Hsa-mir-145 is the top EWS-FLI1-repressed microRNA involved in a positive feedback loop in Ewing's sarcoma.

EWS-FLI1 is a chromosome translocation-derived chimeric transcription factor that has a central and rate-limiting role in the pathogenesis of Ewing's sarcoma. Although the EWS-FLI1 transcriptomic signature has been extensively characterized on the mRNA level, information on its impact on non-coding RNA expression is lacking. We have performed a genome-wide analysis of microRNAs affected by RNAi-mediated silencing of EWS-FLI1 in Ewing's sarcoma cell lines, and differentially expressed between primary Ewing's sarcoma and mesenchymal progenitor cells. Here, we report on the identification of hsa-mir-145 as the top EWS-FLI1-repressed microRNA. Upon knockdown of EWS-FLI1, hsa-mir-145 expression dramatically increases in all Ewing's sarcoma cell lines tested. Vice versa, ectopic expression of the microRNA in Ewing's sarcoma cell lines strongly reduced EWS-FLI1 protein, whereas transfection of an anti-mir to hsa-mir-145 increased the EWS-FLI1 levels. Reporter gene assays revealed that this modulation of EWS-FLI1 protein was mediated by the microRNA targeting the FLI1 3'-untranslated region. Mutual regulations of EWS-FLI1 and hsa-mir-145 were mirrored by an inverse correlation between their expression levels in four of the Ewing's sarcoma cell lines tested. Consistent with the role of EWS-FLI1 in Ewing's sarcoma growth regulation, forced hsa-mir-145 expression halted Ewing's sarcoma cell line growth. These results identify feedback regulation between EWS-FLI1 and hsa-mir-145 as an important component of the EWS-FLI1-mediated Ewing's sarcomagenesis that may open a new avenue to future microRNA-mediated therapy of this devastating malignant disease.

A practical approach to the clinical diagnosis of Ewing's sarcoma/primitive neuroectodermal tumour and other small round cell tumours sharing EWS rearrangement using new fluorescence in situ hybridisation probes for EWSR1 on formalin fixed, paraffin wax embedded tissue.

BACKGROUND: Over 90% of Ewing's sarcoma/primitive neuroectodermal tumour (ES/PNET) cases have the t(11;22) chromosomal rearrangement, which is also found in other small round cell tumours, including desmoplastic small round cell tumour (DSRCT) and clear cell sarcoma (CCS). Although this rearrangement can be analysed by fluorescence in situ hybridisation (FISH) using routinely formalin fixed, paraffin wax embedded (FFPE) tissues when fresh or frozen tissues are not available, a sensitive and convenient detection method is needed for routine clinical diagnosis. AIMS: To investigate the usefulness of newly developed probes for detecting EWS rearrangement resulting from chromosomal translocations using FISH and FFPE tissue in the clinical diagnosis of ES/PNET, DSRCT, and CCS. METHODS: Sixteen ES/PNETs, six DSRCTs, and six CCSs were studied. Three poorly differentiated synovial sarcomas, three alveolar rhabdomyosarcomas, and three neuroblastomas served as negative controls. Interphase FISH analysis was performed on FFPE tissue sections with a commercially available EWSR1 (22q12) dual colour, breakapart rearrangement probe. RESULTS: One fused signal and one split signal of orange and green, demonstrating rearrangement of the EWS gene, was detected in 14 of 16 ES/PNETs, all six DRSCTs, and five of six CCSs, but not in the negative controls. CONCLUSIONS: Interphase FISH using this newly developed probe is sensitive and specific for detecting the EWS gene on FFPE tissues and is of value in the routine clinical diagnosis of ES/PNET, DSRCT, and CCS.

Small GTP-binding protein, Rho, both increased and decreased cellular motility, activation of matrix metalloproteinase 2 and invasion of human osteosarcoma cells.

Rho, a member of the small GTP-binding proteins, and one of its downstream effectors ROCK (Rho-associated coiled-coil forming protein kinase) play an important role in the invasion of tumor cells. Lysophosphatidic acid (LPA) activates Rho and ROCK and promotes the organization of stress fibers and focal adhesions. However, the effect of LPA on tumor cell invasion is still controversial. In the present study, human osteosarcoma cells treated with a high concentration of LPA (high LPA) showed considerable formation of stress fibers and focal adhesions compared to the cells treated with a low concentration of LPA (low LPA). C3 (inhibitor of Rho) or Y27632 (an inhibitor of ROCK) inhibited the effects of LPA, indicating that LPA activates the Rho-ROCK pathway in the cells. In addition, Rho activation assay showed that the activation level of Rho can be altered by changing the concentration of LPA. Low LPA stimulated the motility and invasion of the cells, while high LPA reduced both. The disruption of extracellular matrix (ECM) by matrix metalloproteinase 2 (MMP2) is also critical for tumor cell invasion. MMP2 is activated by membranous type-1 MMP (MT1-MMP) and type-2 tissue inhibitor of MMP (TIMP2). High LPA suppressed the activation of MMP2 through down-regulation of MT1-MMP and TIMP2. C3 and Y27632 reversed the suppression of the activation of MMP2 and expression of MT1-MMP and TIMP2, suggesting the involvement of the Rho-ROCK pathway in ECM degradation. Tyrosine phosphorylation of focal adhesion kinase (FAK) was also required for the invasion of tumor cells to occur. Low LPA enhanced the tyrosine phosphorylation of FAK whereas high LPA reduced it. In conclusion, we suggest that Rho has a dual effect on the invasion of osteosarcoma cells by modulating the motility, the ability to degrade ECM and tyrosine phosphorylation of FAK.

