Non-canonical endogenous expression of voltage-gated sodium channel NaV 1.7 subtype by the TE671 rhabdomyosarcoma cell line.

The human TE671 cell line was originally used as a model of medulloblastoma but has since been reassigned as rhabdomyosarcoma. Despite the characterised endogenous expression of voltage-sensitive sodium currents in these cells, the specific voltage-gated sodium channel (VGSC) subtype underlying these currents remains unknown. To profile the VGSC subtype in undifferentiated TE671 cells, endpoint and quantitative reverse transcription-PCR (qRT-PCR), western blot and whole-cell patch clamp electrophysiology were performed. qRT-PCR profiling revealed that expression of the SCN9A gene was ∼215-fold greater than the SCN4A gene and over 400-fold greater than any of the other VGSC genes, while western blot confirmed that the dominant SCN9A RNA was translated to a protein with a molecular mass of ∼250 kDa. Elicited sodium currents had a mean amplitude of 2.6 ± 0.7 nA with activation and fast inactivation V50 values of -31.9 ± 1.1 and -69.6 ± 1.0 mV, respectively. The currents were completely and reversibly blocked by tetrodotoxin at concentrations greater than 100 nm (IC50  = 22.3 nm). They were also very susceptible to the NaV 1.7 specific blockers Huwentoxin-IV and Protoxin-II with IC50 values of 14.6 nm and 0.8 nm, respectively, characteristic of those previously determined for NaV 1.7. Combined, the results revealed the non-canonical and highly dominant expression of NaV 1.7 in the human TE671 rhabdomyosarcoma cell line. We show that the TE671 cell line is an easy to maintain and cost-effective model for the study of NaV 1.7, a major target for the development of analgesic drugs and more generally for the study of pain. KEY POINTS: Undifferentiated TE671 cells produce a voltage-sensitive sodium current when depolarised. The voltage-gated sodium channel isoform expressed in undifferentiated TE671 cells was previously unknown. Through qRT-PCR, western blot and toxin pharmacology, it is shown that undifferentiated TE671 cells dominantly (>99.5%) express the NaV 1.7 isoform that is strongly associated with pain. The TE671 cell line is, therefore, a very easy to maintain and cost-effective model to study NaV 1.7-targeting drugs.

Chemical Synthesis of a Functional Fluorescent-Tagged α-Bungarotoxin.

α-bungarotoxin is a large, 74 amino acid toxin containing five disulphide bridges, initially identified in the venom of Bungarus multicinctus snake. Like most large toxins, chemical synthesis of α-bungarotoxin is challenging, explaining why all previous reports use purified or recombinant α-bungarotoxin. However, only chemical synthesis allows easy insertion of non-natural amino acids or new chemical functionalities. Herein, we describe a procedure for the chemical synthesis of a fluorescent-tagged α-bungarotoxin. The full-length peptide was designed to include an alkyne function at the amino-terminus through the addition of a pentynoic acid linker. Chemical synthesis of α-bungarotoxin requires hydrazide-based coupling of three peptide fragments in successive steps. After completion of the oxidative folding, an azide-modified Cy5 fluorophore was coupled by click chemistry onto the toxin. Next, we determined the efficacy of the fluorescent-tagged α-bungarotoxin to block acetylcholine (ACh)-mediated currents in response to muscle nicotinic receptor activation in TE671 cells. Using automated patch-clamp recordings, we demonstrate that fluorescent synthetic α-bungarotoxin has the expected nanomolar affinity for the nicotinic receptor. The blocking effect of fluorescent α-bungarotoxin could be displaced by incubation with a 20-mer peptide mimicking the α-bungarotoxin binding site. In addition, TE671 cells could be labelled with fluorescent toxin, as witnessed by confocal microscopy, and this labelling was partially displaced by the 20-mer competitive peptide. We thus demonstrate that synthetic fluorescent-tagged α-bungarotoxin preserves excellent properties for binding onto muscle nicotinic receptors.

Systems Approaches in the Common Metabolomics in Acute Lymphoblastic Leukemia and Rhabdomyosarcoma Cells: A Computational Approach.

