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Objectives To investigate whether acupuncture stimulation affects autonomic nerve function by measuring pupil diameters with electronic pupillography. Methods Two studies were conducted (Experiment 1 and 2) in the Tsukuba University of Technology, Tsukuba, Japan. Experiment 1 examined the responses before, during and after acupuncture. Experiment 2 compared acupuncture and a no acupuncture control in a two period, repeated measurement crossover design. Twelve healthy male university students were recruited for Experiment 1 and nine healthy male university students for Experiment 2. The intervention was superficial acupuncture at the acupuncture point TE5. Following the insertion, gentle repetitive tapping stimulation was applied during the subject's exhalation phase in a sitting position for 90 seconds. The main outcome measures in Experiment I were pupil diameter, heart rate, pulse wave and blood pressure. In both experiments, pupil diameter was measured for three minutes before acupuncture stimulation, during stimulation and for three minutes after simulation. Results In Experiment I, a decrease in pupil diameter was observed after acupuncture stimulation (P=0.18) and a decrease in heart rate was observed during the stimulation (P=0.049). Moreover, a significant decrease of pulse wave amplitude was observed during and after acupuncture stimulation. In Experiment 2, a decrease in pupil diameter occurred after acupuncture stimulation (P=0.007), whereas no change occurred under the no acupuncture control. Conclusions It is thought that the pupillary constriction observed after gentle, superficial acupuncture stimulation may be attributed to an increase of parasympathetic nerve function. Further study is necessary in order to further clarify the duration of the observed response and to elucidate the mechanisms behind it. [ABSTRACT FROM AUTHOR]

Lineage or cell of origin of cancers is often unknown and thus is not a consideration in therapeutic approaches. Alveolar rhabdomyosarcoma (aRMS) is an aggressive childhood cancer for which the cell of origin remains debated. We used conditional genetic mouse models of aRMS to activate the pathognomonic Pax3:Foxo1 fusion oncogene and inactivate p53 in several stages of prenatal and postnatal muscle development. We reveal that lineage of origin significantly influences tumor histomorphology and sensitivity to targeted therapeutics. Furthermore, we uncovered differential transcriptional regulation of the Pax3:Foxo1 locus by tumor lineage of origin, which led us to identify the histone deacetylase inhibitor entinostat as a pharmacological agent for the potential conversion of Pax3:Foxo1-positive aRMS to a state akin to fusion-negative RMS through direct transcriptional suppression of Pax3:Foxo1., Stem Cell and Regenerative Biology

Abstract Background There is not enough evidence to support the efficacy of massage for muscle fatigue despite wide utilization of the modality in various clinical settings. This study investigated the influence of massage application on localized back muscle fatigue. Methods Twenty-nine healthy subjects participated in two experimental sessions (massage and rest conditions). On each test day, subjects were asked to lie in the prone position on a treatment table and perform sustained back extension for 90 seconds. Subjects then either received massage on the lumbar region or rested for a 5 minute duration, then repeated the back extension movement. The median frequency (MDF), mean power frequency (MNF), and root mean square (RMS) amplitude of electromyographic signals during the 90 second sustained lumbar muscle contraction were analyzed. The subjective feeling of fatigue was then evaluated using the Visual Analogue Scale (VAS). Results MDF and MNF significantly declined with time under all conditions. There was no significant difference in MDF, MNF or RMS value change between before and after massage, or between rest and massage conditions. There was a significant increase in fatigue VAS at the end of the 2nd back extension with rest condition. There was a significant difference in fatigue VAS change between massage and rest condition. Conclusions A significant difference was observed between massage and rest condition on VAS for muscle fatigue. On EMG analysis, there were no significant differences to conclude that massage stimulation influenced the myoelectrical muscle fatigue, which is associated with metabolic and electrical changes.

