright

Electron microscopic evidence of a viral nature for osteoclast inclusions in Paget's disease of bone

Circumstantial evidence from electron microscopic and immunological studies support the view that Pagets disease of bone represents a slow virus infection. However, there is only limited information available regarding its electron microscopic, enzyme and immunocytochemical characteristics. Two cases were studied using electron microscopy with particular emphasis on the inclusions in osteoclasts. Detailed ultrastructural and cytochemical studies including immuno-electron microscopy were performed. Some osteoclasts demonstrated specific virus-like structures composed of aggregations of microtubules in the nucleus and cytoplasm. The structures were easily digested by trypsin or protease, and were sensitive to RNase, which provided substantial evidence of a proteinaceous nature and inclusion of ribonucleic acid. Immunocytochemical examination identified binding of anti-respiratory syncytial virus and anti-measles virus antibodies in the tissue obtained from one of the two cases examined. The presence of viral antigens in structures in the cytoplasm of Pagetic osteoclasts supports the theory of paramyxovirus involvement in this disease.

Heterogeneous pattern of gene expression in cloned cell lines established from a rat transplantable osteosarcoma lung metastatic nodule

We have established three cloned cell lines (COS1NR, COS2NR and COS4NR) from the lung metastatic nodule of a highly metastatic variant of rat transplantable osteosarcoma, C-SLM. All three clones shared the same morphological characteristics and tumorigenicity, but their growth rates in vitro and metastatic ability in vivo differed from each other. Single-strand conformation polymorphism (SSCP) analysis revealed all three clones to have the same p53 gene mutation and parent C-SLM tumor. On the other hand, Northern blot analysis showed a different pattern of expression for the genes, c-fos, c-jun, c-Ha-ras, transin (rat stromelysin), bone Gla protein (osteocalsin) and nm23/NDP kinase. These results indicate the presence of a heterogeneous cell population in terms of the different pattern of gene expression in a lung metastatic nodule of rat osteosarcoma and the present newly established cell lines will be useful for further investigation of the biological behavior of osteosarcomas.

Inhibition of Spontaneous Rat Osteosarcoma Lung Metastasis by 3S‐[4‐(Nhydroxyamino)‐2R‐isobutylsuccinyl]amino‐1‐methoxy‐3,4‐dihydrocarbostyril, a Novel Matrix Metalloproteinase Inhibitor

In the present experiment, we examined the effects of OPB‐3206, 3S‐[4‐(N‐hydroxyamino)‐2R isobutylsuccinyl] amino‐1‐methoxy‐3,4‐dihydrocarbostyril, a novel metalloproteinase inhibitor, on the growth and metastasis of transplantable osteosarcomas (spontaneous osteosarcoma, selected lung metastatic lesions; S‐SLM), which were previously established in rats. OPB‐3206 inhibited the activities of interstitial collagenase, gelatinases A and B, and stromelysin in vitro. After oral administration to rats, its serum concentration peaked at 40 min and the drug was no longer detectable at 8 h. When OPB‐3206 was orally administered at 0%, 0.1% and 0.4% in the diet for 4 weeks, starting 7 days after subcutaneous transplantation of osteosarcomas to male Fischer 344 rats, numbers of lung metastatic nodules were significantly reduced by the highest dose, while the growth of subcutaneous tumors was not affected. Zymographic analysis showed the presence of pro matrix metalloproteinase (proMMP)‐2, proMMP‐9 and MMP‐9 activities in S‐SLM. In animals fed 0.4% OPB‐3206, the activity of proMMP‐9 was increased, but that for MMP‐9 had become undetectable. The results thus suggest that OPB‐3206 selectively inhibits lung metastasis of rat transplantable osteosarcomas by inhibiting MMP‐9 activation.

Osteoclast Origin of Giant Cells in Giant Cell Tumors of Bone: Ultrastructural and Cytochemical Study of Six Cases

To clarify the histogenesis of giant cells appearing in giant cell tumors of bone (GCTB), an ultrastructural and cytochemical study of six cases was performed with both acid phosphatase (ACPase) and tartrate-resistant acid phosphatase (TRACPase) as marker enzymes. TRACPase is considered a specific marker for osteoclasts. ACPase was demonstrated in the macrophagelike stromal cells, the multinucleated giant cells, and the infiltrating macrophages. The enzyme reaction was localized in lysosomal dense bodies and Golgi areas. Intense TRACPase activity was demonstrated in the multinucleated giant cells, whereas a weak reaction was found in the macrophagelike stromal cells. The multinucleated giant cells and macrophagelike stromal cells resembled osteoclasts with regard to the subcellular localization of TRACPase. The present results suggest that the giant cells in GCTB are indeed derived from osteoclasts.

