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1.  Truncated SSX Protein Suppresses Synovial Sarcoma Cell Proliferation by Inhibiting the Localization of SS18-SSX Fusion Protein 
PLoS ONE  2013;8(10):e77564.
Synovial sarcoma is a relatively rare high-grade soft tissue sarcoma that often develops in the limbs of young people and induces the lung and the lymph node metastasis resulting in poor prognosis. In patients with synovial sarcoma, specific chromosomal translocation of t(X; 18) (p11.2;q11.2) is observed, and SS18-SSX fusion protein expressed by this translocation is reported to be associated with pathogenesis. However, role of the fusion protein in the pathogenesis of synovial sarcoma has not yet been completely clarified. In this study, we focused on the localization patterns of SS18-SSX fusion protein. We constructed expression plasmids coding for the full length SS18-SSX, the truncated SS18 moiety (tSS18) and the truncated SSX moiety (tSSX) of SS18-SSX, tagged with fluorescent proteins. These plasmids were transfected in synovial sarcoma SYO-1 cells and we observed the expression of these proteins using a fluorescence microscope. The SS18-SSX fusion protein showed a characteristic speckle pattern in the nucleus. However, when SS18-SSX was co-expressed with tSSX, localization of SS18-SSX changed from speckle patterns to the diffused pattern similar to the localization pattern of tSSX and SSX. Furthermore, cell proliferation and colony formation of synovial sarcoma SYO-1 and YaFuSS cells were suppressed by exogenous tSSX expression. Our results suggest that the characteristic speckle localization pattern of SS18-SSX is strongly involved in the tumorigenesis through the SSX moiety of the SS18-SSX fusion protein. These findings could be applied to further understand the pathogenic mechanisms, and towards the development of molecular targeting approach for synovial sarcoma.
PMCID: PMC3793959  PMID: 24130893
2.  Bioavailability of intravenous fosphenytoin sodium in healthy Japanese volunteers 
To compare and evaluate the bioavailability for intravenous fosphenytoin sodium with that of intravenous phenytoin sodium in Japanese subjects. In study 1, healthy Japanese male volunteers received a 30-min infusion of 375 mg fosphenytoin sodium or an equimolar dose of 250 mg phenytoin by a double-blind, crossover method. In study 2, other healthy Japanese male volunteers received a 30-min or 10-min infusion of 563 mg fosphenytoin sodium, followed by a dose of 750 mg after 2 weeks in an unblinded manner. Comparing with 250 mg phenytoin sodium, 375 mg fosphenytoin sodium exhibited lower total plasma phenytoin Cmax, whereas the geometric mean ratio of the AUC of total and free phenyotoin for fosphenytoin sodium at a dose of 375 mg was very similar to phenytoin sodium at a equimolar dose of 250 mg (AUC0–t ratio: 0.98 and 1.02, respectively). Therefore, fosphenytoin is almost completely converted to phenytoin in subjects. Fosphenytoin sodium was rapidly converted to phenytoin at doses of 375, 563, and 750 mg. The maximum concentration (Cmax) of total plasma phenytoin increased in a dose-dependent manner. The area under the plasma concentration–time curve (AUC) increased slightly more than proportionally with the administered dose, and clearance (CL) decreased with increasing dose. Pain and other infusion-site reactions were reported by all 12 subjects with phenytoin sodium, whereas very few symptoms were observed with fosphenytoin sodium. In conclusion, fosphenytoin sodium is considered to be a useful substitute for phenytoin sodium with almost no associated injection-site reactions.
PMCID: PMC3664181  PMID: 22968854
Fosphenytoin sodium injection; Phenytoin; Pharmacokinetics; Safety; Tolerability
3.  Novel Direct Targets of miR-19a Identified in Breast Cancer Cells by a Quantitative Proteomic Approach 
PLoS ONE  2012;7(8):e44095.