Background/aims: To evaluate efficacy and safety of intravitreal aflibercept (IVT-AFL) in Japanese patients with wet age-related macular degeneration (wAMD) from the VIEW 2 trial., Methods: In this double-masked study, patients were randomised to: 0.5 mg IVT-AFL every 4 weeks (0.5q4); 2 mg IVT-AFL every 4 weeks (2q4); 2 mg IVT-AFL every 8 weeks (2q8) after 3 monthly injections; or 0.5 mg ranibizumab every 4 weeks (Rq4). Main efficacy outcomes included vision maintenance and best-corrected visual acuity (BCVA) at week 52., Results: At week 52, all Japanese patients in the IVT-AFL groups (n=70) maintained vision, compared with 96% of Japanese patients (n=23/24) treated with ranibizumab. Japanese patients in all treatment groups showed improvement in BCVA after treatment. The Rq4, 2q4 and 2q8 groups experienced similar gains in BCVA from baseline. The 0.5q4 group had higher gains due to an unexpected drop in BCVA between screening and baseline. Central retinal thickness and mean area of choroidal neovascularisation decreased in all treatment groups with similar magnitude. Ocular treatment-emergent adverse events were balanced across treatment groups., Conclusions: IVT-AFL was effective and well tolerated in Japanese patients. Outcomes in this population were consistent with those in the overall VIEW 2 population., Trial Registration Number: NCT00637377., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Background: Alveolar rhabdomyosarcoma (aRMS) is a myogenic childhood sarcoma frequently associated with a translocation-mediated fusion gene, Pax3:Foxo1a., Methods: We investigated the complementary role of Rb1 loss in aRMS tumor initiation and progression using conditional mouse models., Results: Rb1 loss was not a necessary and sufficient mutational event for rhabdomyosarcomagenesis, nor a strong cooperative initiating mutation. Instead, Rb1 loss was a modifier of progression and increased anaplasia and pleomorphism. Whereas Pax3:Foxo1a expression was unaltered, biomarkers of aRMS versus embryonal rhabdomyosarcoma were both increased, questioning whether these diagnostic markers are reliable in the context of Rb1 loss. Genome-wide gene expression in Pax3:Foxo1a,Rb1 tumors more closely approximated aRMS than embryonal rhabdomyosarcoma. Intrinsic loss of pRb function in aRMS was evidenced by insensitivity to a Cdk4/6 inhibitor regardless of whether Rb1 was intact or null. This loss of function could be attributed to low baseline Rb1, pRb and phospho-pRb expression in aRMS tumors for which the Rb1 locus was intact. Pax3:Foxo1a RNA interference did not increase pRb or improve Cdk inhibitor sensitivity. Human aRMS shared the feature of low and/or heterogeneous tumor cell pRb expression., Conclusions: Rb1 loss from an already low pRb baseline is a significant disease modifier, raising the possibility that some cases of pleomorphic rhabdomyosarcoma may in fact be Pax3:Foxo1a-expressing aRMS with Rb1 or pRb loss of function.

Myf5 is a member of the muscle-specific determination genes and plays a critical role in skeletal muscle development. Whereas the expression of Myf5 during embryonic and fetal myogenesis has been extensively studied, its expression in progenitors that will ultimately give rise to adult satellite cells, the stem cells responsible for muscle repair, is still largely unexplored. To investigate this aspect, we have generated a mouse strain carrying a CreER coding sequence in the Myf5 locus. In this strain, Tamoxifen-inducible Cre activity parallels endogenous Myf5 expression. Combining Myf5(CreER) and Cre reporter alleles, we were able to evaluate the contribution of cells expressing Myf5 at distinct developmental stages to the pool of satellite cells in adult hindlimb muscles. Although it was possible to trace back the origin of some rare satellite cells to a subpopulation of Myf5(+ve) progenitors in the limb buds at the late embryonic stage (∼E12), a significant number of satellite cells arise from cells which expressed Myf5 for the first time at the fetal stage (∼E15). These studies provide direct evidence that adult satellite cells derive from progenitors that first express the myogenic determination gene Myf5 during fetal stages of myogenesis., (Published by Elsevier Inc.)