Downregulation and forced expression of EWS-Fli1 fusion gene results in changes in the expression of G(1)regulatory genes.

Chromosomal translocation t(11;22)(q24:q12) is detected in approximately 90% of tumours of the Ewing family (ET). This translocation results in EWS-Fli1 gene fusion which produces a EWS-Fli1 fusion protein acting as an aberrant transcriptional activator. We previously reported that the inhibition of EWS-Fli1 expression caused the G(0)/G(1)arrest of ET cells. We, therefore, hypothesized that EWS-Fli1 may affect the expression of G(1)regulatory genes. Downregulation of EWS-Fli1 fusion proteins was observed 48 hours after the treatment with EWS-Fli1 antisense oligonucleotides. The expressions of G(1)cyclins, cyclin D1 and cyclin E, were markedly decreased in parallel with the reduction of EWS-Fli1 fusion protein. On the other hand, the expression of p21 and p27, which are important cyclin-dependent kinase inhibitors (CKIs) for G(1)--S transition, was dramatically increased after the treatment with EWS-Fli1 antisense oligonucleotides. RT-PCR analysis showed that alteration of the expressions of the cyclins and CKIs occurred at the mRNA level. Furthermore, transfection of EWS-Fli1 cDNA to NIH3T3 caused transformation of the cells and induction of the expression of cyclin D1 and E. Clinical samples of ET also showed a high level of expression of cyclin D1 mRNA, whereas mRNAs for p21 and p27 were not detected in the samples. These findings strongly suggest that the G(1)--S regulatory genes may be involved in downstream of EWS-Fli1 transcription factor, and that the unbalanced expression of G(1)--S regulatory factors caused by EWS-Fli1 may lead to the tumorigenesis of ET.

Cloning and sequencing of an endoglucanase gene from Scopulariopsis brevicaulis TOF-1212, and its expression in Saccharomyces cerevisiae.

The egI gene, encoding a major endoglucanase (EGI) of Scopulariopsis brevicaulis TOF-1212, was cloned and sequenced. The eglgene consisted of 868 bp with one intron and encoded a protein of 229 amino acids with a calculated molecular mass of 22,392 daltons. The EGI was assigned to a family 45 of glycosyl hydrolases and showed high similarity with other fungal endoglucanases, especially with those of Humicola grisea and Fusarium oxysporum, on the basis of hydrophobic cluster analysis. The egI gene was expressed under the promoter of the phosphoglycerate kinase gene (PGK) in Saccharomyces cerevisiae. The transformed cells were able to secrete the enzyme efficiently in an active form.

A zinc finger transcription factor, alphaA-crystallin binding protein 1, is a negative regulator of the chondrocyte-specific enhancer of the alpha1(II) collagen gene.

Transcription of the type II collagen gene (Col2a1) is regulated by multiple cis-acting sites. The enhancer element, which is located in the first intron, is necessary for high-level and cartilage-specific expression of Col2a1. A mouse limb bud cDNA expression library was screened by the Saccharomyces cerevisiae one-hybrid screening method to identify protein factors bound to the enhancer. A zinc finger protein, alphaA-crystallin binding protein 1 (CRYBP1), which had been reported to bind to the mouse alphaA-crystallin gene promoter, was isolated. We herein demonstrate that CRYBP1 is involved in the negative regulation of Col2a1 enhancer activity. CRYBP1 mRNA expression was downregulated during chondrocyte differentiation in vitro. In situ hybridization analysis of developing mouse cartilage showed that CRYBP1 mRNA was also downregulated during mesenchymal condensation and that CRYBP1 mRNA was highly expressed by hypertrophic chondrocytes, but at very low levels by resting and proliferating chondrocytes. Expression of recombinant CRYBP1 in a transfected rat chondrosarcoma cell line inhibited Col2a1 enhancer activity. Electrophoretic mobility shift assays showed that CRYBP1 bound a specific sequence within the Col2a1 enhancer and inhibited the binding of Sox9, an activator for Col2a1, to the enhancer. Cotransfection of CRYBP1 with Sox9 into BALB/c 3T3 cells inhibited activation of the Col2a1 enhancer by Sox9. These results suggest a novel mechanism that negatively regulates cartilage-specific expression of Col2a1.

Treatment of cervical cord compression, caused by hereditary multiple exostosis, with laminoplasty: a case report.