The miR-17–92 cluster encodes 7 miRNAs inside a single polycistronic transcript, and is known as a group of oncogenic miRNAs that contribute to tumorigenesis in several cancers. However, their direct targets remain unclear, and it has been suggested that a single miRNA is capable of reducing the production of hundreds of proteins. The majority of reports on the identification of miRNA targets are based on computational approaches or the detection of altered mRNA levels, despite the fact that most miRNAs are thought to regulate their targets primarily by translational inhibition in higher organisms. In this study, we examined the target profiles of miR-19a, miR-20a and miR-92-1 in MCF-7 breast cancer cells by a quantitative proteomic strategy to identify their direct targets. A total of 123 proteins were significantly increased after the endogenous miR-19a, miR-20a and miR-92-1 were knocked down, and were identified as potential targets by two-dimensional electrophoresis and a mass spectrometric analysis. Among the upregulated proteins, four (PPP2R2A, ARHGAP1, IMPDH1 and NPEPL1) were shown to have miR-19a or miR-20a binding sites on their mRNAs. The luciferase activity of the plasmids with each binding site was observed to decrease, and an increased luciferase activity was observed in the presence of the specific anti-miRNA-LNA. A Western blot analysis showed the expression levels of IMPDH1 and NPEPL1 to increase after treatment with anti-miR-19a, while the expression levels of PPP2R2A and ARHGAP1 did not change. The expression levels of IMPDH1 and NPEPL1 did not significantly change by anti-miR-19a-LNA at the mRNA level. These results suggest that the IMPDH1 and NPEPL1 genes are direct targets of miR-19a in breast cancer, while the exogenous expression of these genes is not associated with the growth suppression of MCF-7 cells. Furthermore, our proteomic approaches were shown to be valuable for identifying direct miRNA targets.
PMCID: PMC3431339  PMID: 22952885
4.  Population pharmacokinetics of phenytoin after intravenous administration of fosphenytoin sodium in pediatric patients, adult patients, and healthy volunteers 
We performed a population pharmacokinetic analysis of phenytoin after intravenous administration of fosphenytoin sodium in healthy, neurosurgical, and epileptic subjects, including pediatric patients, and determined the optimal dose and infusion rate for achieving the therapeutic range.
We used pooled data obtained from two phase I studies and one phase III study performed in Japan. The population pharmacokinetic analysis was performed using NONMEM software. The optimal dose and infusion rate were determined using simulation results obtained using the final model. The therapeutic range for total plasma phenytoin concentration is 10–20 μg/mL.
We used a linear two-compartment model with conversion of fosphenytoin to phenytoin. Pharmacokinetic parameters of phenytoin, such as total clearance and central and peripheral volume of distribution were influenced by body weight. The dose simulations are as follows. In adult patients, the optimal dose and infusion rate of phenytoin for achieving the therapeutic range was 22.5 mg/kg and 3 mg/kg/min respectively. In pediatric patients, the total plasma concentration of phenytoin was within the therapeutic range for a shorter duration than that in adult patients at 22.5 mg/kg (3 mg/kg/min). However, many pediatric patients showed phenytoin concentration within the toxic range after administration of a dose of 30 mg/kg.
The pharmacokinetics of phenytoin after intravenous administration of fosphenytoin sodium could be described using a linear two-compartment model. The administration of fosphenytoin sodium 22.5 mg/kg at an infusion rate of 3 mg/kg/min was optimal for achieving the desired plasma phenytoin concentration.