Objectives: To determine if acupuncture stimulation elicits a pupillary response under light adaptation and whether there is any difference in the pupillary response between light and dark adaptation environments during acupuncture stimulation., Methods: The participants consisted of 55 healthy individuals who had no known eye diseases or pupil abnormalities. Experiment 1 compared pupillary responses between acupuncture stimulation and no-stimulation groups under light adaptation. Experiment 2 compared pupillary responses to acupuncture between two conditions (dark and light adaptation) with a two-period repeated measurement crossover design. For both experiments the pupil diameter was continuously measured for 3 min before stimulation, during stimulation and for 3 min after stimulation. For all acupuncture stimulation interventions an acupuncture needle was inserted superficially at the TE5 acupuncture point followed by gentle tapping stimulation for 90 s., Results: In experiment 1 the pupil diameter was significantly decreased during (p<0.01) and after stimulation (p<0.0001) compared with the pupil diameter before stimulation under light adaptation. No significant difference was noted in the serial changes in pupil diameter in the no-stimulation group. In experiment 2 the pupil diameter was significantly decreased 90 s after stimulation (p<0.05) and 150 s after stimulation (p<0.05) under light adaptation conditions. Furthermore, the pupil diameter was significantly decreased 120 s after stimulation (p<0.05) and 150 s after stimulation (p<0.01) under dark adaptation conditions. No significant difference in the serial changes in pupil diameter was noted between the groups., Conclusions: This study shows that pupil constriction occurs following acupuncture stimulation under light adaptation and this response is no different from that seen under dark adaptation.

The chimeric PAX3-FKHR transcription factor is present in a majority of alveolar rhabdomyosarcoma (ARMS), an aggressive skeletal muscle cancer of childhood. PAX3-FKHR-mediated aberrant myogenic gene expression resulting in escape from terminal differentiation program is believed to contribute in ARMS development. In skeletal muscle differentiation, activation of AKT pathway leads to myogenic gene activation and terminal differentiation. Here, we report that AKT acts, in part, by modulating PAX3-FKHR transcriptional activity via phosphorylation in the maintenance of the myogenic differentiation blockade in established mouse models of ARMS cells. We observed that low levels of AKT activity are associated with elevated levels of PAX3-FKHR transcriptional activity, and AKT hyperactivation results in PAX3-FKHR phosphorylation coupled with decreased activity once cells are under differentiation-permissible conditions. Subsequent data shows that attenuated AKT activity-associated PAX3-FKHR activity is required to suppress the function of MyoD, a key myogenic regulator of muscle differentiation. Conversely, decreased PAX3-FKHR activity results in the eradication of MyoD expression and subsequent suppression of the myogenic differentiation. Thus, AKT regulation of the PAX3- FKHR suppresses myogenic gene expression in ARMS cells, causing a failure in differentiation. Evidence is presented that provides a novel molecular link between AKT and PAX3-FKHR in maintaining myogenic differentiation blockade in ARMS.

Satellite cells are well known as a postnatal skeletal muscle stem cell reservoir that under injury conditions participate in repair. However, mechanisms controlling satellite cell quiescence and activation are the topic of ongoing inquiry by many laboratories. In this study, we investigated whether loss of the cell cycle regulatory factor, pRb, is associated with the re-entry of quiescent satellite cells into replication and subsequent stem cell expansion. By ablation of Rb1 using a Pax7CreER,Rb1 conditional mouse line, satellite cell number was increased 5-fold over 6 months. Furthermore, myoblasts originating from satellite cells lacking Rb1 were also increased 3-fold over 6 months, while terminal differentiation was greatly diminished. Similarly, Pax7CreER,Rb1 mice exhibited muscle fiber hypotrophy in vivo under steady state conditions as well as a delay of muscle regeneration following cardiotoxin-mediated injury. These results suggest that cell cycle re-entry of quiescent satellite cells is accelerated by lack of Rb1, resulting in the expansion of both satellite cells and their progeny in adolescent muscle. Conversely, that sustained Rb1 loss in the satellite cell lineage causes a deficit of muscle fiber formation. However, we also show that pharmacological inhibition of protein phosphatase 1 activity, which will result in pRb inactivation accelerates satellite cell activation and/or expansion in a transient manner. Together, our results raise the possibility that reversible pRb inactivation in satellite cells and inhibition of protein phosphorylation may provide a new therapeutic tool for muscle atrophy by short term expansion of the muscle stem cells and myoblast pool.