STUDY DESIGN: Case report. OBJECTIVES: Successful excision of the exostosis within the spinal canal. SUMMARY OF BACKGROUND DATA: Myelopathy caused by exostosis within the spinal canal developed in a 13-year-old boy with hereditary multiple exostosis. METHODS: Spinous process-splitting laminoplasty with an ultrasonic knife was performed to remove the mass and minimize the possibility of postlaminectomy kyphosis. RESULTS: The spinal canal exostosis with cervical cord compression was excised successfully with laminoplasty. After surgery there has been no recurrence of tumor, and the stability of the cervical spine has been preserved. CONCLUSION: This is the first report of laminoplasty as a useful surgical approach for intraspinal exostosis to prevent postoperative cervical instability.

[The present state of the 24-hour care system of our hospital and the examination of the significance and problems of the system].

The wishes of patients with terminal cancer are often not realized even if they indicate they want to die at home. We introduced a 24-hour visiting care system from November, 1994, with a view to making it possible for the patient who wants to to stay at home. As a result, the rate of the death at home increased; thus, the 24-hour visiting care system was significant for terminal care at home. On the other hand, from the viewpoint of a medical institution, the lack of sufficient cooperation with doctors and the increase of the burden on the staff were pointed out as problems to be resolved.

[The effect of herbal medicines on the immunodeficient animals by injecting cancer chemotherapeutic agent-special reference to age related recovery of the function].

The acquired immunodeficiency of the host plays an essential role in the occurrence of infections even with low pathogenic bacteria. The increase of cases with MRSA and/or pseudomonas infection is one of the serious problems in hospital management in Japan for the elderly as well as pediatric patients. In the present study, mitomycin C (MMC)-treated hosts were prepared in young, adult and old mice to test the immunopotentiating action of the promising Chinese herbal medicine, Tohki-Rikuoh-Toh (TRT), Hotyu-Ekki-Toh (HET) and Juzen-Taiho-Toh (JTT). The effect of these herbal medicines on organ structure and its function in the MMC-treated hosts is clarified and discussed for medical use. 4-5, 8-10 and over 50 week old male C57BL/6 (Clea Japan Inc.) were injected with MMC at a dosage of 3 to 5 mg/kg to inhibit the bone marrow, thus creating a mouse model with reduced immunopotential. A powder extract of TRT, HET and JTT was administrated orally at a dosage of 500 mg/kg/day for seven consecutive days. The white cell number and the subset analysis were carried out by the FACS method. The bactericidal effect of the host was monitored by NBT reduction test. Peritoneal macrophages were prepared by the adherence technique. The macrophage phagocytic activity was examined by an ACAS system. After the administration of TRT, HET and JTT, the body weights recovered as much as 90%, especially in young animals which had been reduced to 75% of their normal values. After MMC-treatment, with the herbal medicines, HET was good for young mice while JTT was effective for the old ones. As for the effect on B cells, the plaque-forming cells (PFC) of spleen cells were compared among the groups. As a result, PFC in the HET group was 184% and the other two were 80 approximately 95% as compared to 76% in the MMC-treated ones. The number of white blood cells in the MMC-treated mice returned to 80% of their normal value. In addition, the phagocytic activity of macrophages increased to 50% although that of the non-treated group was only 20%. The phagocytic activity also recovered in the JTT and TRT of 131% to 95%, respectively compared to 11% in the MMC-treated control. When TRT, HET and JTT were administered orally to mouse models whose immunopotential had been inhibited, the herbal medicines activated both quantitatively and qualitatively, showing themselves to be effective interstitial medicines. In addition, the data from the animal models showed no side effects, confirming the complete efficacy of the drug. Moreover, there was no direct anti-bactericidal effect from these medicines, suggesting that the immunomodulating action of this medicine is host-mediated. It is interesting that quantitative and qualitative recovery were seen when HET was administered to MMC-treated young hosts while JTT was good for the old. With this investigation, the effective components are still unknown for different generations, and we need to clarify this aspect for better understanding of the efficacy of herbal medicines.

A novel fucosylated glycosphingolipid from the millipede, Parafontaria laminata armigera.

A novel fucosylated glycosphingolipid (GL-3a) was isolated and purified from whole tissues of the millipede, Parafontaria laminata armigera. Its chemical structure was characterized as Man beta 1-4(Fuc alpha 1-3)Glc beta 1-ceramide (I3 alpha Fuc,MlOse2Cer) by gas-liquid chromatography, permethylation study, partial acid hydrolysis, exoglycosidase degradation, TLC/enzyme-immunostaining, negative fast atom bombardment-mass spectrometry and proton nuclear magnetic resonance spectroscopy. This compound was unique in containing a fucose branch linked to the glucose residue of the disaccharide, mannosylglucose. The ceramide moiety was mainly composed of d17:1 (64.3%) and d18:1 (20.0%) sphingoids, and 22:0 (41.8%), 23:0 (16.4%) and 24:0 (15.8%) fatty acids.