Telomerase activity correlates with growth of transplantable osteosarcomas in rats treated with cis-diammine dichloroplatinum or the angiogenesis inhibitor AGM-1470

To determine the role of telomerase activity in the growth of tumors in rats undergoing chemotherapy, a comparison of the volumes of telomerase‐positive transplantable osteosarcomas was made in rats treated with the antineoplastic agent cis‐diammine dichloroplatinum (CDDP) or the angiogenesis inhibitor O‐(chloroacetylcarbamoyl)fumagillol (AGM‐1470). Male F344 rats, 8 weeks old, received transplants of macroscopic lung metastatic nodules into the subcutaneous back space and treatment was started on day 14 thereafter. CDDP was injected i.v. at doses of 0, 0.625, 1.25 and 2.5 mg/kg body weight (b.w.) and AGM‐1470 was administered at total doses of 0, 2.5, 5 and 10 mg/kg b.w. over 2 weeks by osmotic pumps, also implanted into the subcutaneous back space, but remote from the transplanted tumors. On day 28, all animals were killed for measurement of transplanted tumor size and determination of telomerase activities by telomeric repeat amplification protocol (TRAP) assay. The results showed telomerase activity to be highly correlated with the treated/non‐treated (T/C) tumor size ratio (r=0.96, P<0.0001). In a second experiment, CDDP at 2.5 mg/kg b.w. and AGM‐1470 at 10 mg/kg b.w., these being the most effective doses, were given as in the first experiment, and animals were serially killed on days 14, 21, 28, 35 and 42. Tumors in rats treated with CDDP and AGM‐1470 showed 18.2% and 20.5% of the control telomerase activity on days 35 and 21, respectively, when tumor growth was inhibited. However, on day 42, the activities increased to 46.5% and 92.5%, this correlating with re‐growth (r=0.73, P<0.0001). These results suggest that decline of telomerase activity may be involved in tumor growth retardation induced by chemotherapeutic agents. This possibility clearly warrants further mechanistic studies.

Plexiform schwannoma of the ulnar nerve

Plexiform schwannoma is a rare benign neurogenic tumour; we report a case that arose in the ulnar nerve of a 59-year-old woman. Exploration showed a continuous multinodular tumour that involved the ulnar nerve from the hand to the upper arm; the length of the tumour was 35 cm.

Ultrastructural cytochemical demonstration of proteoglycans and calcium in the extracellular matrix of chondroblastomas

To clarify the characteristics of the extracellular matrix of chondroblastomas, six cases were studied under the electron microscope, with special reference to proteoglycans and calcium in the cellular areas. In ruthenium hexammine trichloride (RHT)-stained sections the matrix was observed to be composed of rounded or polygonal fine granules and unbanded thin filaments that appeared to link neighboring granules together. Treatment with potassium-pyroantimonate showed intracellular accumulation of precipitates, mainly localized within the cisternae of the rough endoplasmic reticulum as well as in the extracellular matrix. The presence of calcium in the precipitates was confirmed using x-ray energy dispersive analysis. These findings, similar to characteristic features observed in calcifying systems, support the theory that chondroblastomas are of chondrogenic origin.

Differential expression of cytokines in rat osteosarcoma and malignant fibrous histiocytoma cell lines induced by 4‐(hydroxyamino)quinoline‐1‐oxide

Cytokines are considered to play an important role in tumor pathogenesis and progression, and recent studies have demonstrated that a variety of forms, including interleukins (ILs) and transforming growth factor‐βs (TGF‐βs), may regulate tumors. In the present study, the expression of TGF‐β isoforms and ILs was investigated in cell lines from a rat osteosarcoma and a malignant fibrous histiocytoma (MFH), both established from transplantable tumors induced by 4‐(hydroxyamino) quinoline 1‐oxide (4‐HAQO) in syngeneic F344 male rats. The results of a multiprobe RNase protection assay showed TGF‐β1 expression to be remarkably elevated, with no TGF‐β2 and β3 detectable in MFH cells, while TGF‐β1 and ‐β2 were found to be moderately and TGF‐β3 weakly expressed in osteosarcoma lines. All cell lines of osteosarcomas and MFHs expressed macrophage migration inhibitory factor at similar levels. In contrast to the lack of ILs in the MFH cells, moderate IL‐6 and very weak IL‐1β expression was detected in the osteosarcoma cells. These results suggest that variation in expression pattern of these cytokines in osteosarcomas and MFHs might be involved in differences in histological appearance and biological behavior, including metastatic ability, between these two mesenchyme‐derived tumor types.