Purpose: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood. The alveolar subtype of rhabdomyosarcoma (ARMS) is a paradigm for refractory and incurable solid tumors because more than half of the children at diagnosis have either regional lymph node or distant metastases. These studies follow our previous observation that Interleukin-4 receptor α (IL-4Rα) is upregulated in both human and murine ARMS, and that the IL-4R signaling pathway may be a target for abrogating tumor progression., Experimental Design: By in vitro biochemical and cell biology studies as well as preclinical studies using a genetically engineered mouse model, we evaluated the role of IL-4 and IL-13 in IL-4R-mediated mitogenesis, myodifferentiation, and tumor progression., Results: IL-4 and IL-13 ligands accelerated tumor cell growth and activated STAT6, Akt, or MAPK signaling pathways in the human RMS cell lines, RD and Rh30, as well as in mouse primary ARMS cell cultures. IL-4 and IL-13 treatment also decreased protein expression of myogenic differentiation factors MyoD and Myogenin, indicating a loss of muscle differentiation. Using a genetically engineered mouse model of ARMS, we have shown that inhibition of IL-4R signaling pathway with a neutralizing antibody has a profound effect on the frequency of lymph node and pulmonary metastases, resulting in significant survival extension in vivo., Conclusions: Our results indicate that an IL-4R-dependent signaling pathway regulates tumor cell progression in RMS, and inhibition of this pathway could be a promising adjuvant therapeutic approach., (©2011 AACR.)

Background: Up-regulation of the expression of the gene C7orf24, encoding γ-glutamyl cyclotransferase, is a common event in cancers derived from various tissues, but its involvement in osteosarcomas (OS) has not yet been demonstrated., Materials and Methods: The expression of C7orf24 was analyzed in human OS cell lines and primary tumor samples. The biological effects of C7orf24 on growth, motility, and invasion in the OS cell lines were investigated using siRNA for C7orf24. Genes related to the function of C7orf24 were sought by genome-wide gene expression profiling., Results: The level of C7orf24 expression was much higher in the OS cell lines and OS primary tumors than in normal osteoblasts. Down-regulation of C7orf24 expression inhibited the growth of the cell lines in association with enhancement of cell-clustering. Treatment with C7orf24-siRNA inhibited cell motility and invasion. Gene ontology suggested the function of C7orf24 to be related to cell adhesion and protein transport., Conclusion: C7orf24 is also involved in the growth of OS, and is a potential biomarker for this type of tumor.

Inhibition of the insulin-like growth factor 1 receptor (Igf1r) is an approach being taken in clinical trials to overcome the dismal outcome for metastatic alveolar rhabdomyosarcoma (ARMS), an aggressive muscle cancer of children and young adults. In our study, we address the potential mechanism(s) of Igf1r inhibitor resistance that might be anticipated for patients. Using a genetically engineered mouse model of ARMS, validated for active Igf1r signaling, we show that the prototypic Igf1r inhibitor NVP-AEW541 can inhibit cell growth and induce apoptosis in vitro in association with decreased Akt and Mapk phosphorylation. However, drug resistance in vivo is more common and is accompanied by Igf1r overexpression, Mapk reactivation, and Her2 overexpression. Her2 is found to form heterodimers with Igf1r in resistant primary tumor cell cultures, and stimulation with Igf2 leads to Her2 phosphorylation. The Her2 inhibitor lapatinib cooperates with NVP-AEW541 to reduce Igf1r phosphorylation and to inhibit cell growth even though lapatinib alone has little effect on growth. These results point to the potential therapeutic importance of simultaneous targeting of Igf1r and Her2 to abrogate resistance.

Embryonal rhabdomyosarcoma (eRMS) shows the most myodifferentiation among sarcomas, yet the precise cell of origin remains undefined. Using Ptch1, p53 and/or Rb1 conditional mouse models and controlling prenatal or postnatal myogenic cell of origin, we demonstrate that eRMS and undifferentiated pleomorphic sarcoma (UPS) lie in a continuum, with satellite cells predisposed to giving rise to UPS. Conversely, p53 loss in maturing myoblasts gives rise to eRMS, which have the highest myodifferentiation potential. Regardless of origin, Rb1 loss modifies tumor phenotype to mimic UPS. In human sarcomas that lack pathognomic chromosomal translocations, p53 loss of function is prevalent, whereas Shh or Rb1 alterations likely act primarily as modifiers. Thus, sarcoma phenotype is strongly influenced by cell of origin and mutational profile., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Pituitary adenomas are classified into functioning and nonfunctioning (silent) tumors on the basis of hormone secretion. However, the mechanism of tumorigenesis and the cell of origin for pituitary adenoma subtypes remain to be elucidated. Employing a tamoxifen-inducible mouse model, we demonstrate that a novel postnatal Pax7(+) progenitor cell population in the pituitary gland gives rise to silent corticotroph macro-adenomas when the retinoblastoma tumor suppressor is conditionally deleted. While Pax transcriptional factors are critical for embryonic patterning as well as postnatal stem cell renewal for many organs, we have discovered that Pax7 marks a restricted cell population in the postnatal pituitary intermediate lobe. This Pax7(+) early progenitor cell population is overlapping but ontologically downstream of the Nestin(+) pituitary stem cell population, yet upstream of another newly discovered Myf6(+) late progenitor cell population. Interestingly, the Pax7(+) progenitor cell population is evolutionarily conserved in primates and humans, and Pax7 expression is maintained not only in murine tumors but also in human functioning and silent corticotropinomas. Taken together, our results strongly suggest that human silent corticotroph adenomas may in fact arise from a Pax7 lineage of the intermediate lobe, a region of the human pituitary bearing closer scientific interest as a reservoir of pituitary progenitor cells.