PMCID: PMC3572369  PMID: 22918614
Fosphenytoin sodium injection; Phenytoin; Status epilepticus; Epileptic seizure; Population pharmacokinetics
5.  MET gene amplification or EGFR mutation activate MET in lung cancers untreated with EGFR tyrosine kinase inhibitors 
We analyzed MET protein and copy number in NSCLC with or without EGFR mutations untreated with EGFR tyrosine kinase inhibitors (TKIs). MET copy number was examined in 28 NSCLC and 4 human bronchial epithelial cell lines (HBEC) and 100 primary tumors using quantitative real-time PCR. Positive results were confirmed by array comparative genomic hybridization and fluorescence in-situ hybridization. Total and phospho-MET protein expression was determined in 24 NSCLC and 2 HBEC cell lines using Western blot. EGFR mutations were examined for exon 19 deletions, T790M, and L858R. Knockdown of EGFR with siRNA was performed to examine the relation between EGFR and MET activation. High-level MET amplification was observed in 3 of 28 NSCLC cell lines and in 2 of 100 primary lung tumors that had not been treated with EGFR-TKIs. MET protein was highly expressed and phosphorylated in all the 3 cell lines with high MET amplification. In contrast, 6 NSCLC cell lines showed phospho-MET among 21 NSCLC cell lines without MET amplification (p = 0.042). Furthermore, those 6 cell lines harboring phospho-MET expression without MET amplification were all EGFR mutant (p = 0.0039). siRNA-mediated knockdown of EGFR abolished phospho-MET expression in examined 3 EGFR mutant cell lines of which MET gene copy number was not amplified. By contrast, phospho-MET expression in 2 cell lines with amplified MET gene was not down-regulated by knockdown of EGFR. Our results indicated that MET amplification was present in untreated NSCLC and EGFR mutation or MET amplification activated MET protein in NSCLC.
PMCID: PMC2767331  PMID: 19117057
MET; amplification; EGFR; gefitinib; lung cancer
6.  Increased expression of cytosolic chaperonin CCT in human hepatocellular and colonic carcinoma 
Cell Stress & Chaperones  2001;6(4):345-350.
The chaperonin-containing t-complex polypeptide 1 (CCT) is a hetero-oligomeric molecular chaperone that assists in the folding of actin, tubulin, and other cytosolic proteins. We recently reported that the expression level of CCT is closely correlated with growth rates of mammalian cultured cells. Here we examine the levels of CCT subunits and other molecular chaperones in tumor tissues of patients with hepatocelluar and colonic carcinoma, and compare them with nontumor tissues in the same patients. Expression levels of CCTβ in tumor tissues was significantly higher than in nontumor tissues in all patients with hepatocellular carcinoma (n = 15) and 83% of patients with colonic carcinoma (n = 17). The increased level of CCT expression in colonic cancer cells was confirmed by immunohistochemistry with anti-CCTβ antibody. The levels of CCTβ were highly correlated (r = 0.606) with those of the proliferating cell nuclear antigen (PCNA), which was used as an indicator of cell growth. CCTα gave similar results, although the correlation with PCNA levels was weaker. Other cytosolic and endoplasmic reticulum chaperones also showed higher expression in significant numbers of tumor tissues but less frequently than that observed with CCT. These results suggest that CCT is up-regulated in rapidly proliferating tumor cells in vivo to effectively produce proteins required for growth, and may serve as a useful tumor marker because it is widely distributed in the cytosol.
PMCID: PMC434417  PMID: 11795471
7.  Biochemical Studies on the χ Mutation of Bacteriophage T4: Differential Inhibition of χ+ and χ DNA Synthesis by Mitomycin C 
Journal of Virology  1974;13(1):1-8.
Biochemical studies were carried out to determine the effect of χ mutation on T4 DNA synthesis. The rate and final extent of DNA synthesis are almost the same with T4D- and T4χ-infected cells, although the burst size of T4χ is about one-sixth that of the wild type. The DNA synthesis of T4χ-infected cells is more readily inhibited by mitomycin C than is that of T4 wild type. When mitomycin C was added during active phage growth, DNA synthesis of T4χ halted almost immediately. T4 DNA polymerases isolated from χ+- and χ-infected cells, however, exhibit no difference with regard to their sensitivities to mitomycin C, priming activities with alkylated or ultraviolet light-irradiated templates and other enzymatic properties.
PMCID: PMC355251  PMID: 4359423

Results 1-7 (7)