Acute lymphoblastic leukemia is the most common childhood malignancy. Rhabdomyosarcoma, on the other hand, is a rare type of malignancy which belongs to the primitive neuroectodermal family of tumors. The aim of the present study was to use computational methods in order to examine the similarities and differences of the two different tumors using two cell lines as a model, the T-cell acute lymphoblastic leukemia CCRF-CEM and rhabdomyosarcoma TE-671, and, in particular, similarities of the metabolic pathways utilized by two different cell types in vitro. Both cell lines were studied using microarray technology. Differential expression profile has revealed genes with similar expression, suggesting that there are common mechanisms between the two cell types, where some of these mechanisms are preserved from their ancestor embryonic cells. Expression of identified species was modeled using known functions, in order to find common patterns in metabolism-related mechanisms. Species expression manifested very interesting dynamics, and we were able to model the system with elliptical/helical functions. We discuss the results of our analysis in the context of the commonly occurring genes between the two cell lines and the respective participating pathways as far as extracellular signaling and cell cycle regulation/proliferation are concerned. In the present study, we have developed a methodology, which was able to unravel some of the underlying dynamics of the metabolism-related species of two different cell types. Such approaches could prove useful in understanding the mechanisms of tumor ontogenesis, progression, and proliferation.

The human rhabdomyosarcoma cell line TE671--Towards an innovative production platform for glycosylated biopharmaceuticals.

The market of therapeutic glycoproteins (including coagulation factors, antibodies, cytokines and hormones) is one of the profitable, fast-growing and challenging sectors of the biopharmaceutical industry. Although mammalian cell culture is still expensive and technically complex, the ability to produce desired post-translational modifications, in particular glycosylation, is a major issue. Glycans can influence ligand binding, serum half-life as well as biological activity or product immunogenicity. Aiming to establish a novel production platform for recombinant glycoproteins, the human TE671 cell line was investigated. Since the initial analysis of cell membrane proteins showed a promising glycosylation of TE671 cells for biotechnological purposes, we focused on the recombinant expression of two model glycoproteins of therapeutical relevance. The optimization of the cell transfection procedure and serum-free expression succeeded for the human serine protease inhibitor alpha-1-antitrypsin (A1AT) and the hematopoietic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). N-glycan analyses of both purified proteins by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry provided first fundamental insights into the TE671 glycosylation potential. Besides protein specific pattern, strong distinctions - in particular for N-glycan fucosylation and sialylation - were observed depending on the medium conditions of the respective TE671 cell cultivations. The cell line's ability to synthesize complex and highly sialylated N-glycan structures has been shown. Our results demonstrate the TE671 cell line as a serious alternative to other existing human expression systems.

Osthole induces apoptosis, suppresses cell-cycle progression and proliferation of cancer cells.

BACKGROUND: The aim of the present study was to determine the effects of osthole on cell proliferation and viability, cell-cycle progression and induction of apoptosis in human laryngeal cancer RK33 and human medulloblastoma TE671 cell lines. MATERIALS AND METHODS: Cell viability was measured by means of the MTT method and cell proliferation by the 5-bromo-2-deoxyuridine (BrdU) incorporation assay. Cell-cycle progression was determined by flow cytometry, and induction of apoptosis by release of oligonucleosomes to the cytosol. The gene expression was estimated by a quantitative polymerase chain reaction (qPCR) method. High-performance counter-current chromatography (HPCCC) was applied for isolation of osthole from fruits of Mutellina purpurea. RESULTS: Osthole decreased proliferation and cell viability of cancer cells in a dose-dependent manner. The tested compound induced apoptosis, increased the cell numbers in G1 and decreased cell number in S/G2 phases of the cell cycle, differentially regulating CDKN1A and TP53 gene expression depending on cancer cell type. CONCLUSION: Osthole could be considered as a potential compound for cancer therapy and chemoprevention.

HIV-1 Tat down-regulates expression of mitotic centromere-associated kinesin through inhibiting promoter activity / 第二军医大学学报

Objective: To investigate the regulatory effect of HIV-1 Tat on mitotic centromere-associated kinesin (MCAK) expression and the related mechanism. Methods: Tat-expression TE671 cell model and purified prokaryotically expressed Tat protein were used to verify the down-regulated expression of MCAK using Northern blotting and Western blotting analysis. Various DNA fragments in the promoter region of the MCAK gene were amplified with PCR, linked to the luciferase reporter plasmid, and then transferred into TE671 cells. Luciferase activity analysis was performed to measure the promoter activity of various DNA fragments, so as to determine the active promoter region of MCAK gene. Moreover, HIV-1 Tat protein was co-incubated with TE671 cells, and the promoter activity was detected to analyze the modulating effect of Tat on promoter activity in vitro. Results: The results of Northern blotting and Western blotting analysis indicated that the mRNA and protein levels of MCAK were strongly decreased by Tat protein. The core region of MCAK promoter was located in -399∼+1 bp region, and the promoter activity was strikingly inhibited by Tat protein. Conclusion HIV-1 Tat has a marked inhibitory effect on MCAK expression, which might be related to the decreased promoter activity caused by Tat protein.