In this study, we examined the effect of the difference in time of going to sleep on the numerical values of leukocyte subsets and various hormones. Subjects consisted of 26 healthy adults (15 men, 11 women) with a mean age of 37.6 years. Among the 26 individuals, 12 persons (Group E) were of the habit of going to sleep before midnight consistently, while 14 persons (Group L) were of the habit of staying up late, consistently going to sleep after 2 am. For Group E, it was found that the ratio of lymphocytes was remarkably high in comparison with Group L (Group E 41.6 +/- 2.54%, Group L 31.7 +/- 2.03%, P < 0.01). On the other hand, for Group L it was found that the ratio of granulocytes was remarkably high in comparison with Group E (Group E 53.0 +/- 2.51%, Group L 62.3 +/- 2.22%, P < 0.01). However, no difference was observed in lymphocyte and granulocyte ratios due to the duration of the sleep. As the excessive quantity of granulocytes was not corrected through longer sleep, these findings suggest that the time when first going to sleep is more important than the total hours of sleep achieved.

Bioluminescent reporter genes are sensitive in situ tools for following disease progression in preclinical models, albeit they are subject to scattering and absorption in deep tissues. We have generated a bicistronic Cre/LoxP reporter mouse line that pairs the expression of firefly luciferase with quantifiable expression of a human placental alkaline phosphatase that is secreted into the serum (SeAP). With the use of this dual-modality bioreporter with a novel, inducible Pax7-CreER line for tracking muscle satellite cells, we demonstrate the longitudinal kinetics of muscle stem cell turnover, accounting for a doubling of the signal from satellite cell and progeny every 3.93 wk in the transition from adolescence to early adulthood. We also show that this dual-modality bioreporter can be incorporated in preclinical cancer models, whereby SeAP activity is reflective of tumor burden. Thus, this dual bioreporter permits both spatial localization and accurate quantification of biological processes in vivo even when the tissue of interest is deep within the animal.

Synovial sarcoma of the mediastinum is a rare neoplasm that has overlapping histological and immunophenotypic features with other tumors in the differential diagnosis. We describe a case of this disease. The tumor was located at the right side of the pericardium, where an FDG-PET scan showed an uptake. It was resected, a resection which was complicated by the necessity of partially resecting the pericaridium and right middle lobe which were invaded by the tumor. The doubling time of the main tumor was 11.8 days. The margin of the resected specimen was tumor-free both macro- and microscopically. Reverse transcription-PCR confirmed the diagnosis of synovial sarcoma. The patient rejected chemotherapy or radiation therapy, and had recurrent tumors only one month after the operation. Finally, she opted to have only palliative care and died 79 days after the operation.

The highly aggressive muscle cancer alveolar rhabdomyosarcoma (ARMS) is one of the most common soft tissue sarcoma of childhood, yet the outcome for the unresectable and metastatic disease is dismal and unchanged for nearly three decades. To better understand the pathogenesis of this disease and to facilitate novel preclinical approaches, we previously developed a conditional mouse model of ARMS by faithfully recapitulating the genetic mutations observed in the human disease, i.e., activation of Pax3:Fkhr fusion gene with either p53 or Cdkn2a inactivation. In this report, we show that this model recapitulates the immunohistochemical profile and the rapid progression of the human disease. We show that Pax3:Fkhr expression increases during late preneoplasia but tumor cells undergoing metastasis are under apparent selection for Pax3:Fkhr expression. At a whole-genome level, a cross-species gene set enrichment analysis and metagene projection study showed that our mouse model is most similar to human ARMS when compared with other pediatric cancers. We have defined an expression profile conserved between mouse and human ARMS, as well as a Pax3:Fkhr signature, including the target gene, SKP2. We further identified 7 "druggable" kinases overexpressed across species. The data affirm the accuracy of this genetically engineered mouse model.

The histone deacetylase inhibitor depsipeptide [(1S,4S,7Z,10S, 16E,21R)-7-ethylidene-4,21-bis(propan-2-yl)-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo[8.7.6]tricos-16-ene-3,6,9,19, 22-pentone] (FK228) has attracted a great deal of interest because of its antiproliferative and apoptotic properties in various malignancies. Histone deacetylase inhibitors induce the expression of the multidrug resistance transporter P-glycoprotein (P-gp), and FK228 is a known P-gp substrate. Thus, FK228 seems to induce its own mechanism of drug resistance by up-regulating P-gp. The goal of this study was to establish human FK228-resistant osteosarcoma cell lines and to investigate whether there are mechanisms of FK228 resistance in addition to P-gp up-regulation. After 72 h in culture, the 50% inhibitory concentrations (IC(50)) of FK228 were 4.8 and 991 nM in HOS and HOS/FK8 cells, respectively, and 3.6 and 1420 nM in U2OS and U2OS/FK11 cells, respectively. Increased histone H3 acetylation was observed in FK228-resistant cell lines after a 1-h treatment with 10 nM FK228. Unlike in parental cells, significant P-gp overexpression was detected in FK228-resistant cells, and 10 nM FK228 treatment activated the mitogen-activated protein kinase (MAPK) pathway but did not induce Fas ligand (FasL) up-regulation or c-FLIP down-regulation. However, treatment of FK228-resistant cells with a combination of FK228 and mitogen-activated protein kinase kinase (MEK) inhibitors induced apoptosis, up-regulated FasL, and down-regulated c-FLIP. The expression and function of P-gp were unaltered by treatment with MEK inhibitors. These results indicate that the FK228 resistance of osteosarcoma cells is related to P-gp overexpression and MAPK pathway activation by FK228. MEK or P-gp inhibitors may be useful in overcoming this resistance.

We report the first case of a tumor of the Ewing sarcoma family of tumors arising from the urinary bladder 3 years after chemotherapy for acute lymphoblastic leukemia. A 16-year-old boy complained of macrohematuria and dysuria during the posttreatment follow up of his acute lymphoblastic leukemia. Ultrasonography and computed tomography revealed a 1-cm sized intravesical tumor. The tumor was transurethrally resected with no residual tumor at the margin. Histopathologic analyses revealed a small round blue cell tumor with positive staining for CD99 antibody. EWS-FLI1 fusion transcripts were detected in the tumor tissue by reverse transcriptase polymerase chain reaction. These findings support the diagnosis of Ewing sarcoma family of tumor. After adjuvant multidrug chemotherapy, the patient has shown no evidence of disease for more than 2 years.

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.

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.

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.

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.

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.

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.

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., (Copyright 2004 Wiley-Liss, Inc.)

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.

Background: This study attempted to investigate the influence of massage on the skin and the intramuscular circulatory changes associated with localized muscle fatigue., Material/methods: Twenty-nine healthy male subjects participated in two experimental sessions (massage and rest conditions). Subjects lay prone on the table and were instructed to extend their trunks until the inferior portion of their rib cage no longer rested on the table. Subjects held this position for 90 seconds (Load I). Subjects then either received massage on the lumbar region or rested for 5 minutes, then repeated the same load (Load II). Skin blood flow (SBF), muscle blood volume (MBV), skin temperature (ST), and subjects' subjective feelings of fatigue were evaluated using Visual Analogue Scale (VAS)., Results: An increase of MBV between pre- and post-load II periods was higher after massage than after rest (p<0.05). An increase of SBF at pre- and post-load II was observed only under massage condition. An increase of SBF between post-load I and pre-load II periods was higher after massage than after rest (p<0.05). An increase of ST between post-load I and post-load II periods was greater after massage than after rest (p<0.05). The VAS score was lower with massage than with rest in the post-treatment period (p<0.01)., Conclusions: A significant difference was observed between massage and rest condition on VAS for muscle fatigue. Lumbar massage administration also appeared to have some effect on increasing skin temperature and enhancement of blood flow in local regions.

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.

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.

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.

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.

Background: There is not enough evidence to support the efficacy of massage for muscle fatigue despite wide utilization of the modality in various clinical settings. This study investigated the influence of massage application on localized back muscle fatigue., Methods: Twenty-nine healthy subjects participated in two experimental sessions (massage and rest conditions). On each test day, subjects were asked to lie in the prone position on a treatment table and perform sustained back extension for 90 seconds. Subjects then either received massage on the lumbar region or rested for a 5 minute duration, then repeated the back extension movement. The median frequency (MDF), mean power frequency (MNF), and root mean square (RMS) amplitude of electromyographic signals during the 90 second sustained lumbar muscle contraction were analyzed. The subjective feeling of fatigue was then evaluated using the Visual Analogue Scale (VAS)., Results: MDF and MNF significantly declined with time under all conditions. There was no significant difference in MDF, MNF or RMS value change between before and after massage, or between rest and massage conditions. There was a significant increase in fatigue VAS at the end of the 2nd back extension with rest condition. There was a significant difference in fatigue VAS change between massage and rest condition., Conclusions: A significant difference was observed between massage and rest condition on VAS for muscle fatigue. On EMG analysis, there were no significant differences to conclude that massage stimulation influenced the myoelectrical muscle fatigue, which is associated with metabolic and electrical changes.

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.

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.

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.

Cumulative evidence suggests that immunologic responses are under the regulation of the autonomic nervous system. Since acupuncture has recently been reported to modulate the autonomic nervous system, we investigated the possibility that acupuncture eventually modulates the immune system. In the present study, electro-acupuncture was applied in young volunteer subjects. As for the proportions of granulocytes and lymphocytes in the blood, there were three groups: (1) granulocytosis and lymphocytopenia; (2) granulocytopenia and lymphocytosis; and (3) normal pattern. Interestingly, with the administration of acupuncture, the status of subjects with relatively low levels of granulocytes and high levels of lymphocytes shifted to Group 1, whereas that of subjects with high levels of granulocytes and low levels of lymphocytes shifted to Group 2. In other words, acupuncture tended to normalize the pattern of leukocytes. We confirmed that acupuncture induced parasympathetic nerve stimulation, resulting in a decrease in the heart rate. These results suggest possible mechanisms underlying how acupuncture ameliorates the condition of patients with many chronic diseases.

The effects of electro-acupuncture stimulation (EAS) of two different areas of a hindlimb with different stimulus intensities on sympathoadrenal medullary functions were examined in anesthetized artificially ventilated rats. Two needles of 160 microm diameter and about 5 mm apart were inserted about 5 mm deep into a hindpaw (Chungyang, S42) or a hind leg (Tsusanli, S36) and current of various intensities passed to excite various afferent nerve fiber groups at a repetition rate of 20 Hz and pulse duration of 0.5 ms for 30-60 s. Fiber groups of afferent nerves stimulated in a hindlimb were monitored by recording evoked action potentials from the afferents innervating the areas stimulated. The sympathoadrenal medullary functions were monitored by recording adrenal sympathetic efferent nerve activity and secretion rates of catecholamines from the adrenal medulla. EAS of a hindpaw at a stimulus strength sufficient to excite the group III and IV somatic afferent fibers produced reflex increases in both adrenal sympathetic efferent nerve activity and the secretion rate of catecholamines. EAS of a hind leg at a stimulus strength sufficient to excite the group III and IV afferent fibers produced reflex responses of either increases or decreases in sympathoadrenal medullary functions. All responses of adrenal sympathetic efferent nerve activity were lost after cutting the afferent nerves ipsilateral to the stimulated areas, indicating that the responses are the reflexes whose afferents nerve pathway is composed of hindlimb somatic nerves. It is concluded that electro-acupuncture stimulation of a hindpaw causes an excitatory reflex, while that of a hind leg causes either excitatory or inhibitory reflex of sympathoadrenal medullary functions, even if both group III and IV somatic afferent fibers are stimulated.